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Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery
Accepted Manuscript Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery
Fernando Suaste-Olmos, Claudia Zirión-Martínez, Harumi Takano-Rojas, Leonardo Peraza-Reyes PII: DOI: Reference:
S0167-4889(18)30003-X https://doi.org/10.1016/j.bbamcr.2018.01.003 BBAMCR 18229
To appear in: Received date: Revised date: Accepted date:
21 August 2017 29 December 2017 3 January 2018
Please cite this article as: Fernando Suaste-Olmos, Claudia Zirión-Martínez, Harumi Takano-Rojas, Leonardo Peraza-Reyes , Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbamcr(2018), https://doi.org/10.1016/j.bbamcr.2018.01.003
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ACCEPTED MANUSCRIPT Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery
Fernando Suaste-Olmosa, Claudia Zirión-Martíneza, Harumi Takano-Rojasa,
Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular,
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Leonardo Peraza-Reyesa*
Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de México
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04510, Mexico.
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* Corresponding author:
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Instituto de Fisiología Celular, Universidad Nacional Autónoma de México Ciudad de México 04510, Mexico.
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Tel. (52) (55) 5622 5628
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E-mail address: [email protected]
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ACCEPTED MANUSCRIPT Abstract Peroxisomes are versatile organelles essential for diverse developmental processes. One such process is the meiotic development of Podospora anserina. In this fungus, absence of the docking peroxin PEX13, the RING-finger complex peroxins, or the PTS2 co-receptor
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PEX20 blocks sexual development before meiocyte formation. However, this defect is not
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seen in the absence of the receptors PEX5 and PEX7, or of the docking peroxins PEX14
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and PEX14/17. Here we describe the function of the remaining uncharacterized P. anserina peroxins predictably involved in peroxisome matrix protein import. We show that PEX8, as
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well as the peroxins potentially mediating receptor monoubiquitination (PEX4 and PEX22)
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and membrane dislocation (PEX1, PEX6 and PEX26) are indeed implicated in peroxisome matrix protein import in this fungus. However, we observed that elimination of PEX4 and
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PEX22 affects to different extent the import of distinct PEX5 cargoes, suggesting
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differential ubiquitination-complex requirements for the import of distinct proteins. In addition, we found that elimination of PEX1, PEX6 and PEX26 results in loss of
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peroxisomes, suggesting that these peroxins restrain peroxisome removal in specific
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physiological conditions. Finally, we demonstrate that all analyzed peroxins are required for meiocyte formation, and that PEX20 function in this process depends on its potential
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monoubiquitination target cysteine. Our results suggest that meiotic induction relies on a peroxisome import pathway, which is not dependent on PEX5 or PEX7 but that is driven by an additional cycling receptor. These findings uncover a collection of peroxins implicated in modulating peroxisome activity to facilitate a critical developmental cell fate decision. Keywords. Peroxisome / Meiosis / Sexual development / Fungi / Organelle biogenesis
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ACCEPTED MANUSCRIPT 1. Introduction Peroxisomes are versatile organelles that perform diverse cellular functions essential for development. Peroxisomes have conserved roles in reactive oxygen species (ROS)1 and lipid metabolism, and they execute distinct specialized metabolic functions, which can be
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specific to given organisms [1-3]. In addition, peroxisomes participate in diverse cellular
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functions ranging from intracellular cholesterol transport in mammals [4] to the formation
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of specialized organelles –the Woronin bodies– involved in intercellular channel gating in fungi [5]. Furthermore, peroxisomes are increasingly recognized as signaling organelles
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that play different roles in the orchestration of complex signaling pathways [6].
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Peroxisomes are highly dynamic organelles that adapt their composition, number and arrangement in response to environmental, physiological and developmental cues [7-
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10]. The significance and versatility of peroxisomes is reflected by the diverse
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developmental processes that depend on their function; for example, mammalian processes like audition [11], the development of the nervous system [12], the adipose tissue [13, 14]
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and the skin [15] rely on peroxisomes. Furthermore, these developmental processes can
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involve rather diverse peroxisomal activities. For instance, audition depends on an adaptive proliferation of peroxisomes, which protect the auditory system from noise exposure-
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produced ROS-induced damage [11]; whereas skin formation relies on a correct peroxisome distribution during mitosis, which is critical to define the cell division plane of epidermal progenitor cells and to determine the balance between their proliferation and differentiation [15]. Abbreviations: MCC, Manders correlation coefficient; PCC, Pearson’s correlation coefficient; PMP, peroxisome membrane protein; PTS, peroxisomal-targeting signal; ROS, reactive oxygen species. 1
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ACCEPTED MANUSCRIPT Peroxisome formation and maintenance depend on a group of proteins referred to as peroxins. Although the peroxin composition between species can differ; most peroxins mediating peroxisome biogenesis throughout different eukaryotic lineages are conserved [1]. Peroxisome formation depends on two groups of peroxins, which drive the import of
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proteins into the peroxisome membrane and the peroxisome matrix, respectively. The
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targeting and insertion of peroxisome membrane proteins (PMPs) depend on the conserved
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peroxins Pex3 and Pex19. Pex19 is a receptor and chaperone protein that recognizes most PMPs in the cytosol and subsequently delivers them to peroxisomes by docking on the
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PMP Pex3. An additional membrane peroxin –Pex16– also acts as a receptor for some
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PMPs, including Pex3; however, the function of this peroxin is less conserved [16]. In addition to direct targeting from the cytosol, a number of PMPs traffic through different
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routes to reach peroxisomes, including the endoplasmic reticulum [17] and mitochondria
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[18]. On the other hand, the import of peroxisome matrix proteins is driven by two conserved import pathways, which are mediated by the receptors Pex5 and Pex7. These
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peroxins recognize the peroxisome targeting signals (PTS1 and PTS2, respectively) of
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peroxisome matrix proteins in the cytosol and target them to the organelle [19]. Peroxisome targeting by Pex7 requires accessory proteins known as PTS2 co-receptors that are species
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specific, like Pex20 in most ascomycete fungi, the paralogous Pex18/Pex21 in some yeasts, and a long Pex5 isoform in plants and animals [20]. Following cargo recognition, the receptor/cargo complex is anchored at the peroxisome membrane by interactions with the docking complex, which is composed of Pex13 and Pex14 (and Pex17 in yeasts or Pex14/17 in filamentous fungi), and then the import receptor is inserted into the peroxisome membrane. Import by both receptors depends on the docking complex; however, at least in species possessing PTS2 co-receptors different from Pex5, the 4
ACCEPTED MANUSCRIPT translocation of PTS1 and PTS2 cargoes proceeds through different channels. Actually, in Saccharomyces cerevisiae it has been demonstrated that Pex5 and the PTS2 co-receptor Pex18, respectively, constitute by themselves integral components of two independent channels through which PTS1 and PTS2 proteins are translocated [21, 22]. Both import
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channels also contain Pex14, whereas the PTS2 additionally contains Pex17. The precise
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mechanism of protein translocation is not known, but it is clear that the protein import
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process depends on the export of the receptors (or PTS2 co-receptor) from the peroxisome membrane. Actually, evidence suggests that the protein translocation process is
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mechanistically coupled to the export of the receptor (or PTS2 co-receptor) from the
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membrane (the export-driven protein import model) [23, 24]. However, there are data also indicating that protein translocation is concomitant with the insertion of the import receptor
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into the membrane rather than with its subsequent release from it [25]. The removal of the
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receptors and PTS2 co-receptors from the peroxisome membrane is mediated by the peroxisome receptor export machinery (the exportomer [26]). Export of Pex5 and PTS2 co-
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receptors depends on their ubiquitination. Actually, different types of ubiquitination define
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the fate of these peroxins. Whereas their monoubiquitination facilitates their recycling, their polyubiquitination directs them to degradation by the proteasome. Both types of
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ubiquitination require the RING-finger complex –a peroxisome membrane complex composed of the E3 ubiquitin ligases Pex2, Pex10 and Pex12. Receptor monoubiquitination is catalyzed by specific ubiquitin-conjugating E2 enzymes, like Pex4 along with its membrane-anchor and activator Pex22 in yeasts [27]. The RING-finger complex also interacts with the docking complex via Pex8, producing a larger complex known as the importomer [28]. Ubiquitination primes the receptor for the dislocation machinery, which extracts the receptor from the peroxisome membrane. The dislocation complex is composed 5
ACCEPTED MANUSCRIPT of the dislocases Pex1 and Pex6, two AAA+ ATPases that are anchored to the peroxisome membrane by the tail-anchored PMP Pex26 (Pex15 in yeasts) [29]. Following their release from peroxisomes and their deubiquitination, the receptor/co-receptors are engaged in further rounds of protein import.
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Sexual development of fungi has provided notable examples of diverse
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developmental processes that importantly depend on peroxisomes [30]. One such process is
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the meiotic development of the fungus Podospora anserina (this process is illustrated in Fig. S1A). In this fungus, meiotic development involves a precise modulation at specific
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stages of peroxisome shape, abundance and distribution [8], which is accompanied by
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changes in the functional state of the protein machinery that drives the import of proteins into the organelle [31]. This indicates that peroxisome function during this process is
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defined by a complex modulation of the proteins mediating peroxisome dynamics and
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assembly. Furthermore, in P. anserina both the initiation and progression of meiotic development depend on peroxisomes, albeit on different activities. Nuclear progression
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through meiosis depends on the import receptors PEX5 and PEX7 [32]. In contrast,
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initiation of meiosis depends on specific peroxins that include PEX3 and PEX19, the docking peroxin PEX13, the RING-finger peroxins and the PTS2 co-receptor PEX20, but
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does not require neither of the import receptors (PEX5 and PEX7) nor the docking peroxins PEX14 or PEX14/17 [31-34]. This has led us to propose the existence of an alternative import pathway, which operates in absence of both known import receptors and that is required for meiotic induction. Alternatively, this process could depend on the concerted action of the aforementioned peroxins in a function independent from their activity in peroxisome matrix protein import. To provide further evidence substantiating either of these alternatives, here we investigate the role of the P. anserina peroxisome receptor 6
ACCEPTED MANUSCRIPT export machinery in peroxisome biogenesis and sexual development. By analyzing the function of the two subcomplexes that compose the exportomer –the ubiquitination and the dislocation complexes–, and of the peroxin that links the exportomer to the peroxisome protein import machinery (PEX8), we have obtained strong evidence supporting the
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existence of an alternative peroxisome import pathway required for meiotic development
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initiation.
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2. Material and methods
2.1. Strains and Culture conditions. The P. anserina strains used in this research are
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derived from the "S" wild-type strain. P. anserina ∆pex10 and ∆pex20 strains were
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generated previously [31, 33], and the ∆ku70 strain [35] was kindly provided by Robert
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Debuchy (I2BC, Gif-sur-Yvette, France). P. anserina ∆pex5 strain [32], and the strains expressing GFP-PTS1 or PTS2-GFP in the wild-type genetic context were kindly provided
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by Véronique Berteaux-Lecellier (CRIOBE, CNRS, French Polynesia). Culture media
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consisted on M2 minimal medium containing 1.1% dextrin, 0.3% glucose or 0.05% oleic acid (plus 0.2% TWEEN 40 used as emulsifier) as sole carbon sources. To facilitate the
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growth of pex mutants on oleic acid-based medium, this medium was added with 0.03% of glucose. Ascospores were germinated in G medium supplemented with 0.5% yeast extract, and protoplasts were regenerated on RG medium. When required, media were supplemented with phleomycin (25 g mL-1), geneticin (G418 sulfate, 100 g mL-1), nourseothricin (40 g mL-1) or hygromycin B (75 g mL-1 for pBC-Hygro-derived constructs, or 30 g mL-1 for pUCHygro-derived constructs). P. anserina media and current methods can be found at http://podospora.igmors.u-psud.fr. 7
ACCEPTED MANUSCRIPT 2.2. Nucleic acid isolation, transformation and plasmids. The isolation of genomic DNA and the transformation of P. anserina were performed according to [36]. Streptomyces noursei nat1 gene was obtained from plasmid pAPI509, which is a derivative from pAPI508 [35]. Escherichia coli hph gene was obtained from pBC-Hygro [37] or pUCHygro
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[38] and the Aspergillus nidulans trpC transcription terminator was obtained from
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pMOcosX [39]. Plasmids pAPI509, pMOcosX and pSM334_Genticin were kindly
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provided by Robert Debuchy, and pBC-Hygro by Philippe Silar (Univ. Paris Diderot, Paris, France). Oligonucleotide primers used in this research are shown on Table S1.
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2.3. Gene sequences. PEX1 (Pa_1_6740), PEX4 (Pa_1_9240), PEX6 (Pa_1_9330), PEX8
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(Pa_7_9670), PEX22 (Pa_6_4620) and PEX26 (Pa_7_1760) gene sequences were obtained from the P. anserina genome sequence (http://podospora.igmors.u-psud.fr). The predicted
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protein sequences are available in the GenBank database under accession numbers
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CDP23014.1, CDP23277.1, CDP23286.1, CDP32486.1, CDP30493.1, and CDP31995.1, respectively. FOX2, PEX14 and PEX20 genes were previously reported [31, 33, 40].
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2.4. Gene deletions. Mutant strains deleted for each peroxin gene were generated by
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replacing the corresponding ORF by a selectable marker gene by homologous recombination. PEX1 and PEX4 were replaced by pBC-Hygro-derived hph gene. PEX6,
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PEX8, PEX22 and PEX26 were replaced by the nat1 gene. Gene replacement constructs were generated by double-joint PCR [41] and consisted on the selectable marker gene flanked by 700 bp of the 5′ and 3′ flanking sequences of the respective ORF. For each construct, the 5' fragment was amplified with primers denoted as pexN-5F and pexN-5R (were N designates the corresponding peroxin number, Table S1), the 3' fragment with primers pexN-3F and pexN-3R, and the corresponding selectable marker with primers
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ACCEPTED MANUSCRIPT pexN-hphF and pexN-hphR (for PEX1 and PEX4) or pexN-natF and pexN-natR (for the remaining peroxins). The final fusion PCR products were gel-purified and directly used to transform protoplasts of a ∆ku70 strain. Randomly selected transformants were purified after crosses to the wild-type strain, and the gene deletions were recovered in the KU70+
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genetic background. Correct gene replacements were verified by PCR analyses.
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2.5. Gene complementation analyses. For the gene complementation assays, a given
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mutant was complemented by ectopically introducing a wild-type copy of the corresponding deleted gene. PEX4 and PEX8 alleles were derived from plasmids
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GA0AA328DB09 and GA0AA242CA11 (P. anserina genomic-DNA library, Genoscope,
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France), respectively. The remaining genes were obtained by PCR using genomic DNA as template and the respective pexN-5F / pexN-3R primers. The plasmids or PCR products
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were directly used to co-transform the corresponding mutant strain with the
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pSM334_Genticin vector, and geneticin-resistant transformants were analyzed for peroxisome protein import (tested for GFP-PTS1 and/or FOX2-mCherry) and sexual
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development.
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2.6. Generation of pUC-GFP and pUC-Cherry plasmids. In order to tag PEX14 and FOX2, first we constructed two plasmids that allow fusing the coding sequence of GFP (or
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mCherry) to the 3' end of a given gene at its chromosomal location. For this, we generated a construct consisting on pEGFP1-derived EGFP gene preceded by a sequence coding for a 7-amino-acid linker peptide (as optimized by [42]) and followed by the A. nidulans trpC terminator. This construct was obtained by fusing the trpC terminator (PCR-amplified with primers gfp-trpC and trpC-R) to the 3' end of EGFP gene (amplified with primers lkt-gfp and trp-gfp) by fusion PCR. The 7 amino-acid linker-peptide was added to this construction by including its coding nucleotide sequence as an in-frame overhang on EGFP forward 9
ACCEPTED MANUSCRIPT primer (lkt-gfp). The flanking primers (lkt-gfp and trpC-R) used to obtain this construct contained EcoRI and SacI restriction sites, respectively; which allowed cloning the resulting construct in the corresponding restriction sites of plasmid pUC-Hygro. SacI site of pUC-Hygro is contiguous to the hygromycin B resistance cassette (HygR) of this plasmid;
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therefore, a 2.5kb cassette containing the EGFP gene followed by the HygR selectable
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marker (GFP-HygR) was generated in the resulting plasmid (pUC-GFP). The same strategy
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was followed to produce an equivalent plasmid (pUC-Cherry) containing a mCherry-HygR cassette.
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2.7. Tagging of FOX2 and PEX14. Tagging of PEX14 and FOX2 was done by fusing the
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coding sequence of GFP and mCherry, respectively, to the 3' end of PEX14 and FOX2 coding sequences at their chromosomal locations. To tag PEX14 we generated a construct
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consisting on the GFP-HygR cassette from pUC-GFP flanked by 800 bp of PEX14 stop
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codon-flanking sequences (PEX14::GFP-HygR::PEX14-3'UTR), in which the coding sequence of GFP was fused in-frame to the 3′ end of PEX14 ORF. This construct was
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obtained by fusing the following DNA sequences by double-joint PCR: (i) the last 798 bp
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(excluding the stop codon) of PEX14 ORF 3’ end (amplified with primers pex14-F and lktpex14), (ii) the GFP-HygR cassette from pUC-GFP (amplified with primers pex14-lkt and
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pex14-hph), and (iii) 861 bp of DNA downstream PEX14 stop codon (amplified with primers pex14-3F and pex14-3R). Following the same strategy, an equivalent construct based on the mCherry-HygR cassette was generated for FOX2. In this case, the resulting FOX2::mCherry-HygR::FOX2-3'UTR cassette consisted on the fusion of: (i) the last 680 bp (excluding the stop codon) of FOX2 ORF 3’ end (amplified with primers fox2-F and lktfox2), (ii) the mCherry-HygR cassette from pUC-Cherry (amplified with primers fox-lkt and
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ACCEPTED MANUSCRIPT fox-hph), and (iii) 685 bp of DNA downstream FOX2 stop codon (amplified with primers fox2-3F and fox2-3R). In this case, the resulting construction was cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA), following the provider instructions, to yield plasmid pFS02. Next, we gel-purified the PEX14::GFP-HygR::PEX14-3'UTR PCR
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construct and we recovered the FOX2::mCherry-HygR::FOX2-3'UTR cassette as a NotI
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fragment from plasmid pFS02, and each construct was used to transform cells of a ku70
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strain. HygR transformants were randomly selected and the HygR marker was recovered in
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the KU70+ genetic context after crosses to the wild type. All plasmids were verified by sequencing. FOX2 and PEX14 tagging was verified by PCR analyses and by sequencing.
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2.8. Site directed mutagenesis of PEX20. PEX20 gene containing 770 bp of its ORF 5’ upstream region to 802 bp downstream the stop codon was amplified by PCR using primers
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pex20-F and pex20-3R and cloned into pGEM-T Easy Vector to yield pFS03.
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Complementary mutagenic primers containing selected mutations to change PEX20 conserved cysteine 6 into alanine (primers pex20_C6A-F and pex20_C6A-R) or lysine
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(primers pex20_C6K-F and pex20_C6K-R) were used to amplify pFS03. The obtained PCR
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products were treated with DpnI and used to transform E. coli, and the recovered plasmids (pFS04 for pex20C6A and pFS05 for pex20C6K) were sequenced to verify the DNA
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substitutions. pFS03 and the mutated pFS04 and pFS05 plasmids were respectively used to co-transform P. anserina ∆pex20 cells with the pSM334_Genticin vector, and the obtained transformants were analyzed for sexual development in sexual crosses to a ∆pex20 strain. 2.9. Cytology. Sexual cycle cells were fixed in 7.4% paraformaldehyde and processed for fluorescence microscopy as described before [43]. To inspect sexual development, we analyzed the sexual tissues issued from 15 fruiting bodies (perithecia) in at least three
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ACCEPTED MANUSCRIPT biological replicates per strain (i.e, 45 perithecia / strain). Live-cell imaging was performed with whole individual P. anserina colonies grown for 24h on M2 plates as described in [8], except that agar was replaced by 2% agarose and 0.55% dextrin was used. To analyze peroxisome protein import we used the following proteins: (i) FOX2-mCherry,
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(ii) PEX14-GFP (above), (iii) GFP-PTS1 (GFP fused to the C-terminal PTS1 tripeptide
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SKL [44]) and (iv) PTS2-GFP (GFP fused to the N-terminal PTS2 sequence from P.
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anserina peroxisomal thiolase [32]). Nuclei were stained with DAPI (4',6-diamidino-2-
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phenylindole, dihydrochloride, Molecular Probes, Eugene, OR) (0.5 g mL-1) and cell membranes were stained with the styryl dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(6-
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(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide, Molecular Probes) (7M). 2.10. Microscopy. Imaging was performed on a Nikon Eclipse E600 microscope with a
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cooled Neo Andor sCMOS camera, or on a Zeiss LSM-800 inverted laser scanning
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confocal microscope using a Plan-Apochromat 63x/1.4 oil immersion objective and 405,
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488 and 561nm laser lines. For live-cell confocal microscopy, images from all channels were obtained simultaneously. Maximum intensity projections were done from z-section
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images collected at 0.43 m intervals through entire cell volumes. Images were processed
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using the FIJI package [45] for ImageJ [46] (NIH, Bethesda, USA) or ZEN 2012 (Carl Zeiss, Jena, Germany) software. Colocalization and peroxisome abundance analyses were done on confocal micrographs of growing leading hyphae using ZEN 2012 software. For colocalization analyses, 12-15 hyphae issued from 3 biological replicates (4-5 hyphae/replicate) were analyzed for each strain. For each hypha, a region of interest (ROI) was defined as the cell area extending ≈140μm behind the hyphal apex. Image thresholds were set with the Costes method, and the Pearson's and Manders correlation coefficients
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ACCEPTED MANUSCRIPT were calculated with the corresponding algorithms included in ZEN 2012. P values were calculated using paired two-tailed Student’s t-tests. Line scan analyses were performed on raw pixel data. To determine peroxisome abundance, the number of peroxisomes present in the middle plane area of three adjacent 140μm-long hyphal regions extending behind the
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apex was quantified. For each strain, 15 hyphae issued from 3 biological replicates (5
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hyphae/replicate) were analyzed. P values were calculated using unpaired two-tailed
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Student’s t-tests. To highlight cell outlines, a micrograph was merged to its corresponding
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phase-contrast image colored in blue and contrasted until only the cell outlines were visible.
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3. Results
3.1. P. anserina peroxisomes differ in their protein composition and distribution. To
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analyze the role of the exportomer in peroxisome protein targeting and import, we started
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by generating strains expressing mCherry- or GFP-tagged versions of endogenous peroxisome matrix and membrane proteins. We C-terminally tagged the matrix protein
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FOX2 with mCherry, and the PMP PEX14 with GFP. The function of both proteins in P.
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anserina has been previously studied [31, 33, 40]. FOX2, which catalyzes the second and third steps of the peroxisomal fatty-acid -oxidation pathway, lacks discernible peroxisome
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targeting sequence, but its import into peroxisomes depends on PEX5 [32]. Therefore, FOX2 constitutes a PEX5 cargo with targeting information different from canonical PTS1containing proteins. The import of these latter proteins in P. anserina can be analyzed using a peroxisome-targeted GFP containing a consensus PTS1 sequence (GFP-PTS1) [32, 44]. P. anserina mutant strains devoid of PEX14 or FOX2 are unable to grow or exhibit flimsy mycelial growth, respectively, on oleic acid-containing medium [33, 40]. In contrast,
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ACCEPTED MANUSCRIPT PEX14 or FOX2 tagging did not affect the growth of P. anserina on this medium (Fig. S1B), suggesting that the function of these proteins is not affected by the tagging. FOX2mCherry stained discrete punctae colocalizing with GFP-PTS1 (Fig. 1A) with a high Pearson’s correlation coefficient (PCC, avg. 0.87 ± 0.03 in 12 analyzed hyphae). However,
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the relative intensity of these two fluorescent proteins varied among different peroxisomes.
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A number of peroxisomes that was intensely labeled with FOX2-mCherry presented only
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faint GFP-PTS1 staining (e.g, peroxisome 1 in Fig. 1A), and vice versa (Fig. 1A, peroxisome 3). Manders correlation coefficient (MCC) for GFP (M1 0.788) was similar to
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that of FOX2-mCherry (M2 0.782), suggesting equivalent fractions for these two proteins
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not colocalizing with each other. These data show a high co-occurrence for GFP-PTS1 and
number of peroxisomes might differ.
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FOX2 in hyphal peroxisomes, but also reveal that the relative amount of these proteins in a
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In contrast, the overall colocalization of PEX14-GFP and FOX2-mCherry was relatively low (PCC, av. 0.49 ± 0.06, n=15 hyphae). In addition to peroxisomes that were
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labeled with both FOX2-mCherry and PEX14-GFP, we observed FOX2-mCherry-stained
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peroxisomes devoid of PEX14 labeling (e.g. peroxisome 1 in Fig. 1B), as well as PEX14decorated punctae exhibiting low (peroxisome 2 in Fig. 1B) or not detectable (Fig. 1C)
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FOX2-mCherry labeling. Interestingly, these latter often consisted on small punctae that were more noticeable at the apical part of hyphae (arrows in Fig. 1C). MCC for GFP (M1 0.596) was significantly lower than for mCherry (M2 0.643) (t=5.4, 14 d.f. P=9.23E-05), indicating a larger fraction of PEX14 not colocalizing with FOX2-mCherry than the opposite. In addition, we also observed that PEX14-GFP present in FOX2-mCherry-labeled peroxisomes often exhibited a heterogeneous distribution. This included round-to-elongated PEX14 patches partially overlapping with FOX2-stained peroxisomes (Fig. 1B, peroxisome 14
ACCEPTED MANUSCRIPT 3), uneven PEX14 patches with increasing fluorescent intensity (Fig. 1B, peroxisome 4) and clusters of PEX14 patches distributed along FOX2-labeled peroxisomes (Fig.1C, arrowheads). The latter two distributions were more noticeable in elongated peroxisomes. Furthermore, time-lapse live-cell imaging revealed that these PEX14-GFP-stained patches
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were highly dynamic and redistributed along peroxisomes over time (Movie 1). These
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observations suggest a dynamic PEX14 localization in specific domains of the peroxisome
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membrane. Altogether, the above findings suggest the existence of distinct peroxisome populations in P. anserina hyphae that differ in their protein composition and spatial
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distribution.
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3.2. The exportomer peroxins and PEX8 are required for fatty acid utilization and
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aerial mycelium formation. Next, to investigate the function of the exportomer peroxins, we generated strains deleted for the genes predictably encoding the ubiquitination-complex
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peroxins PEX4 and PEX22, the dislocation-complex peroxins PEX1, PEX6 and PEX26,
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and the peroxin PEX8. Deletion of either of these genes resulted in a phenotype that recapitulates the defects typically observed in P. anserina mutants defective for peroxisome
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biogenesis [31-34], including their incapacity to grow on oleic acid as sole carbon source, a
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reduced formation of aerial mycelium (Fig. S1B), and defective sexual spore (ascospore) pigmentation (assessable in heterozygous sexual crosses to a wild-type strain, shown for
pex8 in Fig. S1C). 3.3. PEX8 is required for peroxisome matrix protein import. Then, we sought to determine the role of these proteins in P. anserina peroxisome biogenesis. First we analyzed the role PEX8. PEX8 is highly conserved in fungi [47], however the function of this protein beyond yeasts has not been addressed. P. anserina PEX8 orthologue 15
ACCEPTED MANUSCRIPT predictably encodes a 691 aa protein (Fig. S2A) containing a canonical PTS1 SKL Cterminal tripeptide, and a N-terminal sequence similar to consensus PTS2 targeting sequences [i.e, R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)] [48]. To study the role of PEX8 in P. anserina peroxisome biogenesis we first tested whether this protein was
T
involved in peroxisome matrix protein import. We observed that, in contrast to the punctate
IP
pattern of GFP-PTS1 (Fig. 2A) and FOX2-mCherry (Fig. 2B) exhibited for peroxisomes in
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wild-type cells, both GFP-PTS1 (Fig. 2C) and FOX2-mCherry (Fig. 2D) remained in the
US
cytosol of pex8 hyphae, indicative of a defective peroxisome import of these two PEX5 cargoes in these cells. The peroxisome import of both GFP-PTS1 and FOX2-mCherry was
AN
restored upon ectopically reintroducing a wild-type copy of PEX8 gene into the pex8 strains (shown for FOX2 in Fig. S3A), corroborating the implication of PEX8 in this
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process.
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Then we investigated if PEX8 was required for the PTS2 peroxisome import
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pathway by studying the localization of a PTS2-containing GFP reporter protein (PTS2GFP). We analyzed PTS2-GFP localization in ascospores of asci (the meiocytes in which
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ascospores are differentiated after meiosis) issued from pex8 to WT heterozygous crosses,
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where the 2:2 segregation allows a direct comparison between WT and pex8 cells. We observed that PTS2-GFP localized to peroxisomes in WT ascospores, but mislocalized to cytosol in pex8 ascospores (Fig. S4A), indicating that PEX8 is also required for the PTS2 peroxisome protein import. Finally, we analyzed the role of PEX8 in PMP import by studying the localization of PEX14. We observed that PEX14-GFP localized to discrete punctae in pex8 hyphae (Fig. 3), which were more heterogeneous in shape than PEX14GFP-stained wild-type peroxisomes, consistent with a peroxisome-remnant labeling. This
16
ACCEPTED MANUSCRIPT shows that the formation of peroxisome membranes and the targeting of, at least, PEX14 into these membranes does not require PEX8. 3.4. PEX4 and PEX22 are differentially required for the import of distinct PEX5 cargoes. Next we studied the role of the proteins potentially involved in receptor
IP
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monoubiquitination. In yeasts, the monoubiquitination of Pex5 and of the PTS2 coreceptors is mediated by Pex4 and its anchor/activator Pex22 [49-55]. P. anserina
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possesses an orthologue of PEX4 and a gene likely coding for a Pex22-like protein (Fig. S2,
US
B and C). Pex22-like proteins are conserved in filamentous ascomycetes and they possess a domain similar to the Pex4-binding motif of Pex22 [47]. Actually, the Pex22-like protein of
AN
Colletotrichum orbiculare is functionally equivalent to S. cerevisiae Pex22 [56] (therefore,
M
we will further refer to this protein as Pex22).
We analyzed the effect of PEX4 and PEX22 deletion on GFP-PTS1 (Fig. 2, E and
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G) and PTS2-GFP (Fig. S4, B-C) localization, and we observed that these two proteins
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mislocalized to cytosol in pex4 and pex22 cells. We also observed that PEX14-GFP localized to discrete punctae in pex4 and pex22 hyphae (Fig. 3, C-D), which were
CE
similar to those observed in ∆pex8 cells. These observations indicate that PEX4 and PEX22
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are involved in the PTS1 and PTS2 peroxisome matrix import pathways, but they are not required for PMP import. Interestingly, however, FOX2-mCherry exhibited a dual localization in pex4 and pex22 hyphae. In these cells, a fraction of FOX2-mCherry localized to cytosol, but discrete punctae resembling peroxisomes were also clearly stained by FOX2-mCherry (Fig. 2, F and H). Double labeling experiments demonstrated that the FOX2-mCherry-labeled punctae present in pex4 and pex22 hyphae also contained PEX14 (shown for pex22 in Fig. 4A and for pex4 in Fig. S5), corroborating the
17
ACCEPTED MANUSCRIPT peroxisomal nature of these structures. In addition, we observed a number of peroxisomes in which PEX14-GFP predominantly decorated the organelle periphery, whereas FOX2mCherry stained its interior (Fig. 4B and Movie 2), suggesting that FOX2-mCherry is actually imported into the matrix of these peroxisomes. We also noticed that the FOX2-
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mCherry-labeled peroxisomes were more abundant in the apical segment of hyphae
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extending ≈140m behind the hyphal tip. Their numbers decreased behind that region
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(between 140 – 280m behind the apex, a region were the first septum is frequently
US
located, i.e. at 271 ± 57m behind the apex for pex4 and at 240 ± 43m for pex22; n=15 hyphae/strain), and they were scarce in the distal parts of hyphae (behind 280m from the
AN
hyphal tip) (Figs. 4C and S5). These differences are likely not the consequence of altered peroxisome movements or distribution, since PEX14-GFP localization exhibited a more
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even peroxisome-remnant distribution along pex4 and pex22 hyphae (shown for pex4
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in Fig. S5). This could indicate a more prominent peroxisomal import of FOX2 in the
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apical cells of pex4 and pex22 hyphae. The ectopic reintroduction of a wild-type allele of PEX4 or PEX22 gene into the respective deletion mutant restored FOX2 peroxisome
CE
import throughout mycelia (Fig. S3), confirming that the described phenotypes are due to
AC
the deletion of these genes. Then we observed that the phenotypes of the single pex4 and
pex22 mutants, and of a pex4pex22 double mutant were alike (Fig. 4D, see also Fig. S1B), consistent with PEX4 and PEX22 working in a common process. These results show that absence of PEX4 and PEX22 prevents the peroxisomal import of GFP-PTS1 and partially inhibits that of FOX2-mCherry. It has been postulated that peroxisome matrix protein import is mechanistically coupled to the export of the receptors from the peroxisome membrane [23]. In S. cerevisiae,
18
ACCEPTED MANUSCRIPT both the mono- and poly-ubiquitinated forms of Pex5 can be released from the peroxisome membrane in a dislocation complex-facilitated process [50]; thus, we hypothesized that the FOX2 import observed in pex4 and pex22 mutants could be promoted by a polyubiquitination-dependent release of PEX5. To test this hypothesis, first we
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corroborated that FOX2-mCherry import into hyphal peroxisomes actually depended on
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PEX5, as indicated by the cytosolic localization of FOX2-mCherry throughout hyphae of a
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pex5 mutant strain (Fig. 4D). Pex5 polyubiquitination depends on the three RING finger
US
peroxins [57-59]. Actually, in S. cerevisiae Pex5 polyubiquitination is mediated by Pex2 acting in close association with Pex10 [59-61]. Thus, we then tested whether the partial
AN
import of FOX2 in pex4 and pex22 cells depended on PEX10. First, we observed that FOX2-mCherry mislocalized to cytosol in pex10 hyphae, with no detectable peroxisome
M
localization (Fig. 4D). Then, we observed that pex10 was epistatic to both pex4 and
ED
pex22 for this phenotype (Fig. 4D). This finding suggests that an ubiquitination system
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depending either, directly or indirectly on PEX10 can partially sustain the peroxisome import of FOX2 in pex4 and pex22 cells.
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Previously, we showed that FOX2 and GFP-PTS1 import differentially depends on
AC
the docking complex at specific stages of sexual development [31]. Therefore, we studied whether this differential requirement for the ubiquitination complex was also observed during sexual development. As shown on figure 4E for a pex22 homozygous sexual cross, FOX2-mCherry also localized both to cytosol and peroxisomes in the dikaryotic stage cells (croziers) of sexual development. In contrast, GFP-PTS1 was predominantly cytosolic. We observed that FOX2 localized to peroxisomes in 151 of 152 (99.3%) analyzed croziers, whereas GFP-PTS1-decorated punctae were only observed in 9 of these cells (5.9%) and in
19
ACCEPTED MANUSCRIPT reduced numbers (Fig. 4E, arrow). These data show a more stringent requirement of PEX4 and PEX22 (only shown for PEX22) for the import of GFP-PTS1 than of FOX2 also during the sexual cycle. The phenotype of pex4 and pex22 mutants during sexual development (see below), however, precluded analyzing the role of these peroxins during meiotic
IP
T
development.
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3.5. Loss of peroxisomes upon elimination of PEX1, PEX6 or PEX26. Next we studied the function of the proteins that predictably constitute the P. anserina peroxisome receptor
US
dislocation complex. P. anserina possesses an orthologue of each PEX1, PEX6 and PEX26. PEX1 predictably encodes a 1236 aa protein containing the two conserved nucleotide-
AN
binding domains (D1 and D2) characteristic of type II AAA+-ATPases (Fig. S2D); whereas
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PEX6 codes for a 1399 aa protein possessing a conserved D2 nucleotide-binding domain but a poorly conserved D1 domain (Fig. S2E). PEX26 predictably consist on a 482 aa
ED
protein possessing a C-terminal trans-membrane segment, which overlaps with a putative
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PEX19-binding domain characteristic of peroxisome tail-anchored proteins (Fig. S2F). To confirm the participation of these proteins in peroxisome biogenesis, we first
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analyzed their involvement in peroxisome matrix protein import. We observed that the
AC
localization of GFP-PTS1, FOX2-mCherry (Fig. 2, I-N) and of PTS2-GFP (Fig. S4, D-F) was cytosolic in pex1, pex6 and pex26 cells, indicating that the three proteins –PEX1, PEX6 and PEX26– are required for the import of peroxisome matrix proteins for both PTS1 and PTS2 pathways. Next we analyzed the effect of PEX1, PEX6 and PEX26 deletion on PEX14 localization. Interestingly, in contrast to the PEX14-GFP punctate distribution observed for WT peroxisomes, or for pex8, pex4 and pex22 peroxisomal remnants, only a reduced number of structures were labeled by PEX14-GFP in pex1, pex6 and
20
ACCEPTED MANUSCRIPT pex26 hyphae (Fig. 3, E-G). These structures consisted in discrete punctae that were often grouped, or in a large dynamic multilobular structure, which could represent tightly clustered structures (shown for pex6 in Fig. 5A and in Movie 3). These PEX14-containing structures were frequently located in the subapical region of hyphae, near to a highly
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vacuolized hyphal region; but they were not observed to colocalize with the
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endosomal/vacuolar membrane dye FM4-64. Importantly, the reintroduction of a wild-type
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allele of the deleted gene into the corresponding deletion mutant (tested for pex1 and
US
pex6, Fig. S3) restored both peroxisome protein-import and abundance, corroborating that both phenotypes were actually caused by these gene deletions.
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Loss of PEX14-GFP-labeled peroxisome remnants in cells devoid of PEX1, PEX6
M
or PEX26 could indicate an involvement for these proteins in peroxisome membrane formation. Alternatively, the elimination of these proteins could increase the degradation of
ED
peroxisomes via pexophagy, as occurs in S. cerevisiae [62]. Actually, we noted that a
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number of small punctate structures was also faintly labeled by PEX14-GFP in pex1,
pex6 and pex26 hyphae. We observed that some of these structures were often
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distributed in the periphery of spherical organelles visible by phase-contrast microscopy
AC
likely representing vacuoles (Fig. 3 E-G, highlighted for pex26 in Fig. 3H), as was further supported by staining cells with FM4-64 (shown for pex1 in Fig. 5B). FM4-64 analyses also revealed diffuse GFP labeling within vacuoles (shown for pex1 and pex6 in Fig. 5, B and C), as well as GFP-labeled punctae confined within vacuolar compartments (Fig. 5C, and Movie 4). Of note, some of these latter punctae appeared to be circumscribed by an FM4-64-labeled intravacuolar membrane (Fig. 5C, arrowhead). These observations suggest that a fraction of PEX14-containing organelles is subjected to vacuolar degradation in
21
ACCEPTED MANUSCRIPT pex1, pex6 and pex26 hyphae. To further inspect whether peroxisome removal was enhanced in P. anserina dislocation-complex mutants, we analyzed PEX14 localization in cells under different metabolic demands. Peroxisomes are degraded via selective autophagy (pexophagy) in response to nutritional availability. For example, in yeasts, a number of
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peroxisomes produced in peroxisome proliferation-inducing conditions are degraded by
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pexophagy when cells are transferred to glucose-containing medium [63]. As in other fungi,
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peroxisome proliferation in P. anserina can be induced by growing cells in oleic acid-based medium [8]. Thus, we compared PEX14-GFP distribution in glucose- and oleic acid-
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containing media. Since pex1, pex6 and pex26 mutants are unable to grow on oleic acid
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as sole carbon source (Fig. S1B), a low concentration of glucose (0.03%) was added to the oleic-acid medium to facilitate the growth of these mutants. In this medium, a substantial
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proliferation of peroxisomes occurred in WT cells (Fig. 5D). Notably, the number of
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PEX14-stained punctae present in pex1, pex6 and pex26 cells relative to dextrin-
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containing medium was markedly increased, reaching levels comparable to those observed for peroxisomes in WT cells (Fig. 5D). This reveals that peroxisome remnants are present
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in pex1, pex6 and pex26 cells under peroxisome proliferation-inducing conditions,
AC
indicating that the dislocation complex is not required for peroxisome membrane biogenesis in P. anserina. In contrast, the number of PEX14-GFP-labeled structures in
pex1, pex6 and pex26 cells grown on glucose as sole carbon source was further reduced (Fig. 5D), consistent with an increased removal of peroxisomes in these metabolic conditions. 3.6. The exportomer peroxins and PEX8 are required for meiocyte formation. Having established the participation of PEX8 and of the exportomer peroxins in peroxisome
22
ACCEPTED MANUSCRIPT biogenesis, we analyzed their requirement for sexual development. Sexual reproduction in P. anserina involves the formation of multicellular fructifications (perithecia) that enclose the fertile tissue (the hymenium) on which karyogamy, meiosis and ascospore differentiation take place (Fig. S1A). The hymenium is issued from fertilization and
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initially consists of syncytial (ascogonial) cells bearing nuclei from both parental origins.
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These cells produce specialized dikaryotic hyphae called croziers (Fig. 6A, cell a), which
CR
undergo karyogamy (Fig. 6A, cell c) and develop into meiocytes (asci, cells d-e in Fig. 6A). Following meiosis, the four resulting nuclei divide mitotically yielding eight nuclei, which
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are packaged by pairs into four ascospores (Fig. 6B).
AN
We analyzed the sexual development of pex8, pex4, pex22, pex1, pex6 and
pex26 mutants both in homozygous and heterozygous (to the wild type) crosses, and we
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observed that all these mutants were sterile in homozygous crosses. In these crosses, all
ED
mutants were able to produce perithecia in which the sexual tissues correctly progressed
PT
until the dikaryotic stage. Crozier cells were properly formed and their nuclear distribution was consistent with an accurate dikaryotic compartmentalization prior to karyogamy (Fig.
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6, C-H). However, these cells did not undergo karyogamy and did not differentiate into asci, consequently, ascospores were never produced. Instead, crorzier cells proliferated
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mitotically rather than entering meiosis, which resulted in the formation of numerous crozier arborescences (Fig. S3B, left panels). For each mutant, this phenotype was shown to be a recessive trait, since asci and ascospores were effectively produced in heterozygous crosses of either mutant to the wild type (e.g, Fig. S4). This phenotype was virtually undistinguishable between mutants, as was from the previously described for pex13,
pex20 and the RING finger-complex mutants [31, 33, 34]. Sexual development was
23
ACCEPTED MANUSCRIPT restored in all tested gene-complemented deletion mutant strains (Fig. S3B), corroborating that the phenotype of these mutants during sexual development was also due to the corresponding gene deletions. These results show that the peroxins of the peroxisome receptor export machinery, as well as the peroxin that presumably links this complex to the
IP
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import machinery, are required for meiocyte formation in P. anserina. 3.7. The potential monoubiquitination target cysteine of PEX20 is required for
CR
meiocyte formation. Altogether, our results suggest that meiocyte formation relies on an
US
alternative peroxisome protein import pathway driven by a cycling receptor whose activity is regulated by monoubiquitination. Since the PTS2 co-receptor PEX20 is required for
AN
meiocyte formation, this peroxin is the most likely candidate to act as such receptor [31].
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To further explore this hypothesis, we tested whether the potential monoubiquitination target amino acid of PEX20 was required for PEX20 function in meiocyte formation.
ED
Monoubiquitination of Pex5 [49, 64] and of yeast PTS2 co-receptors Pex18 [24] and Pex20
PT
[54] takes place at a conserved N-terminal cysteine residue, which is present at position 6 (Cys6) in P. anserina PEX20. Therefore, we generated a PEX20 allele coding for a protein
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in which this cysteine is replaced by alanine (pex20C6A), and we analyzed if the ectopic
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introduction of this allele into a ∆pex20 strain was able to restore sexual development of this mutant. First, to confirm that PEX20+ reintroduction into the ∆pex20 background restores sexual development, we transformed ∆pex20 cells with a wild-type copy of PEX20 gene, and we observed that all recovered transformants (n=8) were able to effectively produce asci and ascospores (Fig. 7C, compare to A and B). In contrast, we observed no restoration of sexual development when ∆pex20 was transformed with the pex20C6A allele (n=8 transformants) (Fig. 7D). In S. cerevisiae, substitution of the conserved Pex5
24
ACCEPTED MANUSCRIPT monoubiquitination target cysteine (Cys6) by lysine, which is artificially polyubiquitinated in yeast cells, results in a functional import receptor; indicating that not the cysteine per se, but the position of this ubiquitination is important for Pex5 function [65]. We performed an equivalent experiment in P. anserina and tested whether the ectopic introduction of a
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pex20C6K allele into the ∆pex20 background was able to restore sexual development.
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However, no restoration of sexual development was observed after transforming ∆pex20
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cells with a pex20C6K allele (n=9 transformants) (Fig. 7E), suggesting that the activity of PEX20 more stringently depends on the monoubiquitination of a cysteine residue. These
US
results indicate that the potential monoubiquitination target cysteine of PEX20 is required
M
peroxisomes is essential for this process.
AN
for PEX20 function in meiotic induction, and suggest that the recycling of PEX20 from
ED
4. Discussion
Peroxisome activity varies depending on the developmental demands of the cell. The
PT
function of this organelle is significantly defined by the composition of the proteins
CE
residing in its luminal compartment. Our research has shown that a specific number of peroxins involved in driving peroxisome matrix protein import is strictly required for the
AC
induction of meiotic development in P. anserina. These peroxins include the PTS2 coreceptor PEX20 but not the import receptors PEX5 or PEX7. Our current research shows that the initiation of meiotic development of this fungus also strictly requires the peroxins of both the peroxisome ubiquitination machinery and the peroxisome receptor dislocation complex, as well as the peroxin that likely bridges the peroxisome protein import and receptor export machineries. These results are consistent with the existence of an
25
ACCEPTED MANUSCRIPT alternative peroxisome import pathway, which is required for meiosis and that operates in absence of the known import receptors. Many cells possess peroxisomes that differ in shape, protein composition and distribution. The existence of different peroxisome populations underscores the dynamic
T
organization of this organelle in a cell, and can be related to the functional specialization of
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a peroxisome subpopulation along cell development [1, 66, 67]. In P. anserina we observed
CR
that vegetative-cell peroxisomes are heterogeneous both in the composition and in the spatial distribution of their proteins. We observed that the peroxisome localization of
US
PEX14, a central component of the peroxisome protein translocation machinery, was either
AN
asymmetric or consisted on scattered patches distributed along peroxisomes, suggesting its localization in specific peroxisome domains. Consistently, recent detailed analyses in
M
human cells have shown that PMPs –including Pex14– are not evenly distributed over the
ED
peroxisome surface, but they rather localize to specific membrane domains [67]. In addition, live-cell imaging analyses revealed that the compartmentalized localization of P.
PT
anserina PEX14 is highly dynamic, suggesting a dynamic reorganization of the membrane
CE
domains that are enriched for this peroxin. In filamentous ascomycetes, two exportomer peroxins –C. orbiculare Pex22 and N. crassa Pex26– also display asymmetric peroxisome
AC
localization [56, 66]. Interestingly, the distribution of these peroxins is related to Woronin body formation, albeit with an opposite pattern. In addition, N. crassa Pex14 occasionally localize to peroxisome foci from where Woronin bodies seemingly emanate [68]. Thus, a fraction of PEX14 could also be associated to Woronin body formation domains. We also obtained evidence for the existence of peroxisomes differing in their protein composition. We observed that the relative amount of two peroxisome matrix proteins in some peroxisomes varied in an inverse relation, suggesting a differential import 26
ACCEPTED MANUSCRIPT competence for these proteins. In N. crassa, the differentiation of highly import competent peroxisomes involves a positive feedback system, which is induced by matrix oligomers of a PTS1-containing protein (HEX1) that promotes the import of further matrix proteins [66]. Interestingly, both implicated proteins in P. anserina are PEX5 cargoes (GFP-PTS1 and the
T
non-PTS1 protein FOX2), but they differ in their targeting information. Thus, it is tempting
IP
to speculate that some peroxisomes exhibit differential import competence towards
CR
different PEX5 cargoes. Actually, we and others previously demonstrated differential docking-complex requirements for the import of distinct PEX5 cargoes both in P. anserina
US
[31] and in N. crassa [69]. Moreover, in our current research we discovered that the import
AN
of these two PEX5 cargoes also exhibit differential exportomer requirements. PTS1 sequences are recognized by the C-terminal TPR domain of Pex5 [70]. In contrast, the
M
targeting of non-PTS1 cargoes, like S. cerevisiae Fox1p and Hansenula polymorpha
ED
alcohol oxidase, depends on their interaction with the N-terminal half of Pex5 [71, 72], which is also the domain mediating receptor docking, membrane insertion and extraction,
PT
and that provides the receptor cargo transport activity [73-75]. Therefore, it is possible that
CE
the import of these different cargoes depends on different configurations of the Pex5 tanslocon, which could differ in their assembly / disassembly requirements.
AC
Our results showing that PEX4 and PEX22 elimination results in different extent of inhibition of the peroxisomal import of GFP-PTS1 and FOX2 suggest that the receptor monoubiquitination machinery is differentially required for the import of distinct PEX5 cargoes. The dynamics of Pex5, and of the PTS2 co-receptors, are defined by different ubiquitination pathways, which include receptor monoubiquitination that facilitates their recycling, and polyubiquitination that promotes their degradation via the proteasome [24, 50, 57, 64, 76, 77]. This latter process has been considered as a quality control system that 27
ACCEPTED MANUSCRIPT prevents receptor accumulation in the peroxisome membrane [57, 58, 78], and it has been referred to as Receptor Accumulation and Degradation in Absence of Recycling (RADAR) [77]. Actually, in the yeast Pichia pastoris, this pathway can partially rescue peroxisome matrix protein import, which is defective when Pex20 recycling is compromised [76]. The
T
observation that the peroxisome import of FOX2 in pex4 and pex22 cells is abrogated
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upon PEX10 elimination is consistent with an equivalent polyubiquitination system
CR
sustaining FOX2 import in these cells. Alternatively, additional ubiquitin-conjugating
US
enzymes could mediate PEX5 mono-ubiquitination in P. anserina, as proposed for Trypanosoma brucei, where peroxisome matrix protein import is only slightly affected by
AN
PEX4 elimination [79]. If a ubiquitination system is involved in alleviating FOX2 import in absence of PEX4 and PEX22, then this system should preferentially act on PEX5
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molecules involved in FOX2 (or non-PTS1) import. Furthermore, such process would be
ED
more plausible under an export-driven import system, in which the export of these ubiquitinated PEX5 molecules exclusively propels the import of their specific cargoes.
PT
Our research also revealed that elimination of the dislocation complex peroxins
CE
results in peroxisome loss in P. anserina. In filamentous ascomycetes, Pex6 (PexF, in A. nidulans) and Pex26 (in N. crassa) were previously shown to be dispensable for
AC
peroxisome membrane formation [66, 80], however whether peroxisome removal was enhanced upon elimination of these peroxins in these fungi was not addressed. In S. cerevisiae the number of peroxisome remnants is reduced in cells lacking of Pex1, Pex6 or Pex15 [62]. Actually, in this yeast, it has been demonstrated that loss of either of these peroxins results in enhanced pexophagy, which is dependent on the pexophagy receptor Atg36 [62]. The precise mechanism accounting for peroxisome loss in P. anserina
28
ACCEPTED MANUSCRIPT dislocation-complex mutants remains to be established; however, our results suggest that this process involves their elimination by pexophagy. Interestingly, it has recently been demonstrated that PEX1 and PEX26 also prevent pexophagy in human cells [81], and that pexophagy is triggered upon PEX1 dysfunction in Arabidopsis thaliana [82]. These
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findings indicate that restraining pexophagy is a conserved function of the dislocation
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complex in animals, plants and fungi.
CR
With the conclusion of this research, we have analyzed the role of all known peroxins predictably (based on orthology relations) involved in peroxisome matrix protein
US
import in P. anserina [31-34]. Notably, with the exception of the import receptors PEX5
AN
and PEX7, and of the docking peroxins PEX14 and PEX14/17, all remaining peroxins are required for the induction of meiotic development in this fungus (Fig. 8). Our findings
M
support a model in which meiocyte formation relies on an alternative peroxisome import
ED
that does not depend on the canonical import receptors. This pathway could be driven by PEX20 as import receptor, and PEX13 could provide its docking factor [31]. Previously we
PT
showed that meiocyte formation depended on the RING-finger complex [33], our current
CE
finding that PEX8 –the peroxin bridging the importomer subcomplexes in S. cerevisiae [28]– is also required for this developmental process further supports a pathway dependent
AC
on the interaction between the peroxisome docking and RING-finger complexes. Actually, data showing that the interaction of Pex8 and Pex13 is critical for Pex8 topogenesis [83] is also consistent with our finding that PEX13 is the only docking peroxin required for meiocyte formation. The RING-finger complex peroxins participate in ubiquitination processes –including the RADAR system and pexophagy signaling [27, 84], additional to peroxisome import receptor monoubiqutination. Our data implicating PEX4 and PEX22 in meiocyte formation now supports the existence of an additional receptor whose dynamics 29
ACCEPTED MANUSCRIPT are regulated by ubiquitination in a similar way to PEX5. Our data also implies a cycling receptor whose activity would critically depend on the AAA ATPase complex. Recently, a paralogue of Pex5 (Pex9) acting as additional import receptor in S. cerevisiae was identified [85, 86]; however, we have not to identify paralogues of PEX5 or PEX7 in the
T
genome of P. anserina. Therefore, PEX20 constitutes the most likely candidate to provide
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an additional import receptor. Consistent with this notion, we now also provided evidence
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suggesting that the monoubiquitination-dependent recycling of PEX20 is essential for induction of meiosis in this fungus. Still, we cannot exclude the possibility of PEX20 acting
US
as co-receptor of another yet unidentified PEX7-unrelated targeting receptor.
AN
In S. cerevisiae, Pex18 is a main constituent of the peroxisome membrane pore through which PTS2 proteins are translocated, which is independent from the PTS1 pore
M
[22]. The activity of Pex18 in S. cerevisiae can be substituted by Pex20 of N. crassa [87],
ED
strongly suggesting the presence of two independent pores also in filamentous ascomycetes. The discovery that in P. anserina PEX20 is required for the initiation of meiotic
PT
development [31] and PEX5 for its progression [32] suggests that the import channels of
development.
CE
this fungus mediate the import of proteins contributing to different stages of meiotic
AC
Nonetheless, the precise peroxisome-dependent developmental process required to initiate meiotic development remains undefined. All peroxin mutants defective for this process are unable to undergo nuclear fusion in their dikaryotic cells. Karyogamy and meiosis are coupled processes in filamentous ascomycetes. Actually, in these fungi, premeiotic S-phase precedes karyogamy and some of the proteins mediating meiosis are incorporated into chromosomes at this stage [88], indicating that the decision to enter meiosis is determined before karyogamy. In P. anserina the frequency of nuclear fusion in 30
ACCEPTED MANUSCRIPT vegetative cells is not affected by pex2 mutation [34], suggesting that PEX2 is not required for the nuclear fusion process per se. In addition, mutants of filamentous acomycetes impaired for karyogamy due to deficient internuclear recongnition at the dikaryotic stage [89] or to defective nuclear envelope fusion [90] are still able to undergo haploid meiosis.
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In contrast, in the peroxin mutants defective for meiocyte formation the dikaryotic cell
IP
nuclei fail to enter the meiotic cycle and continue dividing mitotically. These observations
CR
suggest that rather than karyogamy per se, peroxisomes could be involved in a regulatory process required for cells to leave the mitotic cell cycle and begin meiosis (the
US
mitosis/meiosis decision). Alternatively, peroxisomes could be required for the
AN
differentiation of a cellular state competent to trigger meiosis. Remarkably, recent research has disclosed that peroxisomes are involved in the decision of mammalian epidermal cells
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between entering mitosis or differentiating [15]. These findings underscore an important
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role for peroxisomes in regulatory events that define transcendent cell fate decisions during
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development.
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ACKNOWLEDGMENTS
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We thank all members of the laboratory for assistance and valuable discussions, and Fernando García Hernández (IFC, UNAM, Mexico) for assistance on microscopy. We are much indebted to Jesús Aguirre and Wilhelm Hansberg (IFC, UNAM) for assistance throughout this research, and to Véronique Berteaux-Lecellier (CRIOBE, CNRS), Robert Debuchy (I2BC, Gif-sur-Yvette, France) and Philippe Silar (Univ. Paris Diderot, Paris, France) for kindly providing strains and plasmids. We are grateful to Robert Debuchy for critically reading the manuscript.
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ACCEPTED MANUSCRIPT FUNDING This work was supported by grants UNAM-DGAPA-PAPIIT IA201815 and IA203317.
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CZM and HTR were supported by scholarships from these grants.
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FIGURE LEGENDS Figure 1. Localization of FOX2-mCherry, GFP-PTS1 and PEX14-GFP in P. anserina hyphae. Wild-type hyphae expressing GFP-PTS1 and FOX2-mCherry (A) or FOX2-
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mCherry and PEX14-GFP (B, C) were grown for 24h in M2 medium and analyzed by
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confocal microscopy. In (A-B), insets display enlarged areas showing representative
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peroxisomes, and the lower line graphs display the fluorescence intensity for both mCherry and GFP channels along the lines of the respectively labeled peroxisomes. In (B) note that
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x-axis for line graph 4 is not in the same scale as for peroxisomes 1-3. In (C) note PEX14-
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GFP localization in clusters of patches lying along FOX2-labeled peroxisomes (arrowheads) and in apical punctate structures not colocalizing with FOX2-mCherry
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(arrows). BF: bright field. Scale bar, 5m. Inset scale bar, 2m.
Figure 2. Localization of GFP-PTS1 and FOX2-mCherry in wild-type, Δpex8 and
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exportomer-mutant hyphae. Wild-type, pex8, pex4, pex22, pex1, pex6 or pex26
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strains expressing GFP-PTS1 (left) and FOX2-mCherry (right) were grown in dextrincontaining medium for 24h and analyzed by fluorescence microscopy. GFP-PTS1 and
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FOX2-mCherry localize to peroxisomes in wild-type hyphae (A and B). In contrast, these proteins mislocalize to cytosol in absence of PEX8 (C-D), PEX1 (I-J), PEX6 (K-L), or PEX26 (M-N). GFP-PTS1 is also cytosolic in pex4 (E) and pex22 (G) hyphae, but in these cells FOX2-mCherry localizes both to cytosol and to discrete punctate structures resembling peroxisomes (F and H, arrows). Scale bar, 5m.
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ACCEPTED MANUSCRIPT Figure 3. Localization of PEX14-GFP in wild-type, pex8 and exportomer-mutant hyphae. PEX14-GFP localization in wild-type (A), pex8 (B), pex4 (C), pex22 (D),
pex1 (E), pex6 (F) or pex26 (G-H) mycelia grown for 24h in dextrin-containing medium. Lower panels in (E-H) show corresponding phase-contrast micrographs. Image
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(H) displays a magnification of the boxed area in (G), in which the brightness was digitally
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enhanced; arrowheads indicate the location of phase contrast-visible vacuoles. PEX14-GFP
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decorated wild-type peroxisomes (A) and localized to discrete punctae in pex8 (B), pex4
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(C) and pex22 (D) hyphae. However, the number of comparable PEX14-GFP-labeled punctae in pex1, pex6 and pex26 hyphae was reduced (E-G), and these were clustered
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in the subapical region of hyphae (arrows). In these cells, PEX14-GFP also stained a
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number of small punctae, which were less bright and often localized in the periphery of
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vacuolar organelles (arrowheads). Scale bar, 5m.
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Figure 4. Absence of PEX4 and PEX22 partially inhibits FOX2 peroxisome import. A) Confocal microscopy analysis of FOX2-mCherry and PEX14-GFP localization in pex22
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cells. B) Detail of PEX14-GFP and FOX2-mCherry localization in a pex22 hypha. C)
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FOX2-mCherry localization along different regions of pex22 hyphae. Lower histogram, quantitation of FOX2-labeled peroxisomes in pex4 and pex22 hyphae. Values are mean ± SD; n = 15. *P=0.01 **P=0.0019, ***P<0.0001 by unpaired Student’s t test. D) FOX2mCherry localization in wild-type (WT), pex5, pex4, pex22 or pex10 strains, and in
pex4pex22, pex4pex10 or pex22pex10 double mutant strains. FOX2-mCherry localizes to both cytosol and peroxisomes in pex4, pex22 and pex4pex22, but only to cytosol in pex5, pex10, pex4pex10 and pex22pex10 cells. E) Maximum-intensity 45
ACCEPTED MANUSCRIPT projections of GFP-PTS1 and FOX2-mCherry localization in dikaryotic crozier cells issued from pex22 homozygous sexual crosses. FOX2-mCherry labeled the cytosol and peroxisomes of pex22 croziers, while GFP-PTS1 was predominantly cytosolic, few GFPPTS1-stained peroxisomes were observed in these cells (arrow). Bottom panels show
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corresponding mCherry/GFP (left) and DAPI/bright field (BF, right) merge micrographs.
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Scale bar in A, C-E: 5m; scale bar in B: 1m.
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Figure 5. PEX14-containing peroxisome remnants are lost upon elimination of the dislocation complex peroxins. A) Confocal microscopy analysis of PEX14-GFP in a
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pex6 hypha stained with FM4-64. S, indicate Spitzenkörpers; BF, bright field. Note the predominant localization of PEX14 in a large subapical structure (arrow). B)
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Epifluorescence microscopy analysis of PEX14-GFP localization in a pex1 hypha stained
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with FM4-64. Right panels display magnifications of the boxed areas at left. PC, phase
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contrast. Note the punctate localization of PEX14-GFP in the periphery of phase-contrastand FM4-64-detectable vacuoles (arrows), as well as of diffuse GFP labeling within these
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vacuoles. C) Detail of PEX14-GFP localization in pex6 hyphae stained with FM4-64.
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PEX14-GFP-labeleded punctae (right and middle panels, arrows) and diffuse GFP staining (left panels) are detected in the lumen of FM4-64-labeled vacuoles. Note a luminal PEX14GFP-stained patch encircled by an FM4-64-labeled internal membrane (arrowhead). D) PEX14-GFP localization in wild-type, pex1, pex6 or pex26 hyphae grown for 24h in media containing glucose (0.3%) (left) or oleic acid (OA) plus low glucose (0.03%) (right). Cell outlines are highlighted in blue. Scale bar in A, B and D: 5m; scale bar in C: 2m.
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ACCEPTED MANUSCRIPT Figure 6. PEX8 and the exportomer peroxins are required for meiocyte formation. Sexual cycle cells issued from homozygous crosses of the indicated genotypes were stained with DAPI and analyzed by fluorescence microscopy (left panels). Right panels, corresponding DAPI/bright field merge micrographs. In (B) only the DAPI micrograph is
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shown. Small lettering indicates progressive developmental stages. Sexual crosses of WT
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strains produce dinucleate dikaryotic croziers (A, crozier a, arrowheads with different
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shading indicate opposite mating-type nuclei), which divide to produce tetranucleate croziers. In these croziers, two opposite mating-type nuclei undergo karyogamy (c, arrow)
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and the two remaining (arrowheads) engage in the formation of a new crozier. Upon
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karyogamy the upper crozier cell differentiates into an ascus (note that elongation in the upper part of crozier c) and immediately enters meiosis (d and e show asci at different
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stages of first meiotic prophase). Ultimately, the meiotic-derived nuclei are
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compartmentalized into four dinucleate ascospores (B). In contrast, sexual development does not progress beyond the dikaryotic stage in homozygous crosses ofpex8 (C), pex4
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(D), pex22 (E), pex1 (F), pex6 (G) and pex26 (H) mutants. In these mutants, croziers
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are properly formed and their nuclear divisions and movements take place correctly, yielding croziers that posses two pairs of nuclei (b, compare to dinuceated corziers in a);
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however, these croziers do not undergo karyogamy and their nuclei divide mitotically rather than entering meiosis. Scale bar, 5m.
Figure 7. Cys6 of PEX20 is required for meiocyte formation. Mature fruiting bodies from wild-type sexual crosses contain 4-spored asci (A), while those from ∆pex20 mutant strains contain only crozier-shaped dikaryotic cells (B). The ectopic introduction of a wild-
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ACCEPTED MANUSCRIPT type allele of PEX20 into ∆pex20 restores asci and ascospore formation (C). In contrast, the introduction of a pex20C6A (D) or a pex20C6K (E) allele was unable to restore asci formation of ∆pex20 strains. Scale bar 10μm.
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Figure 8. Model for peroxisome matrix protein import in P. anserina. Peroxisome
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matrix protein import in P. anserina is mediated by the two conserved pathways, which are
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driven by the receptor PEX5, and by the receptor/co-receptor couple PEX7/PEX20, respectively (only the peroxin numbers are indicated). In addition, most P. anserina
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peroxins are required for initiation of meiotic development (red shading). The only
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exceptions are the two import receptors (PEX5 and PEX7) and the docking components PEX14 and PEX14/17 (green shading). These data suggest the existence of an alternative
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further details). Ub, ubiquitin.
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import pathway, which could depend on PEX20 as import receptor (please see the text for
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ACCEPTED MANUSCRIPT Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery
Highlights
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P. anserina peroxisomes differ in their protein composition and distribution
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The import of distinct proteins differs in its ubiquitination-complex requirements
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The peroxisome dislocation complex restrains peroxisome removal in P. anserina
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Meiotic induction in P. anserina requires PEX8 and the exportomer peroxins
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Meiotic induction requires the PEX20 cysteine that is potentially monoubiquitinated
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Graphics Abstract
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Fernando Suaste-Olmos, Claudia Zirión-Martínez, Harumi Takano-Rojas, Leonardo Peraza-Reyes PII: DOI: Reference:
S0167-4889(18)30003-X https://doi.org/10.1016/j.bbamcr.2018.01.003 BBAMCR 18229
To appear in: Received date: Revised date: Accepted date:
21 August 2017 29 December 2017 3 January 2018
Please cite this article as: Fernando Suaste-Olmos, Claudia Zirión-Martínez, Harumi Takano-Rojas, Leonardo Peraza-Reyes , Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbamcr(2018), https://doi.org/10.1016/j.bbamcr.2018.01.003
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ACCEPTED MANUSCRIPT Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery
Fernando Suaste-Olmosa, Claudia Zirión-Martíneza, Harumi Takano-Rojasa,
Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular,
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a
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Leonardo Peraza-Reyesa*
Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de México
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04510, Mexico.
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* Corresponding author:
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Instituto de Fisiología Celular, Universidad Nacional Autónoma de México Ciudad de México 04510, Mexico.
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Tel. (52) (55) 5622 5628
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E-mail address: [email protected]
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ACCEPTED MANUSCRIPT Abstract Peroxisomes are versatile organelles essential for diverse developmental processes. One such process is the meiotic development of Podospora anserina. In this fungus, absence of the docking peroxin PEX13, the RING-finger complex peroxins, or the PTS2 co-receptor
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PEX20 blocks sexual development before meiocyte formation. However, this defect is not
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seen in the absence of the receptors PEX5 and PEX7, or of the docking peroxins PEX14
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and PEX14/17. Here we describe the function of the remaining uncharacterized P. anserina peroxins predictably involved in peroxisome matrix protein import. We show that PEX8, as
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well as the peroxins potentially mediating receptor monoubiquitination (PEX4 and PEX22)
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and membrane dislocation (PEX1, PEX6 and PEX26) are indeed implicated in peroxisome matrix protein import in this fungus. However, we observed that elimination of PEX4 and
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PEX22 affects to different extent the import of distinct PEX5 cargoes, suggesting
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differential ubiquitination-complex requirements for the import of distinct proteins. In addition, we found that elimination of PEX1, PEX6 and PEX26 results in loss of
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peroxisomes, suggesting that these peroxins restrain peroxisome removal in specific
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physiological conditions. Finally, we demonstrate that all analyzed peroxins are required for meiocyte formation, and that PEX20 function in this process depends on its potential
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monoubiquitination target cysteine. Our results suggest that meiotic induction relies on a peroxisome import pathway, which is not dependent on PEX5 or PEX7 but that is driven by an additional cycling receptor. These findings uncover a collection of peroxins implicated in modulating peroxisome activity to facilitate a critical developmental cell fate decision. Keywords. Peroxisome / Meiosis / Sexual development / Fungi / Organelle biogenesis
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ACCEPTED MANUSCRIPT 1. Introduction Peroxisomes are versatile organelles that perform diverse cellular functions essential for development. Peroxisomes have conserved roles in reactive oxygen species (ROS)1 and lipid metabolism, and they execute distinct specialized metabolic functions, which can be
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specific to given organisms [1-3]. In addition, peroxisomes participate in diverse cellular
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functions ranging from intracellular cholesterol transport in mammals [4] to the formation
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of specialized organelles –the Woronin bodies– involved in intercellular channel gating in fungi [5]. Furthermore, peroxisomes are increasingly recognized as signaling organelles
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that play different roles in the orchestration of complex signaling pathways [6].
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Peroxisomes are highly dynamic organelles that adapt their composition, number and arrangement in response to environmental, physiological and developmental cues [7-
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10]. The significance and versatility of peroxisomes is reflected by the diverse
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developmental processes that depend on their function; for example, mammalian processes like audition [11], the development of the nervous system [12], the adipose tissue [13, 14]
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and the skin [15] rely on peroxisomes. Furthermore, these developmental processes can
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involve rather diverse peroxisomal activities. For instance, audition depends on an adaptive proliferation of peroxisomes, which protect the auditory system from noise exposure-
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produced ROS-induced damage [11]; whereas skin formation relies on a correct peroxisome distribution during mitosis, which is critical to define the cell division plane of epidermal progenitor cells and to determine the balance between their proliferation and differentiation [15]. Abbreviations: MCC, Manders correlation coefficient; PCC, Pearson’s correlation coefficient; PMP, peroxisome membrane protein; PTS, peroxisomal-targeting signal; ROS, reactive oxygen species. 1
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ACCEPTED MANUSCRIPT Peroxisome formation and maintenance depend on a group of proteins referred to as peroxins. Although the peroxin composition between species can differ; most peroxins mediating peroxisome biogenesis throughout different eukaryotic lineages are conserved [1]. Peroxisome formation depends on two groups of peroxins, which drive the import of
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proteins into the peroxisome membrane and the peroxisome matrix, respectively. The
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targeting and insertion of peroxisome membrane proteins (PMPs) depend on the conserved
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peroxins Pex3 and Pex19. Pex19 is a receptor and chaperone protein that recognizes most PMPs in the cytosol and subsequently delivers them to peroxisomes by docking on the
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PMP Pex3. An additional membrane peroxin –Pex16– also acts as a receptor for some
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PMPs, including Pex3; however, the function of this peroxin is less conserved [16]. In addition to direct targeting from the cytosol, a number of PMPs traffic through different
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routes to reach peroxisomes, including the endoplasmic reticulum [17] and mitochondria
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[18]. On the other hand, the import of peroxisome matrix proteins is driven by two conserved import pathways, which are mediated by the receptors Pex5 and Pex7. These
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peroxins recognize the peroxisome targeting signals (PTS1 and PTS2, respectively) of
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peroxisome matrix proteins in the cytosol and target them to the organelle [19]. Peroxisome targeting by Pex7 requires accessory proteins known as PTS2 co-receptors that are species
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specific, like Pex20 in most ascomycete fungi, the paralogous Pex18/Pex21 in some yeasts, and a long Pex5 isoform in plants and animals [20]. Following cargo recognition, the receptor/cargo complex is anchored at the peroxisome membrane by interactions with the docking complex, which is composed of Pex13 and Pex14 (and Pex17 in yeasts or Pex14/17 in filamentous fungi), and then the import receptor is inserted into the peroxisome membrane. Import by both receptors depends on the docking complex; however, at least in species possessing PTS2 co-receptors different from Pex5, the 4
ACCEPTED MANUSCRIPT translocation of PTS1 and PTS2 cargoes proceeds through different channels. Actually, in Saccharomyces cerevisiae it has been demonstrated that Pex5 and the PTS2 co-receptor Pex18, respectively, constitute by themselves integral components of two independent channels through which PTS1 and PTS2 proteins are translocated [21, 22]. Both import
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channels also contain Pex14, whereas the PTS2 additionally contains Pex17. The precise
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mechanism of protein translocation is not known, but it is clear that the protein import
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process depends on the export of the receptors (or PTS2 co-receptor) from the peroxisome membrane. Actually, evidence suggests that the protein translocation process is
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mechanistically coupled to the export of the receptor (or PTS2 co-receptor) from the
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membrane (the export-driven protein import model) [23, 24]. However, there are data also indicating that protein translocation is concomitant with the insertion of the import receptor
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into the membrane rather than with its subsequent release from it [25]. The removal of the
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receptors and PTS2 co-receptors from the peroxisome membrane is mediated by the peroxisome receptor export machinery (the exportomer [26]). Export of Pex5 and PTS2 co-
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receptors depends on their ubiquitination. Actually, different types of ubiquitination define
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the fate of these peroxins. Whereas their monoubiquitination facilitates their recycling, their polyubiquitination directs them to degradation by the proteasome. Both types of
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ubiquitination require the RING-finger complex –a peroxisome membrane complex composed of the E3 ubiquitin ligases Pex2, Pex10 and Pex12. Receptor monoubiquitination is catalyzed by specific ubiquitin-conjugating E2 enzymes, like Pex4 along with its membrane-anchor and activator Pex22 in yeasts [27]. The RING-finger complex also interacts with the docking complex via Pex8, producing a larger complex known as the importomer [28]. Ubiquitination primes the receptor for the dislocation machinery, which extracts the receptor from the peroxisome membrane. The dislocation complex is composed 5
ACCEPTED MANUSCRIPT of the dislocases Pex1 and Pex6, two AAA+ ATPases that are anchored to the peroxisome membrane by the tail-anchored PMP Pex26 (Pex15 in yeasts) [29]. Following their release from peroxisomes and their deubiquitination, the receptor/co-receptors are engaged in further rounds of protein import.
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Sexual development of fungi has provided notable examples of diverse
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developmental processes that importantly depend on peroxisomes [30]. One such process is
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the meiotic development of the fungus Podospora anserina (this process is illustrated in Fig. S1A). In this fungus, meiotic development involves a precise modulation at specific
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stages of peroxisome shape, abundance and distribution [8], which is accompanied by
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changes in the functional state of the protein machinery that drives the import of proteins into the organelle [31]. This indicates that peroxisome function during this process is
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defined by a complex modulation of the proteins mediating peroxisome dynamics and
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assembly. Furthermore, in P. anserina both the initiation and progression of meiotic development depend on peroxisomes, albeit on different activities. Nuclear progression
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through meiosis depends on the import receptors PEX5 and PEX7 [32]. In contrast,
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initiation of meiosis depends on specific peroxins that include PEX3 and PEX19, the docking peroxin PEX13, the RING-finger peroxins and the PTS2 co-receptor PEX20, but
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does not require neither of the import receptors (PEX5 and PEX7) nor the docking peroxins PEX14 or PEX14/17 [31-34]. This has led us to propose the existence of an alternative import pathway, which operates in absence of both known import receptors and that is required for meiotic induction. Alternatively, this process could depend on the concerted action of the aforementioned peroxins in a function independent from their activity in peroxisome matrix protein import. To provide further evidence substantiating either of these alternatives, here we investigate the role of the P. anserina peroxisome receptor 6
ACCEPTED MANUSCRIPT export machinery in peroxisome biogenesis and sexual development. By analyzing the function of the two subcomplexes that compose the exportomer –the ubiquitination and the dislocation complexes–, and of the peroxin that links the exportomer to the peroxisome protein import machinery (PEX8), we have obtained strong evidence supporting the
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existence of an alternative peroxisome import pathway required for meiotic development
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initiation.
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2. Material and methods
2.1. Strains and Culture conditions. The P. anserina strains used in this research are
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derived from the "S" wild-type strain. P. anserina ∆pex10 and ∆pex20 strains were
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generated previously [31, 33], and the ∆ku70 strain [35] was kindly provided by Robert
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Debuchy (I2BC, Gif-sur-Yvette, France). P. anserina ∆pex5 strain [32], and the strains expressing GFP-PTS1 or PTS2-GFP in the wild-type genetic context were kindly provided
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by Véronique Berteaux-Lecellier (CRIOBE, CNRS, French Polynesia). Culture media
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consisted on M2 minimal medium containing 1.1% dextrin, 0.3% glucose or 0.05% oleic acid (plus 0.2% TWEEN 40 used as emulsifier) as sole carbon sources. To facilitate the
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growth of pex mutants on oleic acid-based medium, this medium was added with 0.03% of glucose. Ascospores were germinated in G medium supplemented with 0.5% yeast extract, and protoplasts were regenerated on RG medium. When required, media were supplemented with phleomycin (25 g mL-1), geneticin (G418 sulfate, 100 g mL-1), nourseothricin (40 g mL-1) or hygromycin B (75 g mL-1 for pBC-Hygro-derived constructs, or 30 g mL-1 for pUCHygro-derived constructs). P. anserina media and current methods can be found at http://podospora.igmors.u-psud.fr. 7
ACCEPTED MANUSCRIPT 2.2. Nucleic acid isolation, transformation and plasmids. The isolation of genomic DNA and the transformation of P. anserina were performed according to [36]. Streptomyces noursei nat1 gene was obtained from plasmid pAPI509, which is a derivative from pAPI508 [35]. Escherichia coli hph gene was obtained from pBC-Hygro [37] or pUCHygro
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[38] and the Aspergillus nidulans trpC transcription terminator was obtained from
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pMOcosX [39]. Plasmids pAPI509, pMOcosX and pSM334_Genticin were kindly
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provided by Robert Debuchy, and pBC-Hygro by Philippe Silar (Univ. Paris Diderot, Paris, France). Oligonucleotide primers used in this research are shown on Table S1.
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2.3. Gene sequences. PEX1 (Pa_1_6740), PEX4 (Pa_1_9240), PEX6 (Pa_1_9330), PEX8
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(Pa_7_9670), PEX22 (Pa_6_4620) and PEX26 (Pa_7_1760) gene sequences were obtained from the P. anserina genome sequence (http://podospora.igmors.u-psud.fr). The predicted
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protein sequences are available in the GenBank database under accession numbers
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CDP23014.1, CDP23277.1, CDP23286.1, CDP32486.1, CDP30493.1, and CDP31995.1, respectively. FOX2, PEX14 and PEX20 genes were previously reported [31, 33, 40].
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2.4. Gene deletions. Mutant strains deleted for each peroxin gene were generated by
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replacing the corresponding ORF by a selectable marker gene by homologous recombination. PEX1 and PEX4 were replaced by pBC-Hygro-derived hph gene. PEX6,
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PEX8, PEX22 and PEX26 were replaced by the nat1 gene. Gene replacement constructs were generated by double-joint PCR [41] and consisted on the selectable marker gene flanked by 700 bp of the 5′ and 3′ flanking sequences of the respective ORF. For each construct, the 5' fragment was amplified with primers denoted as pexN-5F and pexN-5R (were N designates the corresponding peroxin number, Table S1), the 3' fragment with primers pexN-3F and pexN-3R, and the corresponding selectable marker with primers
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ACCEPTED MANUSCRIPT pexN-hphF and pexN-hphR (for PEX1 and PEX4) or pexN-natF and pexN-natR (for the remaining peroxins). The final fusion PCR products were gel-purified and directly used to transform protoplasts of a ∆ku70 strain. Randomly selected transformants were purified after crosses to the wild-type strain, and the gene deletions were recovered in the KU70+
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genetic background. Correct gene replacements were verified by PCR analyses.
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2.5. Gene complementation analyses. For the gene complementation assays, a given
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mutant was complemented by ectopically introducing a wild-type copy of the corresponding deleted gene. PEX4 and PEX8 alleles were derived from plasmids
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GA0AA328DB09 and GA0AA242CA11 (P. anserina genomic-DNA library, Genoscope,
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France), respectively. The remaining genes were obtained by PCR using genomic DNA as template and the respective pexN-5F / pexN-3R primers. The plasmids or PCR products
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were directly used to co-transform the corresponding mutant strain with the
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pSM334_Genticin vector, and geneticin-resistant transformants were analyzed for peroxisome protein import (tested for GFP-PTS1 and/or FOX2-mCherry) and sexual
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development.
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2.6. Generation of pUC-GFP and pUC-Cherry plasmids. In order to tag PEX14 and FOX2, first we constructed two plasmids that allow fusing the coding sequence of GFP (or
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mCherry) to the 3' end of a given gene at its chromosomal location. For this, we generated a construct consisting on pEGFP1-derived EGFP gene preceded by a sequence coding for a 7-amino-acid linker peptide (as optimized by [42]) and followed by the A. nidulans trpC terminator. This construct was obtained by fusing the trpC terminator (PCR-amplified with primers gfp-trpC and trpC-R) to the 3' end of EGFP gene (amplified with primers lkt-gfp and trp-gfp) by fusion PCR. The 7 amino-acid linker-peptide was added to this construction by including its coding nucleotide sequence as an in-frame overhang on EGFP forward 9
ACCEPTED MANUSCRIPT primer (lkt-gfp). The flanking primers (lkt-gfp and trpC-R) used to obtain this construct contained EcoRI and SacI restriction sites, respectively; which allowed cloning the resulting construct in the corresponding restriction sites of plasmid pUC-Hygro. SacI site of pUC-Hygro is contiguous to the hygromycin B resistance cassette (HygR) of this plasmid;
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therefore, a 2.5kb cassette containing the EGFP gene followed by the HygR selectable
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marker (GFP-HygR) was generated in the resulting plasmid (pUC-GFP). The same strategy
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was followed to produce an equivalent plasmid (pUC-Cherry) containing a mCherry-HygR cassette.
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2.7. Tagging of FOX2 and PEX14. Tagging of PEX14 and FOX2 was done by fusing the
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coding sequence of GFP and mCherry, respectively, to the 3' end of PEX14 and FOX2 coding sequences at their chromosomal locations. To tag PEX14 we generated a construct
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consisting on the GFP-HygR cassette from pUC-GFP flanked by 800 bp of PEX14 stop
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codon-flanking sequences (PEX14::GFP-HygR::PEX14-3'UTR), in which the coding sequence of GFP was fused in-frame to the 3′ end of PEX14 ORF. This construct was
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obtained by fusing the following DNA sequences by double-joint PCR: (i) the last 798 bp
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(excluding the stop codon) of PEX14 ORF 3’ end (amplified with primers pex14-F and lktpex14), (ii) the GFP-HygR cassette from pUC-GFP (amplified with primers pex14-lkt and
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pex14-hph), and (iii) 861 bp of DNA downstream PEX14 stop codon (amplified with primers pex14-3F and pex14-3R). Following the same strategy, an equivalent construct based on the mCherry-HygR cassette was generated for FOX2. In this case, the resulting FOX2::mCherry-HygR::FOX2-3'UTR cassette consisted on the fusion of: (i) the last 680 bp (excluding the stop codon) of FOX2 ORF 3’ end (amplified with primers fox2-F and lktfox2), (ii) the mCherry-HygR cassette from pUC-Cherry (amplified with primers fox-lkt and
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ACCEPTED MANUSCRIPT fox-hph), and (iii) 685 bp of DNA downstream FOX2 stop codon (amplified with primers fox2-3F and fox2-3R). In this case, the resulting construction was cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA), following the provider instructions, to yield plasmid pFS02. Next, we gel-purified the PEX14::GFP-HygR::PEX14-3'UTR PCR
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construct and we recovered the FOX2::mCherry-HygR::FOX2-3'UTR cassette as a NotI
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fragment from plasmid pFS02, and each construct was used to transform cells of a ku70
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strain. HygR transformants were randomly selected and the HygR marker was recovered in
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the KU70+ genetic context after crosses to the wild type. All plasmids were verified by sequencing. FOX2 and PEX14 tagging was verified by PCR analyses and by sequencing.
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2.8. Site directed mutagenesis of PEX20. PEX20 gene containing 770 bp of its ORF 5’ upstream region to 802 bp downstream the stop codon was amplified by PCR using primers
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pex20-F and pex20-3R and cloned into pGEM-T Easy Vector to yield pFS03.
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Complementary mutagenic primers containing selected mutations to change PEX20 conserved cysteine 6 into alanine (primers pex20_C6A-F and pex20_C6A-R) or lysine
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(primers pex20_C6K-F and pex20_C6K-R) were used to amplify pFS03. The obtained PCR
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products were treated with DpnI and used to transform E. coli, and the recovered plasmids (pFS04 for pex20C6A and pFS05 for pex20C6K) were sequenced to verify the DNA
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substitutions. pFS03 and the mutated pFS04 and pFS05 plasmids were respectively used to co-transform P. anserina ∆pex20 cells with the pSM334_Genticin vector, and the obtained transformants were analyzed for sexual development in sexual crosses to a ∆pex20 strain. 2.9. Cytology. Sexual cycle cells were fixed in 7.4% paraformaldehyde and processed for fluorescence microscopy as described before [43]. To inspect sexual development, we analyzed the sexual tissues issued from 15 fruiting bodies (perithecia) in at least three
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ACCEPTED MANUSCRIPT biological replicates per strain (i.e, 45 perithecia / strain). Live-cell imaging was performed with whole individual P. anserina colonies grown for 24h on M2 plates as described in [8], except that agar was replaced by 2% agarose and 0.55% dextrin was used. To analyze peroxisome protein import we used the following proteins: (i) FOX2-mCherry,
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(ii) PEX14-GFP (above), (iii) GFP-PTS1 (GFP fused to the C-terminal PTS1 tripeptide
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SKL [44]) and (iv) PTS2-GFP (GFP fused to the N-terminal PTS2 sequence from P.
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anserina peroxisomal thiolase [32]). Nuclei were stained with DAPI (4',6-diamidino-2-
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phenylindole, dihydrochloride, Molecular Probes, Eugene, OR) (0.5 g mL-1) and cell membranes were stained with the styryl dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(6-
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(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide, Molecular Probes) (7M). 2.10. Microscopy. Imaging was performed on a Nikon Eclipse E600 microscope with a
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cooled Neo Andor sCMOS camera, or on a Zeiss LSM-800 inverted laser scanning
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confocal microscope using a Plan-Apochromat 63x/1.4 oil immersion objective and 405,
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488 and 561nm laser lines. For live-cell confocal microscopy, images from all channels were obtained simultaneously. Maximum intensity projections were done from z-section
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images collected at 0.43 m intervals through entire cell volumes. Images were processed
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using the FIJI package [45] for ImageJ [46] (NIH, Bethesda, USA) or ZEN 2012 (Carl Zeiss, Jena, Germany) software. Colocalization and peroxisome abundance analyses were done on confocal micrographs of growing leading hyphae using ZEN 2012 software. For colocalization analyses, 12-15 hyphae issued from 3 biological replicates (4-5 hyphae/replicate) were analyzed for each strain. For each hypha, a region of interest (ROI) was defined as the cell area extending ≈140μm behind the hyphal apex. Image thresholds were set with the Costes method, and the Pearson's and Manders correlation coefficients
12
ACCEPTED MANUSCRIPT were calculated with the corresponding algorithms included in ZEN 2012. P values were calculated using paired two-tailed Student’s t-tests. Line scan analyses were performed on raw pixel data. To determine peroxisome abundance, the number of peroxisomes present in the middle plane area of three adjacent 140μm-long hyphal regions extending behind the
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apex was quantified. For each strain, 15 hyphae issued from 3 biological replicates (5
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hyphae/replicate) were analyzed. P values were calculated using unpaired two-tailed
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Student’s t-tests. To highlight cell outlines, a micrograph was merged to its corresponding
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phase-contrast image colored in blue and contrasted until only the cell outlines were visible.
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3. Results
3.1. P. anserina peroxisomes differ in their protein composition and distribution. To
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analyze the role of the exportomer in peroxisome protein targeting and import, we started
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by generating strains expressing mCherry- or GFP-tagged versions of endogenous peroxisome matrix and membrane proteins. We C-terminally tagged the matrix protein
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FOX2 with mCherry, and the PMP PEX14 with GFP. The function of both proteins in P.
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anserina has been previously studied [31, 33, 40]. FOX2, which catalyzes the second and third steps of the peroxisomal fatty-acid -oxidation pathway, lacks discernible peroxisome
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targeting sequence, but its import into peroxisomes depends on PEX5 [32]. Therefore, FOX2 constitutes a PEX5 cargo with targeting information different from canonical PTS1containing proteins. The import of these latter proteins in P. anserina can be analyzed using a peroxisome-targeted GFP containing a consensus PTS1 sequence (GFP-PTS1) [32, 44]. P. anserina mutant strains devoid of PEX14 or FOX2 are unable to grow or exhibit flimsy mycelial growth, respectively, on oleic acid-containing medium [33, 40]. In contrast,
13
ACCEPTED MANUSCRIPT PEX14 or FOX2 tagging did not affect the growth of P. anserina on this medium (Fig. S1B), suggesting that the function of these proteins is not affected by the tagging. FOX2mCherry stained discrete punctae colocalizing with GFP-PTS1 (Fig. 1A) with a high Pearson’s correlation coefficient (PCC, avg. 0.87 ± 0.03 in 12 analyzed hyphae). However,
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the relative intensity of these two fluorescent proteins varied among different peroxisomes.
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A number of peroxisomes that was intensely labeled with FOX2-mCherry presented only
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faint GFP-PTS1 staining (e.g, peroxisome 1 in Fig. 1A), and vice versa (Fig. 1A, peroxisome 3). Manders correlation coefficient (MCC) for GFP (M1 0.788) was similar to
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that of FOX2-mCherry (M2 0.782), suggesting equivalent fractions for these two proteins
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not colocalizing with each other. These data show a high co-occurrence for GFP-PTS1 and
number of peroxisomes might differ.
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FOX2 in hyphal peroxisomes, but also reveal that the relative amount of these proteins in a
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In contrast, the overall colocalization of PEX14-GFP and FOX2-mCherry was relatively low (PCC, av. 0.49 ± 0.06, n=15 hyphae). In addition to peroxisomes that were
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labeled with both FOX2-mCherry and PEX14-GFP, we observed FOX2-mCherry-stained
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peroxisomes devoid of PEX14 labeling (e.g. peroxisome 1 in Fig. 1B), as well as PEX14decorated punctae exhibiting low (peroxisome 2 in Fig. 1B) or not detectable (Fig. 1C)
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FOX2-mCherry labeling. Interestingly, these latter often consisted on small punctae that were more noticeable at the apical part of hyphae (arrows in Fig. 1C). MCC for GFP (M1 0.596) was significantly lower than for mCherry (M2 0.643) (t=5.4, 14 d.f. P=9.23E-05), indicating a larger fraction of PEX14 not colocalizing with FOX2-mCherry than the opposite. In addition, we also observed that PEX14-GFP present in FOX2-mCherry-labeled peroxisomes often exhibited a heterogeneous distribution. This included round-to-elongated PEX14 patches partially overlapping with FOX2-stained peroxisomes (Fig. 1B, peroxisome 14
ACCEPTED MANUSCRIPT 3), uneven PEX14 patches with increasing fluorescent intensity (Fig. 1B, peroxisome 4) and clusters of PEX14 patches distributed along FOX2-labeled peroxisomes (Fig.1C, arrowheads). The latter two distributions were more noticeable in elongated peroxisomes. Furthermore, time-lapse live-cell imaging revealed that these PEX14-GFP-stained patches
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were highly dynamic and redistributed along peroxisomes over time (Movie 1). These
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observations suggest a dynamic PEX14 localization in specific domains of the peroxisome
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membrane. Altogether, the above findings suggest the existence of distinct peroxisome populations in P. anserina hyphae that differ in their protein composition and spatial
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distribution.
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3.2. The exportomer peroxins and PEX8 are required for fatty acid utilization and
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aerial mycelium formation. Next, to investigate the function of the exportomer peroxins, we generated strains deleted for the genes predictably encoding the ubiquitination-complex
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peroxins PEX4 and PEX22, the dislocation-complex peroxins PEX1, PEX6 and PEX26,
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and the peroxin PEX8. Deletion of either of these genes resulted in a phenotype that recapitulates the defects typically observed in P. anserina mutants defective for peroxisome
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biogenesis [31-34], including their incapacity to grow on oleic acid as sole carbon source, a
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reduced formation of aerial mycelium (Fig. S1B), and defective sexual spore (ascospore) pigmentation (assessable in heterozygous sexual crosses to a wild-type strain, shown for
pex8 in Fig. S1C). 3.3. PEX8 is required for peroxisome matrix protein import. Then, we sought to determine the role of these proteins in P. anserina peroxisome biogenesis. First we analyzed the role PEX8. PEX8 is highly conserved in fungi [47], however the function of this protein beyond yeasts has not been addressed. P. anserina PEX8 orthologue 15
ACCEPTED MANUSCRIPT predictably encodes a 691 aa protein (Fig. S2A) containing a canonical PTS1 SKL Cterminal tripeptide, and a N-terminal sequence similar to consensus PTS2 targeting sequences [i.e, R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)] [48]. To study the role of PEX8 in P. anserina peroxisome biogenesis we first tested whether this protein was
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involved in peroxisome matrix protein import. We observed that, in contrast to the punctate
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pattern of GFP-PTS1 (Fig. 2A) and FOX2-mCherry (Fig. 2B) exhibited for peroxisomes in
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wild-type cells, both GFP-PTS1 (Fig. 2C) and FOX2-mCherry (Fig. 2D) remained in the
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cytosol of pex8 hyphae, indicative of a defective peroxisome import of these two PEX5 cargoes in these cells. The peroxisome import of both GFP-PTS1 and FOX2-mCherry was
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restored upon ectopically reintroducing a wild-type copy of PEX8 gene into the pex8 strains (shown for FOX2 in Fig. S3A), corroborating the implication of PEX8 in this
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process.
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Then we investigated if PEX8 was required for the PTS2 peroxisome import
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pathway by studying the localization of a PTS2-containing GFP reporter protein (PTS2GFP). We analyzed PTS2-GFP localization in ascospores of asci (the meiocytes in which
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ascospores are differentiated after meiosis) issued from pex8 to WT heterozygous crosses,
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where the 2:2 segregation allows a direct comparison between WT and pex8 cells. We observed that PTS2-GFP localized to peroxisomes in WT ascospores, but mislocalized to cytosol in pex8 ascospores (Fig. S4A), indicating that PEX8 is also required for the PTS2 peroxisome protein import. Finally, we analyzed the role of PEX8 in PMP import by studying the localization of PEX14. We observed that PEX14-GFP localized to discrete punctae in pex8 hyphae (Fig. 3), which were more heterogeneous in shape than PEX14GFP-stained wild-type peroxisomes, consistent with a peroxisome-remnant labeling. This
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ACCEPTED MANUSCRIPT shows that the formation of peroxisome membranes and the targeting of, at least, PEX14 into these membranes does not require PEX8. 3.4. PEX4 and PEX22 are differentially required for the import of distinct PEX5 cargoes. Next we studied the role of the proteins potentially involved in receptor
IP
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monoubiquitination. In yeasts, the monoubiquitination of Pex5 and of the PTS2 coreceptors is mediated by Pex4 and its anchor/activator Pex22 [49-55]. P. anserina
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possesses an orthologue of PEX4 and a gene likely coding for a Pex22-like protein (Fig. S2,
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B and C). Pex22-like proteins are conserved in filamentous ascomycetes and they possess a domain similar to the Pex4-binding motif of Pex22 [47]. Actually, the Pex22-like protein of
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Colletotrichum orbiculare is functionally equivalent to S. cerevisiae Pex22 [56] (therefore,
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we will further refer to this protein as Pex22).
We analyzed the effect of PEX4 and PEX22 deletion on GFP-PTS1 (Fig. 2, E and
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G) and PTS2-GFP (Fig. S4, B-C) localization, and we observed that these two proteins
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mislocalized to cytosol in pex4 and pex22 cells. We also observed that PEX14-GFP localized to discrete punctae in pex4 and pex22 hyphae (Fig. 3, C-D), which were
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similar to those observed in ∆pex8 cells. These observations indicate that PEX4 and PEX22
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are involved in the PTS1 and PTS2 peroxisome matrix import pathways, but they are not required for PMP import. Interestingly, however, FOX2-mCherry exhibited a dual localization in pex4 and pex22 hyphae. In these cells, a fraction of FOX2-mCherry localized to cytosol, but discrete punctae resembling peroxisomes were also clearly stained by FOX2-mCherry (Fig. 2, F and H). Double labeling experiments demonstrated that the FOX2-mCherry-labeled punctae present in pex4 and pex22 hyphae also contained PEX14 (shown for pex22 in Fig. 4A and for pex4 in Fig. S5), corroborating the
17
ACCEPTED MANUSCRIPT peroxisomal nature of these structures. In addition, we observed a number of peroxisomes in which PEX14-GFP predominantly decorated the organelle periphery, whereas FOX2mCherry stained its interior (Fig. 4B and Movie 2), suggesting that FOX2-mCherry is actually imported into the matrix of these peroxisomes. We also noticed that the FOX2-
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mCherry-labeled peroxisomes were more abundant in the apical segment of hyphae
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extending ≈140m behind the hyphal tip. Their numbers decreased behind that region
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(between 140 – 280m behind the apex, a region were the first septum is frequently
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located, i.e. at 271 ± 57m behind the apex for pex4 and at 240 ± 43m for pex22; n=15 hyphae/strain), and they were scarce in the distal parts of hyphae (behind 280m from the
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hyphal tip) (Figs. 4C and S5). These differences are likely not the consequence of altered peroxisome movements or distribution, since PEX14-GFP localization exhibited a more
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even peroxisome-remnant distribution along pex4 and pex22 hyphae (shown for pex4
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in Fig. S5). This could indicate a more prominent peroxisomal import of FOX2 in the
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apical cells of pex4 and pex22 hyphae. The ectopic reintroduction of a wild-type allele of PEX4 or PEX22 gene into the respective deletion mutant restored FOX2 peroxisome
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import throughout mycelia (Fig. S3), confirming that the described phenotypes are due to
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the deletion of these genes. Then we observed that the phenotypes of the single pex4 and
pex22 mutants, and of a pex4pex22 double mutant were alike (Fig. 4D, see also Fig. S1B), consistent with PEX4 and PEX22 working in a common process. These results show that absence of PEX4 and PEX22 prevents the peroxisomal import of GFP-PTS1 and partially inhibits that of FOX2-mCherry. It has been postulated that peroxisome matrix protein import is mechanistically coupled to the export of the receptors from the peroxisome membrane [23]. In S. cerevisiae,
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ACCEPTED MANUSCRIPT both the mono- and poly-ubiquitinated forms of Pex5 can be released from the peroxisome membrane in a dislocation complex-facilitated process [50]; thus, we hypothesized that the FOX2 import observed in pex4 and pex22 mutants could be promoted by a polyubiquitination-dependent release of PEX5. To test this hypothesis, first we
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corroborated that FOX2-mCherry import into hyphal peroxisomes actually depended on
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PEX5, as indicated by the cytosolic localization of FOX2-mCherry throughout hyphae of a
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pex5 mutant strain (Fig. 4D). Pex5 polyubiquitination depends on the three RING finger
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peroxins [57-59]. Actually, in S. cerevisiae Pex5 polyubiquitination is mediated by Pex2 acting in close association with Pex10 [59-61]. Thus, we then tested whether the partial
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import of FOX2 in pex4 and pex22 cells depended on PEX10. First, we observed that FOX2-mCherry mislocalized to cytosol in pex10 hyphae, with no detectable peroxisome
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localization (Fig. 4D). Then, we observed that pex10 was epistatic to both pex4 and
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pex22 for this phenotype (Fig. 4D). This finding suggests that an ubiquitination system
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depending either, directly or indirectly on PEX10 can partially sustain the peroxisome import of FOX2 in pex4 and pex22 cells.
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Previously, we showed that FOX2 and GFP-PTS1 import differentially depends on
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the docking complex at specific stages of sexual development [31]. Therefore, we studied whether this differential requirement for the ubiquitination complex was also observed during sexual development. As shown on figure 4E for a pex22 homozygous sexual cross, FOX2-mCherry also localized both to cytosol and peroxisomes in the dikaryotic stage cells (croziers) of sexual development. In contrast, GFP-PTS1 was predominantly cytosolic. We observed that FOX2 localized to peroxisomes in 151 of 152 (99.3%) analyzed croziers, whereas GFP-PTS1-decorated punctae were only observed in 9 of these cells (5.9%) and in
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ACCEPTED MANUSCRIPT reduced numbers (Fig. 4E, arrow). These data show a more stringent requirement of PEX4 and PEX22 (only shown for PEX22) for the import of GFP-PTS1 than of FOX2 also during the sexual cycle. The phenotype of pex4 and pex22 mutants during sexual development (see below), however, precluded analyzing the role of these peroxins during meiotic
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development.
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3.5. Loss of peroxisomes upon elimination of PEX1, PEX6 or PEX26. Next we studied the function of the proteins that predictably constitute the P. anserina peroxisome receptor
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dislocation complex. P. anserina possesses an orthologue of each PEX1, PEX6 and PEX26. PEX1 predictably encodes a 1236 aa protein containing the two conserved nucleotide-
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binding domains (D1 and D2) characteristic of type II AAA+-ATPases (Fig. S2D); whereas
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PEX6 codes for a 1399 aa protein possessing a conserved D2 nucleotide-binding domain but a poorly conserved D1 domain (Fig. S2E). PEX26 predictably consist on a 482 aa
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protein possessing a C-terminal trans-membrane segment, which overlaps with a putative
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PEX19-binding domain characteristic of peroxisome tail-anchored proteins (Fig. S2F). To confirm the participation of these proteins in peroxisome biogenesis, we first
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analyzed their involvement in peroxisome matrix protein import. We observed that the
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localization of GFP-PTS1, FOX2-mCherry (Fig. 2, I-N) and of PTS2-GFP (Fig. S4, D-F) was cytosolic in pex1, pex6 and pex26 cells, indicating that the three proteins –PEX1, PEX6 and PEX26– are required for the import of peroxisome matrix proteins for both PTS1 and PTS2 pathways. Next we analyzed the effect of PEX1, PEX6 and PEX26 deletion on PEX14 localization. Interestingly, in contrast to the PEX14-GFP punctate distribution observed for WT peroxisomes, or for pex8, pex4 and pex22 peroxisomal remnants, only a reduced number of structures were labeled by PEX14-GFP in pex1, pex6 and
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ACCEPTED MANUSCRIPT pex26 hyphae (Fig. 3, E-G). These structures consisted in discrete punctae that were often grouped, or in a large dynamic multilobular structure, which could represent tightly clustered structures (shown for pex6 in Fig. 5A and in Movie 3). These PEX14-containing structures were frequently located in the subapical region of hyphae, near to a highly
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vacuolized hyphal region; but they were not observed to colocalize with the
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endosomal/vacuolar membrane dye FM4-64. Importantly, the reintroduction of a wild-type
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allele of the deleted gene into the corresponding deletion mutant (tested for pex1 and
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pex6, Fig. S3) restored both peroxisome protein-import and abundance, corroborating that both phenotypes were actually caused by these gene deletions.
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Loss of PEX14-GFP-labeled peroxisome remnants in cells devoid of PEX1, PEX6
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or PEX26 could indicate an involvement for these proteins in peroxisome membrane formation. Alternatively, the elimination of these proteins could increase the degradation of
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peroxisomes via pexophagy, as occurs in S. cerevisiae [62]. Actually, we noted that a
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number of small punctate structures was also faintly labeled by PEX14-GFP in pex1,
pex6 and pex26 hyphae. We observed that some of these structures were often
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distributed in the periphery of spherical organelles visible by phase-contrast microscopy
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likely representing vacuoles (Fig. 3 E-G, highlighted for pex26 in Fig. 3H), as was further supported by staining cells with FM4-64 (shown for pex1 in Fig. 5B). FM4-64 analyses also revealed diffuse GFP labeling within vacuoles (shown for pex1 and pex6 in Fig. 5, B and C), as well as GFP-labeled punctae confined within vacuolar compartments (Fig. 5C, and Movie 4). Of note, some of these latter punctae appeared to be circumscribed by an FM4-64-labeled intravacuolar membrane (Fig. 5C, arrowhead). These observations suggest that a fraction of PEX14-containing organelles is subjected to vacuolar degradation in
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ACCEPTED MANUSCRIPT pex1, pex6 and pex26 hyphae. To further inspect whether peroxisome removal was enhanced in P. anserina dislocation-complex mutants, we analyzed PEX14 localization in cells under different metabolic demands. Peroxisomes are degraded via selective autophagy (pexophagy) in response to nutritional availability. For example, in yeasts, a number of
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peroxisomes produced in peroxisome proliferation-inducing conditions are degraded by
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pexophagy when cells are transferred to glucose-containing medium [63]. As in other fungi,
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peroxisome proliferation in P. anserina can be induced by growing cells in oleic acid-based medium [8]. Thus, we compared PEX14-GFP distribution in glucose- and oleic acid-
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containing media. Since pex1, pex6 and pex26 mutants are unable to grow on oleic acid
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as sole carbon source (Fig. S1B), a low concentration of glucose (0.03%) was added to the oleic-acid medium to facilitate the growth of these mutants. In this medium, a substantial
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proliferation of peroxisomes occurred in WT cells (Fig. 5D). Notably, the number of
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PEX14-stained punctae present in pex1, pex6 and pex26 cells relative to dextrin-
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containing medium was markedly increased, reaching levels comparable to those observed for peroxisomes in WT cells (Fig. 5D). This reveals that peroxisome remnants are present
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in pex1, pex6 and pex26 cells under peroxisome proliferation-inducing conditions,
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indicating that the dislocation complex is not required for peroxisome membrane biogenesis in P. anserina. In contrast, the number of PEX14-GFP-labeled structures in
pex1, pex6 and pex26 cells grown on glucose as sole carbon source was further reduced (Fig. 5D), consistent with an increased removal of peroxisomes in these metabolic conditions. 3.6. The exportomer peroxins and PEX8 are required for meiocyte formation. Having established the participation of PEX8 and of the exportomer peroxins in peroxisome
22
ACCEPTED MANUSCRIPT biogenesis, we analyzed their requirement for sexual development. Sexual reproduction in P. anserina involves the formation of multicellular fructifications (perithecia) that enclose the fertile tissue (the hymenium) on which karyogamy, meiosis and ascospore differentiation take place (Fig. S1A). The hymenium is issued from fertilization and
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initially consists of syncytial (ascogonial) cells bearing nuclei from both parental origins.
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These cells produce specialized dikaryotic hyphae called croziers (Fig. 6A, cell a), which
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undergo karyogamy (Fig. 6A, cell c) and develop into meiocytes (asci, cells d-e in Fig. 6A). Following meiosis, the four resulting nuclei divide mitotically yielding eight nuclei, which
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are packaged by pairs into four ascospores (Fig. 6B).
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We analyzed the sexual development of pex8, pex4, pex22, pex1, pex6 and
pex26 mutants both in homozygous and heterozygous (to the wild type) crosses, and we
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observed that all these mutants were sterile in homozygous crosses. In these crosses, all
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mutants were able to produce perithecia in which the sexual tissues correctly progressed
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until the dikaryotic stage. Crozier cells were properly formed and their nuclear distribution was consistent with an accurate dikaryotic compartmentalization prior to karyogamy (Fig.
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6, C-H). However, these cells did not undergo karyogamy and did not differentiate into asci, consequently, ascospores were never produced. Instead, crorzier cells proliferated
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mitotically rather than entering meiosis, which resulted in the formation of numerous crozier arborescences (Fig. S3B, left panels). For each mutant, this phenotype was shown to be a recessive trait, since asci and ascospores were effectively produced in heterozygous crosses of either mutant to the wild type (e.g, Fig. S4). This phenotype was virtually undistinguishable between mutants, as was from the previously described for pex13,
pex20 and the RING finger-complex mutants [31, 33, 34]. Sexual development was
23
ACCEPTED MANUSCRIPT restored in all tested gene-complemented deletion mutant strains (Fig. S3B), corroborating that the phenotype of these mutants during sexual development was also due to the corresponding gene deletions. These results show that the peroxins of the peroxisome receptor export machinery, as well as the peroxin that presumably links this complex to the
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import machinery, are required for meiocyte formation in P. anserina. 3.7. The potential monoubiquitination target cysteine of PEX20 is required for
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meiocyte formation. Altogether, our results suggest that meiocyte formation relies on an
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alternative peroxisome protein import pathway driven by a cycling receptor whose activity is regulated by monoubiquitination. Since the PTS2 co-receptor PEX20 is required for
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meiocyte formation, this peroxin is the most likely candidate to act as such receptor [31].
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To further explore this hypothesis, we tested whether the potential monoubiquitination target amino acid of PEX20 was required for PEX20 function in meiocyte formation.
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Monoubiquitination of Pex5 [49, 64] and of yeast PTS2 co-receptors Pex18 [24] and Pex20
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[54] takes place at a conserved N-terminal cysteine residue, which is present at position 6 (Cys6) in P. anserina PEX20. Therefore, we generated a PEX20 allele coding for a protein
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in which this cysteine is replaced by alanine (pex20C6A), and we analyzed if the ectopic
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introduction of this allele into a ∆pex20 strain was able to restore sexual development of this mutant. First, to confirm that PEX20+ reintroduction into the ∆pex20 background restores sexual development, we transformed ∆pex20 cells with a wild-type copy of PEX20 gene, and we observed that all recovered transformants (n=8) were able to effectively produce asci and ascospores (Fig. 7C, compare to A and B). In contrast, we observed no restoration of sexual development when ∆pex20 was transformed with the pex20C6A allele (n=8 transformants) (Fig. 7D). In S. cerevisiae, substitution of the conserved Pex5
24
ACCEPTED MANUSCRIPT monoubiquitination target cysteine (Cys6) by lysine, which is artificially polyubiquitinated in yeast cells, results in a functional import receptor; indicating that not the cysteine per se, but the position of this ubiquitination is important for Pex5 function [65]. We performed an equivalent experiment in P. anserina and tested whether the ectopic introduction of a
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pex20C6K allele into the ∆pex20 background was able to restore sexual development.
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However, no restoration of sexual development was observed after transforming ∆pex20
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cells with a pex20C6K allele (n=9 transformants) (Fig. 7E), suggesting that the activity of PEX20 more stringently depends on the monoubiquitination of a cysteine residue. These
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results indicate that the potential monoubiquitination target cysteine of PEX20 is required
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peroxisomes is essential for this process.
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for PEX20 function in meiotic induction, and suggest that the recycling of PEX20 from
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4. Discussion
Peroxisome activity varies depending on the developmental demands of the cell. The
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function of this organelle is significantly defined by the composition of the proteins
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residing in its luminal compartment. Our research has shown that a specific number of peroxins involved in driving peroxisome matrix protein import is strictly required for the
AC
induction of meiotic development in P. anserina. These peroxins include the PTS2 coreceptor PEX20 but not the import receptors PEX5 or PEX7. Our current research shows that the initiation of meiotic development of this fungus also strictly requires the peroxins of both the peroxisome ubiquitination machinery and the peroxisome receptor dislocation complex, as well as the peroxin that likely bridges the peroxisome protein import and receptor export machineries. These results are consistent with the existence of an
25
ACCEPTED MANUSCRIPT alternative peroxisome import pathway, which is required for meiosis and that operates in absence of the known import receptors. Many cells possess peroxisomes that differ in shape, protein composition and distribution. The existence of different peroxisome populations underscores the dynamic
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organization of this organelle in a cell, and can be related to the functional specialization of
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a peroxisome subpopulation along cell development [1, 66, 67]. In P. anserina we observed
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that vegetative-cell peroxisomes are heterogeneous both in the composition and in the spatial distribution of their proteins. We observed that the peroxisome localization of
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PEX14, a central component of the peroxisome protein translocation machinery, was either
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asymmetric or consisted on scattered patches distributed along peroxisomes, suggesting its localization in specific peroxisome domains. Consistently, recent detailed analyses in
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human cells have shown that PMPs –including Pex14– are not evenly distributed over the
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peroxisome surface, but they rather localize to specific membrane domains [67]. In addition, live-cell imaging analyses revealed that the compartmentalized localization of P.
PT
anserina PEX14 is highly dynamic, suggesting a dynamic reorganization of the membrane
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domains that are enriched for this peroxin. In filamentous ascomycetes, two exportomer peroxins –C. orbiculare Pex22 and N. crassa Pex26– also display asymmetric peroxisome
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localization [56, 66]. Interestingly, the distribution of these peroxins is related to Woronin body formation, albeit with an opposite pattern. In addition, N. crassa Pex14 occasionally localize to peroxisome foci from where Woronin bodies seemingly emanate [68]. Thus, a fraction of PEX14 could also be associated to Woronin body formation domains. We also obtained evidence for the existence of peroxisomes differing in their protein composition. We observed that the relative amount of two peroxisome matrix proteins in some peroxisomes varied in an inverse relation, suggesting a differential import 26
ACCEPTED MANUSCRIPT competence for these proteins. In N. crassa, the differentiation of highly import competent peroxisomes involves a positive feedback system, which is induced by matrix oligomers of a PTS1-containing protein (HEX1) that promotes the import of further matrix proteins [66]. Interestingly, both implicated proteins in P. anserina are PEX5 cargoes (GFP-PTS1 and the
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non-PTS1 protein FOX2), but they differ in their targeting information. Thus, it is tempting
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to speculate that some peroxisomes exhibit differential import competence towards
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different PEX5 cargoes. Actually, we and others previously demonstrated differential docking-complex requirements for the import of distinct PEX5 cargoes both in P. anserina
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[31] and in N. crassa [69]. Moreover, in our current research we discovered that the import
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of these two PEX5 cargoes also exhibit differential exportomer requirements. PTS1 sequences are recognized by the C-terminal TPR domain of Pex5 [70]. In contrast, the
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targeting of non-PTS1 cargoes, like S. cerevisiae Fox1p and Hansenula polymorpha
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alcohol oxidase, depends on their interaction with the N-terminal half of Pex5 [71, 72], which is also the domain mediating receptor docking, membrane insertion and extraction,
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and that provides the receptor cargo transport activity [73-75]. Therefore, it is possible that
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the import of these different cargoes depends on different configurations of the Pex5 tanslocon, which could differ in their assembly / disassembly requirements.
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Our results showing that PEX4 and PEX22 elimination results in different extent of inhibition of the peroxisomal import of GFP-PTS1 and FOX2 suggest that the receptor monoubiquitination machinery is differentially required for the import of distinct PEX5 cargoes. The dynamics of Pex5, and of the PTS2 co-receptors, are defined by different ubiquitination pathways, which include receptor monoubiquitination that facilitates their recycling, and polyubiquitination that promotes their degradation via the proteasome [24, 50, 57, 64, 76, 77]. This latter process has been considered as a quality control system that 27
ACCEPTED MANUSCRIPT prevents receptor accumulation in the peroxisome membrane [57, 58, 78], and it has been referred to as Receptor Accumulation and Degradation in Absence of Recycling (RADAR) [77]. Actually, in the yeast Pichia pastoris, this pathway can partially rescue peroxisome matrix protein import, which is defective when Pex20 recycling is compromised [76]. The
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observation that the peroxisome import of FOX2 in pex4 and pex22 cells is abrogated
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upon PEX10 elimination is consistent with an equivalent polyubiquitination system
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sustaining FOX2 import in these cells. Alternatively, additional ubiquitin-conjugating
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enzymes could mediate PEX5 mono-ubiquitination in P. anserina, as proposed for Trypanosoma brucei, where peroxisome matrix protein import is only slightly affected by
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PEX4 elimination [79]. If a ubiquitination system is involved in alleviating FOX2 import in absence of PEX4 and PEX22, then this system should preferentially act on PEX5
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molecules involved in FOX2 (or non-PTS1) import. Furthermore, such process would be
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more plausible under an export-driven import system, in which the export of these ubiquitinated PEX5 molecules exclusively propels the import of their specific cargoes.
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Our research also revealed that elimination of the dislocation complex peroxins
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results in peroxisome loss in P. anserina. In filamentous ascomycetes, Pex6 (PexF, in A. nidulans) and Pex26 (in N. crassa) were previously shown to be dispensable for
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peroxisome membrane formation [66, 80], however whether peroxisome removal was enhanced upon elimination of these peroxins in these fungi was not addressed. In S. cerevisiae the number of peroxisome remnants is reduced in cells lacking of Pex1, Pex6 or Pex15 [62]. Actually, in this yeast, it has been demonstrated that loss of either of these peroxins results in enhanced pexophagy, which is dependent on the pexophagy receptor Atg36 [62]. The precise mechanism accounting for peroxisome loss in P. anserina
28
ACCEPTED MANUSCRIPT dislocation-complex mutants remains to be established; however, our results suggest that this process involves their elimination by pexophagy. Interestingly, it has recently been demonstrated that PEX1 and PEX26 also prevent pexophagy in human cells [81], and that pexophagy is triggered upon PEX1 dysfunction in Arabidopsis thaliana [82]. These
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findings indicate that restraining pexophagy is a conserved function of the dislocation
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complex in animals, plants and fungi.
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With the conclusion of this research, we have analyzed the role of all known peroxins predictably (based on orthology relations) involved in peroxisome matrix protein
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import in P. anserina [31-34]. Notably, with the exception of the import receptors PEX5
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and PEX7, and of the docking peroxins PEX14 and PEX14/17, all remaining peroxins are required for the induction of meiotic development in this fungus (Fig. 8). Our findings
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support a model in which meiocyte formation relies on an alternative peroxisome import
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that does not depend on the canonical import receptors. This pathway could be driven by PEX20 as import receptor, and PEX13 could provide its docking factor [31]. Previously we
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showed that meiocyte formation depended on the RING-finger complex [33], our current
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finding that PEX8 –the peroxin bridging the importomer subcomplexes in S. cerevisiae [28]– is also required for this developmental process further supports a pathway dependent
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on the interaction between the peroxisome docking and RING-finger complexes. Actually, data showing that the interaction of Pex8 and Pex13 is critical for Pex8 topogenesis [83] is also consistent with our finding that PEX13 is the only docking peroxin required for meiocyte formation. The RING-finger complex peroxins participate in ubiquitination processes –including the RADAR system and pexophagy signaling [27, 84], additional to peroxisome import receptor monoubiqutination. Our data implicating PEX4 and PEX22 in meiocyte formation now supports the existence of an additional receptor whose dynamics 29
ACCEPTED MANUSCRIPT are regulated by ubiquitination in a similar way to PEX5. Our data also implies a cycling receptor whose activity would critically depend on the AAA ATPase complex. Recently, a paralogue of Pex5 (Pex9) acting as additional import receptor in S. cerevisiae was identified [85, 86]; however, we have not to identify paralogues of PEX5 or PEX7 in the
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genome of P. anserina. Therefore, PEX20 constitutes the most likely candidate to provide
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an additional import receptor. Consistent with this notion, we now also provided evidence
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suggesting that the monoubiquitination-dependent recycling of PEX20 is essential for induction of meiosis in this fungus. Still, we cannot exclude the possibility of PEX20 acting
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as co-receptor of another yet unidentified PEX7-unrelated targeting receptor.
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In S. cerevisiae, Pex18 is a main constituent of the peroxisome membrane pore through which PTS2 proteins are translocated, which is independent from the PTS1 pore
M
[22]. The activity of Pex18 in S. cerevisiae can be substituted by Pex20 of N. crassa [87],
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strongly suggesting the presence of two independent pores also in filamentous ascomycetes. The discovery that in P. anserina PEX20 is required for the initiation of meiotic
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development [31] and PEX5 for its progression [32] suggests that the import channels of
development.
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this fungus mediate the import of proteins contributing to different stages of meiotic
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Nonetheless, the precise peroxisome-dependent developmental process required to initiate meiotic development remains undefined. All peroxin mutants defective for this process are unable to undergo nuclear fusion in their dikaryotic cells. Karyogamy and meiosis are coupled processes in filamentous ascomycetes. Actually, in these fungi, premeiotic S-phase precedes karyogamy and some of the proteins mediating meiosis are incorporated into chromosomes at this stage [88], indicating that the decision to enter meiosis is determined before karyogamy. In P. anserina the frequency of nuclear fusion in 30
ACCEPTED MANUSCRIPT vegetative cells is not affected by pex2 mutation [34], suggesting that PEX2 is not required for the nuclear fusion process per se. In addition, mutants of filamentous acomycetes impaired for karyogamy due to deficient internuclear recongnition at the dikaryotic stage [89] or to defective nuclear envelope fusion [90] are still able to undergo haploid meiosis.
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In contrast, in the peroxin mutants defective for meiocyte formation the dikaryotic cell
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nuclei fail to enter the meiotic cycle and continue dividing mitotically. These observations
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suggest that rather than karyogamy per se, peroxisomes could be involved in a regulatory process required for cells to leave the mitotic cell cycle and begin meiosis (the
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mitosis/meiosis decision). Alternatively, peroxisomes could be required for the
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differentiation of a cellular state competent to trigger meiosis. Remarkably, recent research has disclosed that peroxisomes are involved in the decision of mammalian epidermal cells
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between entering mitosis or differentiating [15]. These findings underscore an important
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role for peroxisomes in regulatory events that define transcendent cell fate decisions during
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development.
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ACKNOWLEDGMENTS
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We thank all members of the laboratory for assistance and valuable discussions, and Fernando García Hernández (IFC, UNAM, Mexico) for assistance on microscopy. We are much indebted to Jesús Aguirre and Wilhelm Hansberg (IFC, UNAM) for assistance throughout this research, and to Véronique Berteaux-Lecellier (CRIOBE, CNRS), Robert Debuchy (I2BC, Gif-sur-Yvette, France) and Philippe Silar (Univ. Paris Diderot, Paris, France) for kindly providing strains and plasmids. We are grateful to Robert Debuchy for critically reading the manuscript.
31
ACCEPTED MANUSCRIPT FUNDING This work was supported by grants UNAM-DGAPA-PAPIIT IA201815 and IA203317.
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CZM and HTR were supported by scholarships from these grants.
J.J. Smith, J.D. Aitchison, Peroxisomes take shape, Nat Rev Mol Cell Biol 14
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FIGURE LEGENDS Figure 1. Localization of FOX2-mCherry, GFP-PTS1 and PEX14-GFP in P. anserina hyphae. Wild-type hyphae expressing GFP-PTS1 and FOX2-mCherry (A) or FOX2-
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mCherry and PEX14-GFP (B, C) were grown for 24h in M2 medium and analyzed by
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confocal microscopy. In (A-B), insets display enlarged areas showing representative
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peroxisomes, and the lower line graphs display the fluorescence intensity for both mCherry and GFP channels along the lines of the respectively labeled peroxisomes. In (B) note that
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x-axis for line graph 4 is not in the same scale as for peroxisomes 1-3. In (C) note PEX14-
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GFP localization in clusters of patches lying along FOX2-labeled peroxisomes (arrowheads) and in apical punctate structures not colocalizing with FOX2-mCherry
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(arrows). BF: bright field. Scale bar, 5m. Inset scale bar, 2m.
Figure 2. Localization of GFP-PTS1 and FOX2-mCherry in wild-type, Δpex8 and
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exportomer-mutant hyphae. Wild-type, pex8, pex4, pex22, pex1, pex6 or pex26
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strains expressing GFP-PTS1 (left) and FOX2-mCherry (right) were grown in dextrincontaining medium for 24h and analyzed by fluorescence microscopy. GFP-PTS1 and
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FOX2-mCherry localize to peroxisomes in wild-type hyphae (A and B). In contrast, these proteins mislocalize to cytosol in absence of PEX8 (C-D), PEX1 (I-J), PEX6 (K-L), or PEX26 (M-N). GFP-PTS1 is also cytosolic in pex4 (E) and pex22 (G) hyphae, but in these cells FOX2-mCherry localizes both to cytosol and to discrete punctate structures resembling peroxisomes (F and H, arrows). Scale bar, 5m.
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ACCEPTED MANUSCRIPT Figure 3. Localization of PEX14-GFP in wild-type, pex8 and exportomer-mutant hyphae. PEX14-GFP localization in wild-type (A), pex8 (B), pex4 (C), pex22 (D),
pex1 (E), pex6 (F) or pex26 (G-H) mycelia grown for 24h in dextrin-containing medium. Lower panels in (E-H) show corresponding phase-contrast micrographs. Image
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(H) displays a magnification of the boxed area in (G), in which the brightness was digitally
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enhanced; arrowheads indicate the location of phase contrast-visible vacuoles. PEX14-GFP
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decorated wild-type peroxisomes (A) and localized to discrete punctae in pex8 (B), pex4
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(C) and pex22 (D) hyphae. However, the number of comparable PEX14-GFP-labeled punctae in pex1, pex6 and pex26 hyphae was reduced (E-G), and these were clustered
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in the subapical region of hyphae (arrows). In these cells, PEX14-GFP also stained a
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number of small punctae, which were less bright and often localized in the periphery of
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vacuolar organelles (arrowheads). Scale bar, 5m.
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Figure 4. Absence of PEX4 and PEX22 partially inhibits FOX2 peroxisome import. A) Confocal microscopy analysis of FOX2-mCherry and PEX14-GFP localization in pex22
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cells. B) Detail of PEX14-GFP and FOX2-mCherry localization in a pex22 hypha. C)
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FOX2-mCherry localization along different regions of pex22 hyphae. Lower histogram, quantitation of FOX2-labeled peroxisomes in pex4 and pex22 hyphae. Values are mean ± SD; n = 15. *P=0.01 **P=0.0019, ***P<0.0001 by unpaired Student’s t test. D) FOX2mCherry localization in wild-type (WT), pex5, pex4, pex22 or pex10 strains, and in
pex4pex22, pex4pex10 or pex22pex10 double mutant strains. FOX2-mCherry localizes to both cytosol and peroxisomes in pex4, pex22 and pex4pex22, but only to cytosol in pex5, pex10, pex4pex10 and pex22pex10 cells. E) Maximum-intensity 45
ACCEPTED MANUSCRIPT projections of GFP-PTS1 and FOX2-mCherry localization in dikaryotic crozier cells issued from pex22 homozygous sexual crosses. FOX2-mCherry labeled the cytosol and peroxisomes of pex22 croziers, while GFP-PTS1 was predominantly cytosolic, few GFPPTS1-stained peroxisomes were observed in these cells (arrow). Bottom panels show
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corresponding mCherry/GFP (left) and DAPI/bright field (BF, right) merge micrographs.
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Scale bar in A, C-E: 5m; scale bar in B: 1m.
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Figure 5. PEX14-containing peroxisome remnants are lost upon elimination of the dislocation complex peroxins. A) Confocal microscopy analysis of PEX14-GFP in a
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pex6 hypha stained with FM4-64. S, indicate Spitzenkörpers; BF, bright field. Note the predominant localization of PEX14 in a large subapical structure (arrow). B)
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Epifluorescence microscopy analysis of PEX14-GFP localization in a pex1 hypha stained
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with FM4-64. Right panels display magnifications of the boxed areas at left. PC, phase
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contrast. Note the punctate localization of PEX14-GFP in the periphery of phase-contrastand FM4-64-detectable vacuoles (arrows), as well as of diffuse GFP labeling within these
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vacuoles. C) Detail of PEX14-GFP localization in pex6 hyphae stained with FM4-64.
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PEX14-GFP-labeleded punctae (right and middle panels, arrows) and diffuse GFP staining (left panels) are detected in the lumen of FM4-64-labeled vacuoles. Note a luminal PEX14GFP-stained patch encircled by an FM4-64-labeled internal membrane (arrowhead). D) PEX14-GFP localization in wild-type, pex1, pex6 or pex26 hyphae grown for 24h in media containing glucose (0.3%) (left) or oleic acid (OA) plus low glucose (0.03%) (right). Cell outlines are highlighted in blue. Scale bar in A, B and D: 5m; scale bar in C: 2m.
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ACCEPTED MANUSCRIPT Figure 6. PEX8 and the exportomer peroxins are required for meiocyte formation. Sexual cycle cells issued from homozygous crosses of the indicated genotypes were stained with DAPI and analyzed by fluorescence microscopy (left panels). Right panels, corresponding DAPI/bright field merge micrographs. In (B) only the DAPI micrograph is
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shown. Small lettering indicates progressive developmental stages. Sexual crosses of WT
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strains produce dinucleate dikaryotic croziers (A, crozier a, arrowheads with different
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shading indicate opposite mating-type nuclei), which divide to produce tetranucleate croziers. In these croziers, two opposite mating-type nuclei undergo karyogamy (c, arrow)
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and the two remaining (arrowheads) engage in the formation of a new crozier. Upon
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karyogamy the upper crozier cell differentiates into an ascus (note that elongation in the upper part of crozier c) and immediately enters meiosis (d and e show asci at different
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stages of first meiotic prophase). Ultimately, the meiotic-derived nuclei are
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compartmentalized into four dinucleate ascospores (B). In contrast, sexual development does not progress beyond the dikaryotic stage in homozygous crosses ofpex8 (C), pex4
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(D), pex22 (E), pex1 (F), pex6 (G) and pex26 (H) mutants. In these mutants, croziers
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are properly formed and their nuclear divisions and movements take place correctly, yielding croziers that posses two pairs of nuclei (b, compare to dinuceated corziers in a);
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however, these croziers do not undergo karyogamy and their nuclei divide mitotically rather than entering meiosis. Scale bar, 5m.
Figure 7. Cys6 of PEX20 is required for meiocyte formation. Mature fruiting bodies from wild-type sexual crosses contain 4-spored asci (A), while those from ∆pex20 mutant strains contain only crozier-shaped dikaryotic cells (B). The ectopic introduction of a wild-
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ACCEPTED MANUSCRIPT type allele of PEX20 into ∆pex20 restores asci and ascospore formation (C). In contrast, the introduction of a pex20C6A (D) or a pex20C6K (E) allele was unable to restore asci formation of ∆pex20 strains. Scale bar 10μm.
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Figure 8. Model for peroxisome matrix protein import in P. anserina. Peroxisome
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matrix protein import in P. anserina is mediated by the two conserved pathways, which are
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driven by the receptor PEX5, and by the receptor/co-receptor couple PEX7/PEX20, respectively (only the peroxin numbers are indicated). In addition, most P. anserina
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peroxins are required for initiation of meiotic development (red shading). The only
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exceptions are the two import receptors (PEX5 and PEX7) and the docking components PEX14 and PEX14/17 (green shading). These data suggest the existence of an alternative
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further details). Ub, ubiquitin.
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import pathway, which could depend on PEX20 as import receptor (please see the text for
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ACCEPTED MANUSCRIPT Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery
Highlights
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P. anserina peroxisomes differ in their protein composition and distribution
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The import of distinct proteins differs in its ubiquitination-complex requirements
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The peroxisome dislocation complex restrains peroxisome removal in P. anserina
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Meiotic induction in P. anserina requires PEX8 and the exportomer peroxins
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Meiotic induction requires the PEX20 cysteine that is potentially monoubiquitinated
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Graphics Abstract
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