Identification of the guanine nucleotide exchange factor for SAR1 in the filamentous fungal model Aspergillus nidulans

BBA - Molecular Cell Research 1866 (2019) 118551

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Identification of the guanine nucleotide exchange factor for SAR1 in the filamentous fungal model Aspergillus nidulans

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Ignacio Bravo-Plaza, Miguel Hernández-González1, Mario Pinar, J. Fernando Díaz, ⁎ Miguel A. Peñalva Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain

A R T I C LE I N FO

A B S T R A C T

Keywords: ER exit ERES, ER exit sites COPII Exocytosis Protein production SAR1

In spite of its basic and applied interest, the regulation of ER exit by filamentous fungi is insufficiently understood. In previous work we isolated a panel of conditional mutations in sarA encoding the master GTPase SarASAR1 in A. nidulans and demonstrated its key role in exocytosis and hyphal morphogenesis. However, the SAR1 guanine nucleotide exchange factor (GEF), Sec12, has not been characterized in any filamentous fungus, largely due to the fact that SEC12 homologues share little amino acid sequence identity beyond a GGGGxxxxGϕxN motif involved in guanine nucleotide exchange. Here we demonstrate that AN11127 encodes A. nidulans Sec12, which is an essential protein that localizes to the ER and that, when overexpressed, rescues the growth defect resulting from a hypomorphic sarA6ts mutation at 37 °C. Using purified, bacterially expressed proteins we demonstrate that the product of AN11127 accelerates nucleotide exchange on SarASAR1, but not on its closely related GTPase ArfAARF1, as expected for a bona fide GEF. The unequivocal characterization of A. nidulans Sec12 paves the way for the tailored modification of ER exit in a model organism that is closely related to industrial species of filamentous fungi.

1. Introduction Cells of ascomycetes are surrounded by an external cell wall allowing them to thrive under different osmotic conditions, making exocytosis, which is required to deliver cell-wall modifying enzymes and its substrates to the periphery of the cell, essential. The filamentous ascomycete Aspergillus nidulans is a genetic [1–4] and cell biological model [5,6] that is closely related to the industrial species A. niger and A. oryzae and to the devastating fungal pathogen A. fumigatus. A. nidulans grows by apical extension, implying that the secretory pathway has adapted to meet the demands imposed by the rapid rates of polarized growth (~1 μm/min) [7]. Studies on the late steps of the secretory pathway revealed that the process by which the trans-Golgi network (TGN) tears off into secretory vesicles occurs by maturation [8], with the RabERAB11 GTPase and its activating guanine nucleotide exchange factor (GEF) TRAPPII playing a major role [7,9]. Rapid endocytic recycling involving the TGN as intermediate station crucially contributes to maintain apical extension [10]. In contrast, the earliest steps of the secretory pathway are insufficiently understood, despite the

fact that the ER/Golgi interface plays a crucial role in apical extension and hyphal morphogenesis [11]. From an applied point of view, the ER/Golgi interface plays crucial roles in the secretion of extracellular enzymes such as those produced with biotechnological purposes in the industrial species A. niger and Trichoderma ressei. For example, highlevel enzyme production induces ER stress [12,13] and activates ERassociated degradation [14], jeopardizing productivity. Indeed ER exiting appears to be the limiting step for the secretion of an endogenous A. nidulans inulinase used as model cargo [15]. In addition, blocking traffic between the ER and the Golgi with conditional mutations in genes encoding the early Golgi regulators SedVSed5 and RabORAB1 result in tip swelling [16]. In spite of this applied and basic importance, the regulation of traffic across the ER/Golgi interface has been insufficiently studied in filamentous fungi. The key regulators of this traffic are the essential small GTPases ARF1 and SAR1, designated ArfAARF1 and SarASAR1, respectively in A. nidulans [11,17]. ArfAARF1 plays roles in the early Golgi and late Golgi (= TGN) cisternae, activated by its GEFs GeaAGea1 and HypBSec7, respectively [18]. SarASAR1 belongs to the SAR1 family of

Abbreviations: ER, endoplasmic reticulum; GEF, guanine nucleotide exchange factor; TGN, trans-Golgi network; TMD, transmembrane domain ⁎ Corresponding author. E-mail address: [email protected] (M.A. Peñalva). 1 The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. https://doi.org/10.1016/j.bbamcr.2019.118551 Received 12 June 2019; Received in revised form 2 August 2019; Accepted 11 August 2019 Available online 02 September 2019 0167-4889/ © 2019 Elsevier B.V. All rights reserved.

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Sec12-expressing gpdAmini-sec12 plasmid (p2280), the same DNA fragment was cloned into p1660 [37] with NcoI/XmaI ends.

GTPases regulating the budding of COPII vesicles from the ER [19,20]. COPII vesicles bud from ribosome-free regions of the ER denoted ER exit sites (ERES) or ‘transitional ER’ (tER) [21]. The biogenesis of COPII/ERES involves, in addition to SAR1 and the COPII coat components themselves, lysophospholipids [22], the scaffold Sec16 [20,23] and, in higher cells, regulators of COPII carrier size such as TANGO1/ cTAGE5 [24,25]. Prominent amongst ERES components is Sec12, a type II single-TMD protein first identified in S. cerevisiae [26–28] that mediates nucleotide exchange on SAR1, thereby activating this GTPase and initiating the assembly of COPII coats [20]. Despite its central role in secretion, no Sec12 homologue has been characterized in any filamentous fungus, almost certainly due to the fact that the protein shows little amino acid sequence conservation (mammalian and yeast Sec12 share 18% identity only [29]). Here we report the unambiguous identification of the A. nidulans gene encoding Sec12, an essential protein that localizes to the ER and that specifically mediates guanine nucleotide exchange on SarASAR1.

2.3. Purification of bacterially-expressed proteins To obtain the cytosolic domain of Sec12, the AN11127 coding sequence was PCR-amplified from A. nidulans cDNA and cloned into pGEM-T Easy to generate plasmid p2228. The sequence coding for the cytosolic domain of Sec12 (residues 1 through 458) was amplified from p2228 by PCR using oligonucleotides IBP119 and IBP120 and cloned into pET21 to generate plasmid p2262, which drives expression of the Sec12 cytosolic domain with a C-terminal 6-His tag. pDvi-SarA-His10 (p2157) is a plasmid expressing the complete coding region of SarA [11]. To obtain purified 20Δ-SarA lacking the 20 N-terminal residues conforming the amphipathic helix, the corresponding coding sequence was amplified by PCR from p2157 with primers IBP140/MHG_49 and cloned as an NcoI fragment into pDvi to yield plasmid p2263. The coding region of 15Δ-ArfA, also lacking the 15-residue N-terminal amphipathic helix was amplified by PCR from cDNA with primers IBP172/IBP173 and similarly cloned in pDvi. Sec12-His6, SarA-His10 and ArfA-His10 were expressed in E. coli BL21 (DE3) cells incubated for 20 h at 15 °C in the presence of 0.1 mM IPTG. Frozen bacterial pellets were resuspended in lysis buffer (25 mM HEPES, 500 mM KCl and 30 mM imidazole pH 7.5) containing 200 μg/ ml lysozyme (Sigma Aldrich), 1 μg/ml DNAse I (Roche) and EDTA-free protease inhibitor (Roche). After incubation for 1 h on ice, cells were lysed by high-pressure cell disruption with a French Press. Cleared lysates (25 ml) were rotated with 0.5 ml of Ni Sepharose™ High Performance beads (GE Healthcare) for 1 h at 4 °C, followed by three washing steps in a buffer containing 25 mM HEPES pH 7.5, 500 mM KCl and increasing imidazole concentration (40, 70 and 100 mM). For SarASAR1 and ArfAARF1purification 1 mM MgCl2 was added to all buffers. Proteins were eluted with 500 mM imidazole-containing buffer and exchanged with PD-10 Sephadex G-25 columns (GE Healthcare) to HKM buffer (50 mM HEPES pH 7.4, 120 mM KAc and 1 mM MgCl2).

2. Material and methods 2.1. Aspergillus media, strains and plasmids Standard A. nidulans media described by Cove [30] were used for strain propagation, conidiospore production and growth tests [15,31]. Genetic techniques [2] and transformation [32] have been described. Strains are listed in S1 Table. Primers are listed in S2 Table. E. coli expression plasmids and integrative A. nidulans plasmids detailed under the appropriate subheadings below are briefly summarized in S3 Table. 2.2. A. nidulans DNA constructs The sec12Δ∷pyrG deletion cassette was assembled by fusion PCR [3,33]. Genomic regions upstream and downstream sec12 ORF were amplified with primers IBP223/IBP224 and IBP225/IBP226, respectively. The A. fumigatus pyrG gene was amplified with primers IBP132/ IBP133. Endogenous tagging of Sec63 with C-terminal mRFP has been described [34]. The cassette for endogenous C-terminal tagging of sec13 (AN4317) with mCherry consisted of five PCR-generated DNA segments that were assembled by fusion PCR [35] in the following order: (i) 900 bp DNA fragment corresponding to the 3′ end of the Sec13 open reading frame lacking the stop codon (primers IBP1/IBP2); (ii) the mCherry coding sequence preceded by a (Gly-Ala)5 linker -encoding DNA fragment matching the phase of the Sec13 C-terminus (primers IBP3/IBP12; template plasmid p1827); (iii) A DNA fragment of 220 bp of the sec13 3′-UTR region (primers IBP5/IBP6); (iv) A DNA fragment encoding A. fumigatus pyrG (primers IBP7/IBP8; template plasmid p1530); and (v) a 900 bp fragment of the sec13 downstream region (primers IBP9/IBP10). The fusion PCR to join these five fragments used primers IBP1 and IBP10. The cassette for endogenous N-terminal GFP tagging of sec12 consisted of a 900 bp fragment of the upstream region of the gene (primers IBP13/IBP14) followed by the A. fumigatus pyrG gene (primers IBP15/IBP16), a 192 bp region that lies upstream AN11127 start codon that we predicted to include the promoter plus the 5′-UTR region (primers IBP17/IBP18), the DNA sequence for A. nidulans codon-optimized GFP with DNA coding for a C-terminal (GlyAla)5 linker (primers IBP19/MHG_238, template plasmid p2246) and a 900 bp sequence encoding the 300 N-terminal residues of Sec12 (primers IBP20/IBP21). Fragments were assembled by fusion PCR with primers IBP13 and IBP21. Correct integration events for the sec13∷mCherry-pyrGAf, pyrGAf-GFP∷sec12 and sec12Δ∷pyrGAf were diagnosed by PCR with external primer pairs IBP24/IBP25, IBP52/IBP53 and IBP227/IBP228, respectively. To construct the integrative plasmid gpdAmini-gfp-sec12 (p2279) driving expression of GFP-Sec12 under the control of gpdAmini promoter, sec12 was PCR-amplified with primers IBP174/IBP175 and cloned into p1881 [36] with NsiI/XmaI ends. To construct the similar untagged

2.4. Activation assays of ArfAARF1 and SarASAR1 Activation kinetics of GTPases was monitored by the increases in tryptophan fluorescence associated with the conformational changes induced by the binding of GTP to their nucleotide pockets, as described [38–40]. Fluorescence was measured at 340 nm on a Jobin Yvon-Spex FluoroMax2 fluorimeter (Horiba), using 290 nm excitation light. Reactions were carried out at 37 °C in a total volume of 1 ml of 50 mM HEPES pH 7.4, 120 mM KAc and 1 mM MgCl2 containing 0.67 μM SarASAR1 or ArfAARF1 and, when indicated, the cytosolic domain of Sec12. Reactions were initiated by addition of 50 μM GTP. Fluorescence data were fitted to mono-exponential plots that were used to deduce the kobs of the reactions in the presence of Sec12; we denoted kbasal to the rate constant of the spontaneous reactions that took place in the absence of Sec12 and kexchange = kobs-kbasal to the rate constant of the GTP-loading reaction attributable to Sec12, which increases linearly with the concentration of GEF. Thus kexchange = (Kcat/Km) [Sec12], which was used to determine the catalytic efficiency. 2.5. Microscopy Hyphae cultured in pH 6.8 ‘watch minimal medium’ (WMM) [41] at 28 °C using 8-well chambers (IBIDI GmbH, Martinriesd, Germany) were imaged by epifluorescence microscopy as detailed previously [7], using a Leica DMI6000 B inverted microscope driven by Metamorph Premier software (Molecular Dynamics) and equipped with Leica 63×/1.4 N.A. Plan Apochromatic objective and Hamamatsu ORCA-ER (CCD) or Hamamatsu Flash (CMOS) cameras. For colocalization experiments images were acquired simultaneously in the GFP and red (mRFP, mCherry) channels with a Photometrics Dual-View or a Hamamatsu Gemini beam 2

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SarASAR1 [11]. sarA6, one such thermosensitive allele, markedly restricts growth at 37 °C. sarA6 results in a single Ser186Pro substitution in the C-terminal α-helix of the GTPase fold. This amino acid change does not substantially affect SarASAR1 levels at 30 °C, but pronouncedly reduces them at 37 °C, indicating that Ser186Pro results in protein misfolding/destabilization [11]. Thus we sought to compensate the markedly impaired growth phenotype of the sarA6 mutant by moderately overexpressing Sec12 using the gpdAm promoter (gpdA ‘mini’ is a truncated version of the strong, ‘full’ gpdA promoter) [37]. To this end we used an integrative plasmid carrying a truncated pyroA gene as selective marker, which targets recombination to a mutated pyroA4 locus of the recipient strain (Fig. 2A). Single cross-over events reconstruct a functional pyroA gene and incorporate the gpdAm-driven transgene, whereas double cross-over events convert pyroA4 into a pyroA+ allele without incorporating the transgene (Fig. 2, left). We obtained two classes of transformants, one that grew as sarA6 at 37 °C and a second that showed more vigorous growth (Fig. 2, right). By DNA genotyping (S2 Fig.) these classes were shown to correspond to the double and single cross-over events, respectively. Thus these data are consistent with AN11127 positively contributing to the physiological role of sarA.

splitter. Images were deconvolved with appropriate settings using Huygens Professional 14.06 software for Windows 7. Dual-View channels were aligned with Metamorph using fluorescent TetraSpeck microspheres, (0.5 μm, Molecular Probes) as references. Once converted to 8-bit inverted greyscale of RGB, images were annotated with CorelDraw (Corel, Ottawa, Canada). 4D (x, y, z, t) image series made with maximal intensity projections of the Z-stacks were assembled with the ‘review multidimensional acquisition’ plugin of Metamorph. Time series were converted to QuickTime movies with Metamorph and compressed with ImageJ mpeg-4 for QuickTime. For Pearson's colocalization analysis, Z-stacks of images in the GFP-Sec12 and Sec63-mRFP channels acquired with a Gemini beam splitter were deconvolved with Huygens. Pearson's coefficients were determined with the Coloc 2 plugin of FIJI (ImageJ 1.52), using the corresponding maximal intensity projections and manually traced ROIs covering the apex-proximal region of the hyphae. 3. Results and discussion 3.1. Identification and genetic characterization of A. nidulans Sec12 A pioneer study predicted that the cytosolic domain of yeast Sec12p forms a seven-blade β-propeller fold [42] and uncovered as the most informative diagnostic feature of Sec12 homologues a GGGGxxxxGϕxN sequence motif (where ϕ is a hydrophobic amino acid), present in a predicted loop between the second and the third propeller blade [42]. X-ray crystallography confirmed both the overall fold of the cytosolic domain and the existence of this loop, denoted the K-loop because it is stabilized by a potassium ion [43]. The K-loop is critical for mediating nucleotide exchange on Sar1 [43] through an as yet undetermined mechanism that might be akin to that of RCC1 (also a seven-blade beta propeller containing this loop motif) acting as the GEF for the GTPase Ran that mediates nuclear import [44]. An in silico study had identified a putative A. fumigatus Sec12-encoding gene using Psi-BLAST [45]. The only A. nidulans orthologue of this gene is AN11127, which encodes a single-TMD type II transmembrane protein containing a predicted cytosolic 458-residue domain and a lumenal 186-residue domain (Fig. 1A). The cytosolic domain sequence contains a diagnostic GGGGxxxxGϕxN motif (Fig. 1A) suggesting that this gene actually encodes Sec12. Two additional pieces of in silico evidence strongly suggested that this was indeed the case. Firstly, whereas globally considered the amino acid sequences of Sec12p and the AN11127 product showed a marginal level of identity outside of the K-loop (S1 Fig.), a convincing 27% amino acid identity was found when only the 212 N-terminal amino acids were considered. Secondly, mammalian Sec12 had been initially described as a prolactin regulatory element binding protein (PREBP), the founding member of the PTHR23284 PREBP family composed of Sec12 homologues. AN11127 matches the PTHR23284 PREBP family profile. However, S1 Fig. illustrates the high degree of Sec12 amino acid sequence divergence existing between A. nidulans and related filamentous ascomycetes, including its close relative A. oryzae, showing that in silico analyses are insufficient for the unambiguous identification of the protein. Therefore we set out to demonstrate genetically, biochemically and subcellularly that AN11227 encodes A. nidulans Sec12. The Sar1- and Sec12-dependent events mediating exit from the ER are conserved across eukaryotes [46]. Because A. nidulans SarASar1 is essential, ablation of its GEF should result in lethality. Thus, as a first step to validate that AN11127 encodes Sec12 we used heterokaryon rescue to demonstrate that conidiospores lacking AN11127 are unviable (Fig. 1B), consistent with AN11127, the only A. nidulans gene distantly resembling yeast Sec12p, encoding the as yet uncharacterized SarASar1 GEF. Hereafter we will denote AN11127 as sec12. To provide functional evidence that sec12 encodes a protein positively regulating SarASar1 we used a genetic approach. We have previously characterized a panel of ts mutations in the gene encoding

3.2. Sec12 localizes to the ER Reassured by this genetic evidence we next studied the subcellular localization of Sec12 endogenously tagged with GFP in its N-terminus. The tagged allele supported growth, indicating that GFP-Sec12 retains function, but no fluorescence was detected in recombinant cells, indicating that sec12 is expressed at very low levels. Thus we expressed GFP-Sec12 under the control of moderately strong gpdAm promoter and selected transformants carrying a single-copy integration of the construct. We confirmed that GFP-Sec12 is functional after deleting the endogenous copy of sec12 in one such transformant and demonstrating that the resulting strain, in which GFP-Sec12 was the only Sec12 present, grew as the wild-type (S3 Fig.). Like in Saccharomyces cerevisiae [47,48], the A. nidulans ER comprises two different ‘domains’, the nuclear envelopes (NEs), formed by two sheets of membranes contacting only at the nuclear pores, and the so denoted peripheral ER, which includes all ER structures excepting the NEs [11,49]. The peripheral ER is a prominent network of membranes largely localized underneath the plasma membrane. This ‘cortical’ ER is connected with the NEs by tubular structures, forming a convoluted network. Neither the A. nidulans NEs [50] nor the peripheral ER [49] disassemble during mitosis. GFP-Sec12, driven by the gpdAm promoter, clearly localized to the convoluted network of structures formed the peripheral tubules and cortical strands of the A. nidulans ER and labeled strongly the NEs (Fig. 3A) (note that A. nidulans hyphal tip cells contain several evenly spaced nuclei, whose NEs are indicated by arrowheads in Fig. 3A–B). The network is best appreciated with successive optical sections of a GFP-Sec12 Z-stack that has been deconvolved to minimize blur (Fig. 3B). We used 4D (x, y, z, time) microscopy to document that the localization of Sec12 to the ER is homogeneous both over time and within a whole population of hyphae filmed growing by apical extension for 30 min (Movie 1). The behavior of Sec12-labeled ER over a 3.5 min period, imaged in 3D (x, y, time) with a time resolution of 0.5 fps, is shown on Movie 2. GFP-Sec12 images also showed that the fusion protein is not substantially polarized, contrasting with the behavior of the endogenously mRFP-tagged translocon component Sec63 (Fig. 4A) and of the endogenously mCherry-tagged COPII vesicle component Sec13 (Fig. 5A), which show polarization towards the tip when expressed at physiological levels form endogenously tagged alleles. This polarization of Sec63 (protein translocation into the ER) and Sec13 (ER exit) indicates that the secretory ER is most active in the tip-proximal region, fulfilling the needs imposed by continuous growth by apical extension [37,49]. This lack of polarization of GFP-Sec12 might result from its overexpression (see discussion below). To establish that the cortical and 3

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Fig. 1. A. nidulans sec12 is essential. (A) Scheme showing the topology of Sec12 regions relative to the ER membrane and localization of the N-terminal conserved region that includes the GGGGxxxxGϕxN K-loop motif. Numbers indicate amino acid residues. The single TMD region lies between residues 459 and 480. (B) A pyrimidine auxotroph carrying a pyrG89 mutation was transformed with a sec12Δ∷pyrGAf deletion cassette (pyrGAf is the wild-type A. fumigatus pyrG gene, which fully complements the pyrimidine auxotrophy resulting from the pyrG89 mutation). The resulting transformants were heterokaryons containing in the same cell both untransformed nuclei (pyrG− sec12+, green in the scheme) and transformed ones (sec12Δ∷pyrGAf, red), which provided functional Sec12 and PyrG products, respectively, thereby permitting growth (see scheme). These heterokaryons were allowed to produce uninucleate asexual spores, denoted conidiospores, onto which individual nuclei segregate. When these conidiospores were plated on medium supplemented with pyrimidines, auxotrophic pyrG− sec12+ ones gave rise to a confluent lawn of fungal colonies (top). However, neither these auxotrophic pyrG− sec12+ conidiospores nor prototrophic sec12Δ∷pyrGAf ones grew on pyrimidine-less medium, indicating that sec12 is essential. The bottom autoradiography shows a Southern blot analysis of primary transformants discriminating spontaneously forming diploid clones (examples are strains DP1-2 in the Southern blot) from heterokaryotic transformants (examples are strains HK1-HK4). In these HKs the sec12Δ∷pyrGAf band is substoichiometric to the wild-type sec12+ band.

Fig. 2. Rescue of sarA6 by moderate overexpression of Sec12. A plasmid carrying a gpdAm∷sec12 transgene was targeted to the pyroA locus by homologous recombination. Pyridoxine auxotrophs can only be generated by the indicated single blue crossover or by the also indicated double crossover (blue plus red) between the non-complementing alleles present in the plasmid and the genome. Double crossover corrects the mutation in the recipient chromosome without incorporating the plasmid, with the resulting transformants serving as isogenic negative controls for those strains that incorporate the transgene. The right panel shows the colony growth phenotypes of the wild type, of an untransformed strain carrying sarA6 and of sarA6 pyroA+ transformants carrying (underlined and bold) or not the gpdAm∷sec12 transgene (see also S2 Fig.).

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Fig. 3. A. nidulans Sec12 localizes to the ER. (A) Maximal intensity projection of a deconvolved Z-stack of a hypha expressing GFP-Sec12 showing a convoluted network of peripheral ER strands and the nuclear envelope ER. (B) Successive optical sections, separated by 0.5 μm, of a deconvolved Z-stack of a cell expressing GFP-Sec12. For both panels the positions of the nuclear envelopes (N.E.) are indicated with arrows.

cisternae [37,52]. Figs. 3 and 4 strongly indicate that A. nidulans Sec12 labels the ER membranes more or less uniformly. To confirm that Sec12 does not concentrate on discrete ER spots containing COPII biogenesis proteins we tagged the outer COPII coat component Sec13 endogenously with mCherry. Like the GFP-tagged inner COPII component Sec23 [37], Sec13-mCherry, expressed at physiological levels as the only source of the protein, localizes to ERES, which appear as punctate intracellular structures in fluorescence images. In addition to ERES, Sec13 labels the nuclear envelopes faintly, as reported (Sec13 is also a component of the nuclear pore complexes [53]) (Fig. 5A). In middle planes of N = 9 hyphae, 89% of 338 Sec13-labeled ERES overlapped or were closely associated with peripheral ER strands and the ER in the nuclear envelopes (Fig. 5B) (Movie 4, shows the ‘merge’ channel of the different planes of the Z-stack). However, GFP-Sec12 does not concentrate on these ERES. Nevertheless, we note that Sec12, although functionally tagged with GFP, is overexpressed, which might result in this integral membrane protein diffusing away of its physiological localization. For example, the discrete organization of P. pastoris Sec12 in punctate ER-associated structures requires the scaffold protein Sec16 [21] and Sec16 can be saturated by an excess of Sec12 [54]. Thus the homogeneous distribution of overexpressed Sec12 in A. nidulans does not support any firm conclusion as to whether in this particular regard

peripheral strands to which GFP-Sec12 localizes represent ER, we analyzed the colocalization of GFP-Sec12 and Sec63-mRFP in the 20–30 μm apicalmost regions where the two proteins are similarly abundant (Fig. 4B) (Movie 3, constructed with the sections of the Zstack). The two proteins essentially colocalized, showing a Pearson's coefficient of 0.85 ± 0.04 S. D. (N = 8 cells) (Fig. 4C). Therefore the subcellular localization of Sec12 is consistent with its being the GEF for SarASar1. 3.3. Sec12 does not concentrate on ERES The budding of COPII-coated vesicles bound for the Golgi occurs at specialized ER domains termed transitional ER or ERES (ER exit sites), which can be visualized with COPII coat components labeled with fluorescent proteins. In S. cerevisiae Sec12 distributes across the whole nuclear and peripheral ER, whereas in Pichia pastoris Sec12 concentrates in a small number of discrete ERES containing early and late components of the COPII biogenesis machinery [51]. These findings are interesting because the distinct activation of Sec12 at a few discrete spots of the ER has been suggested to underlie the fact that P. pastoris forms Golgi stacks in their vicinity, whereas S. cerevisiae and A. nidulans, which have numerous and ubiquitous ERES, have dispersed Golgi 5

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Fig. 4. A. nidulans Sec12 labels the ER uniformly. (A) Maximal intensity projections of a deconvolved Z-stack of a cell expressing Sec63-mRFP (endogenously tagged) and GFP-Sec12. The positions of the nuclear envelopes (N.E.) are indicated on the Sec12 channel. The inset is magnified 2.5 x relative to the images on the left. (B) Middle plane of a cell. The two channels were acquired simultaneously with a Gemini beam splitter. (C) Maximal intensity projections of deconvolved Z-stacks of simultaneously acquired GFP and mRFP channels were used to calculate the Pearson's coefficients in N = 8 hyphal tip cells.

A. nidulans resembles one or the other yeast.

cytosolic domain of the former is able to promote nucleotide exchange on the GTPase in vitro [43]. Unlike RAS and RAB GTPases, which are recruited to membranes by lipidated C-terminal motifs, ARF family GTPases insert an N-terminal amphipathic helix that protrudes into the target membrane upon loading with GTP [55–57], coordinating activation with membrane recruitment. However ARF/SARs lacking the N-

3.4. A. nidulans Sec12 mediates guanine nucleotide exchange on SarASAR1 To demonstrate that Sec12 specifically mediates nucleotide exchange on SarASar1 we exploited the fact that the seven-blade propeller 6

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Fig. 5. ER exit sites are associated with the GFP-Sec12 ER. (A) Simultaneous imaging in live cells of GFP-Sec12 and ERES labeled with endogenously tagged Sec13-mCherry. Images are maximal intensity projections of a deconvolved Z-stack. The inset on the right is magnified 2 x relative to the source image. (B) Deconvolved images of a middle plane of a cell expressing GFP-Sec12 and Sec13-mCherry. The two channels were acquired simultaneously with a Gemini beam splitter. The Sec12 channel shows the labeling of the nuclear ER (N.E.) and the prominent peripheral ER located between the apex and the apicalmost nucleus. Most Sec13 ERES are closely associated with both nuclear and peripheral ER, as illustrated by the quantitation on the right, derived from N = 9 cells.

RerARER1 is a COPI adaptor facilitating the retrieval to the ER of integral membrane proteins that ‘leak’ to the Golgi, including a proportion of Sec12 itself [61]. Accordingly RerARER1 forms a cytosolic haze in strains carrying early Golgi-disrupting ts mutations impairing SedVSed5 and RabORAB1 [9], which dissipate the early Golgi preventing the budding of early Golgi-derived retrograde COPI vesicles (COPI localizes to the early Golgi [62,63]). In contrast, it accumulates in the ER in SarASAR1impairing mutants preventing its COPII-dependent anterograde trafficking to the Golgi [11]. We have recently shown, by exploiting its glycosylation pattern as a diagnostic tool to track an inulinase across the exocytic pathway, that ER exit is the limiting step in the secretion of the enzyme [15]. ER exit takes place at ERES, which are membrane domains formed by a network of protein interactions at the cytosolic side of the ER that generate COPII-coated carriers [54]. Sec16 contributes crucially to the organization of ERES [21] by mediating the recruitment of Sec12 to them [64] and scaffolding the SAR1-dependent assembly of the COPII coat [65,66]. These findings suggest a strategy to improve the production of extracellular enzymes: up-regulating ERES by increasing the availability of active SAR1. Indeed a recent report showed that moderate expression of Sec16p increases the production of other hydrolases by S. cerevisiae [67]. Our identification of the SAR1 GEF Sec12 in a model filamentous ascomycete and its industrial protein-producing relatives opens the way to manipulate this protein alone or in combination with Sec16 to pursue this strategy further. We note that we have demonstrated that increased levels of Sec12 augment the levels of active SarASAR1 by partially rescuing the growth defect of a hypomorphic sarA6 allele with Sec12 overexpression. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbamcr.2019.118551.

terminal helix can be activated by their GEFs in the absence of biological membranes [58]. Thus, we purified bacterially-expressed Histagged constructs 15Δ-ArfAARF1 and 20Δ-SarASAR1 lacking the 15 and 20 N-terminal residues, respectively, that formed the N-terminal amphipathic helices (Fig. 6A, left). In parallel, we purified to homogeneity a recombinant protein containing residues 1–458 (i.e.: the complete cytosolic domain) of Sec12 fused to a C-terminal His tag (Fig. 6A, right). These proteins were used in nucleotide exchange assays in which the conformational switch that takes place in the presence of excess GTP results in an increase in tryptophan fluorescence [40]. Fig. 6B shows that although both ArfAARF1 and SarASAR1 constructs displayed spontaneous activation by GTP, the addition of Sec12 strongly accelerated this reaction in the case of SarASAR1 but not of ArfAARF1. Increases in tryptophan fluorescence were fitted to mono-exponential curves that were used to derive kobs rate constants at different concentration of the GEFs and in the absence of GEF (kbasal). A plot of (kobs-kbasal) values vs. GEF concentration (Fig. 6C) was used to estimate that the catalytic efficiency (Kcat/Km) of Sec12 on 20Δ-SarASAR1 is 2.1 × 104 M−1 s−1. Taken together these data biochemically establish that AN11127/Sec12 acts as GEF for SarASAR1.

4. Conclusions This study expands our studies on SarASAR1, the key regulator controlling the biogenesis of COPII vesicles [11], with the characterization of its cognate GEF. In spite of its basic and applied relevance, very few studies have addressed the regulation of ER exit in filamentous ascomycetes. In a pioneering study, the genes encoding SAR1 homologues in Trichoderma reesei and A. niger were isolated and shown to complement yeast sar1 mutants [59]. In N. crassa, ER adaptors incorporating CBH-1 and CBH-2 cellulases into COPII vesicles have been identified [60]. We have also made extensive use of RerARer1 as a subcellular marker localizing to the early Golgi in the steady state [16]. 7

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Fig. 6. Sec12 promotes GDP exchange on SarASAR1. (A) Coomassie-stained SDS-PAGE gels of purified proteins used in GDP exchange assays. Gels were loaded with 5 μg of 20Δ-SarA and 15Δ-ArfA, and with 10 μg of the Sec12 cytosolic domain. (B) Activation of SarASAR1 and ArfAARF1 lacking the Nterminal amphipathic helixes by the cytosolic domain of Sec12 was measured by tryptophan fluorescence kinetics in the presence of 50 μM GTP or, as controls, GDP. These plots were used to determine the kobs values for the activation reactions from mono-exponential curves. The values for spontaneous GTP loading obtained in the absence of Sec12 were denoted kbasal. (C) kobs-kbasal values obtained, in triplicate, using the indicated concentrations of Sec12 cytosolic domain were used to deduce Kcat/Km from least square fittings of the datasets. Error bars indicate S.D.

Transparency document

65090R and RTI2018-093344-B-I00 to M.A.P. and BFU2016-75319-R to J.F.D.; and by grant S2017/BMD-3691 InGEMICS-CM funded by the Comunidad de Madrid (Spain) and European Structural and Investment Funds. We thank Elena Reoyo and Ana Alonso for Technical assistance. I. B.-P. and M. H.-G. were holders of ‘Formación de Personal Investigador’ pre-doctoral contracts (Ministerio de Ciencia, Innovación y Universidades). M.A.P. and J.F.D. are members of the WhiteBiotech (industrial biotechnology) interdepartmental unit of the Centro de Investigaciones Biológicas.

The Transparency document associated with this article can be found, in online version. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authors' contributions Acknowledgments Conception and design of the study, M.P.S., M.H.G. J.F.D. and M.A.P.; Data acquisition, I.B.P., M.H.G., M.P.S. and M.A.P.; Data analysis and interpretation, M.P.S., J.F.D. and M.A.P.; Writing the draft

This work was supported by Spain's ‘Ministerio de Ciencia, Innovación y Universidades’ (MCIU/AEI/FEDER, UE) grants BIO20158

BBA - Molecular Cell Research 1866 (2019) 118551

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manuscript, M.A.P.; Review & editing of the draft manuscript, all five authors.

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