Role of AAA+-proteins in peroxisome biogenesis and function

    Role of AAA+ -Proteins in Peroxisome Biogenesis and Function Immanuel Grimm, Ralf Erdmann, Wolfgang Girzalsky PII: DOI: Reference:

S0167-4889(15)00347-X doi: 10.1016/j.bbamcr.2015.10.001 BBAMCR 17688

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

18 August 2015 30 September 2015 3 October 2015

Please cite this article as: Immanuel Grimm, Ralf Erdmann, Wolfgang Girzalsky, Role of AAA+ -Proteins in Peroxisome Biogenesis and Function, BBA - Molecular Cell Research (2015), doi: 10.1016/j.bbamcr.2015.10.001

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ACCEPTED MANUSCRIPT Role of AAA+-Proteins in Peroxisome Biogenesis and Function

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Immanuel Grimm, Ralf Erdmann*, Wolfgang Girzalsky*

D-44780 Bochum, Germany

*

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Correspondence to: Dr. Wolfgang Girzalsky

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Abteilung für Systembiochemie, Medizinische Fakultät der Ruhr-Universität Bochum,

Institut für Biochemie und Pathobiochemie

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Ruhr-Universität Bochum

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Universitätsstr. 150

Tel.

49-234-322-7979 49-234-321-4266

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Fax.

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D-44780 Bochum

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email. [email protected]

Prof. Dr. Ralf Erdmann Institut für Biochemie und Pathobiochemie Ruhr-Universität Bochum Universitätsstr. 150 D-44780 Bochum Tel.

49-234-322-7979

Fax.

49-234-321-4266

email. [email protected]

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Abstract Mutations in the PEX1 gene, which encodes a protein required for peroxisome biogenesis, are

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the most common cause of the Zellweger spectrum diseases. The recognition that Pex1p

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shares a conserved ATP-binding domain with p97 and NSF led to the discovery of the

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extended family of AAA+-type ATPases. So far, four AAA+-type ATPases are related to peroxisome function. Pex6p functions together with Pex1p in peroxisome biogenesis, ATAD1/Msp1p plays a role in membrane protein targeting and a member of the LON-family

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of proteases is associated with peroxisomal quality control. This review summarizes the

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current knowledge on the AAA+-proteins involved in peroxisome biogenesis and function.

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Keywords: AAA+-ATPase, peroxisome, Pex1p, Pex6p, ATAD1/MSP1, Lon

Abbreviations: AAA+, extended ATPases associated with various cellular activities; ATAD1,

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ATPase family AAA domain-containing protein 1; ERAD, endoplasmic reticulum associated degradation; Msp1p, mitochondrial sorting of proteins 1; NSF, N-ethylmaleimide-sensitive

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factor; NTD, N-terminal domain; Pex, peroxin; PTS, peroxisomal targeting signal; RING, really interesting new genes; Ubc, ubiquitin conjugating enzyme

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1. Introduction Peroxisomes are organelles that can be found in all nucleated cells. The number and

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morphology of these organelles varies significantly among different cell types, tissues or

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species. The enzyme equipment of the peroxisomal matrix is adapted to the cellular demands

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and metabolic requirements of the cell. Accordingly, peroxisomes are considered to be multipurpose organelles that contribute to the adaptation of cells to different environmental conditions [1]. A general function of peroxisomes is the -oxidation of fatty acids, initiated by

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specialized oxidases and accompanied by the detoxification of produced hydrogen-peroxide

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by catalases, which was eponymous for peroxisomes [2]. While in yeast and most other organisms fatty acids are exclusively metabolized in peroxisomes, the peroxisomal -

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oxidation is specialized on branched and very long chain fatty acids (VLCFA) in human cells,

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complementing the mitochondrial fatty acid degradation [3-6]. Biosynthesis of bile acids [7] and ether-lipids [8] are only two examples for metabolic functions of peroxisomes in humans.

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A more detailed view on peroxisomal functions in plants, fungi and mammals can be found elsewhere (reference to appropriate reviews by this special issue).

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The importance of functional peroxisomes for the human organism is highlighted by severe diseases, resumed as peroxisomal biogenesis disorders (PBDs). Mutations in genes coding for proteins required for peroxisomal biogenesis, so-called peroxins, manifest in mild to severe symptoms summarized as Zellweger syndrome spectrum (ZSS) or Zellweger spectrum disorders (ZSD) [9]. In accordance to the severity of symptoms, the ZSS is categorized in three

disease

forms:

The

infantile

Refsum

disease

(IRD)[10],

the

neonatale

adrenoleukodystrophy (NALD)[11] and the Zellweger syndrome (ZS)[12, 13], with latter representing the severest form of PBDs. Affected patients suffer on heterogenic symptoms with neurologic, hepatic and renal dysfunctions, hypertonia and characteristic facial abnormalities. These patients hardly survive their first year of life. Relevant for diagnostic purpose, patients exhibit increased levels of VLCFA, phytanic and pristanic acids

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accompanied with reduced levels of plasmalogens [14]. In corresponding cell lines, peroxisomal matrix proteins are mislocalized to the cytosol and peroxisomes either appear as

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empty membrane structures (“ghosts”) or are fully absent [15]. The underlying genetic defect

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lies in mutation of PEX genes coding for proteins, required for peroxisome biogenesis [16].

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Although mutations in different PEX genes were identified to cause PBDs, around 60 % of Zellweger patients exhibit mutations in PEX1 and further 16 % in PEX6 [17]. In particular, the by far most abundant Pex1pG843D variation impairs the binding between Pex1p and

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Pex6p [18-20]. Interestingly, Pex1p was the first peroxin identified in the early 90s [21] and

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shares a highly conserved ATP-binding domain with NSF (N-ethylmaleimide-sensitive factor) [22] and VCP (valosin-containing protein) [23]. This led to a classification of a new family of ATPases; the AAA-type ATPases [21, 24, 25], which also included the second peroxisomal

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AAA-peroxin Pex6p [26].

In this review, we first provide an overview on common features of the extended family of

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AAA+-ATPases and then describe the function of members of this family in peroxisome biogenesis and function. These include Msp1p/ATAD1 and Lon-proteases, and emphasis will

Pex6p.

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be on the function and molecular architecture of the peroxisomal AAA+-proteins Pex1p and

2. Classification of AAA+-ATPases and their general principle of function AAA+-ATPases (extended ATPases associated with various cellular activities) represent a heterogeneous family of molecular motors involved in a wide range of cellular processes. These proteins in general can be located in the cytosol or organellar matrices, or they are anchored at distinct membrane sides by transmembrane segments. AAA+-ATPases may contact a variety of adaptor proteins, ubiquitin markers or degradation signal sequences (degrons), which guide the motor proteins to their defined places of activity (reviewed in [27,

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28]. In line with this remarkable variability, AAA+-proteins function in protease associated quality control, membrane fusion- and protein translocation but also fulfill further specialized

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functions like the transport of targets on microtubule cables or the initiation of DNA

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replication, recombination and transcription events (reviewed in [29]). A common

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mechanistic feature of AAA+-ATPases is their ability to transform the energy of ATP hydrolysis on specialized segments, domains and/or associated adaptor proteins to further transduce wide conformational changes within their different target structures (reviewed in

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[30]).

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Initially, sequence alignments of Pex1p, NSF and p97 revealed a ~230 amino acid comprising core domain to represent a new class of ATPases, named AAA-ATPases [21, 25, 31]. Based on further sequence and structural comparisons, this group was widely extended [32] and to

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date comprises more than 30.000 AAA+-type proteins throughout all kingdoms of life [33]. The basic defining structural feature of AAA+-ATPases is a distinct -helical extension (C-

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terminal domain or -domain), which is C-terminally linked to an ASCE (additional strand conserved E) ATP binding domain (or /-domain)(Fig. 1A). The broad range of AAA+-

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ATPases is further classified in diverse clades, based on presence or absence of small extensions, inclusions or special homologous segments as the SRH (second region of homology) [34-38] within the ASCE-domain [39-41]. The C-terminal domain is frequently involved in intra-molecule contacts (by its sensor 2 arginine) (reviewed in [33]) and intermolecule contacts of oligomerized AAA+-complexes and participates in concerted movements during the ATP-cycle (reviewed in [42]). The ASCE domain on the other hand coordinates the - and -phosphates of ATP together with an Mg2+ ion by a hydrogen bond network. This network includes charged residues of the p-loop strand (called Walker A motif) and the Walker B motif [43] as well as a polar residue at the end of an additional -strand (Sensor 1) positioned in between the two Walker motifs (reviewed in [33]). Typically, AAA+ proteins act as oligomers and most of them are organized in hexameric ring shaped complexes,

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surrounding a central pore (Fig. 1A). Within this AAA+-ring, the ATP binding sites are located at the interfaces of the protomers (reviewed in [30, 33]), allowing inter-molecule

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communication, which is discussed to enable coordinated ATP binding and hydrolysis events

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(reviewed in [42]). Regarding to this communication, arginine residues of the C-terminal

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domain (Sensor 2) and/or arginine fingers at the ASCE-domain exterior can additionally participate in the hydrogen-bond network and sense the nucleotide states of the protomers (reviewed in [44], [45]) (Fig. 1A). While the sensor 2 arginine makes intra-molecule contacts

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between the C-terminal domain and the ASCE domain, latter arginine fingers reach the nucleotide binding side of the neighboring protomer to stabilize complex formation and

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promote ATP hydrolysis events. Most ring shaped AAA+-complexes, especially protease associated AAA+-proteins, are believed to thread substrates through their central pore.

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Therefore, the energy of ATP hydrolysis is converted into movements of substrate interacting loops positioned at the inner surface of their rings [46-49]. Other AAA+-ATPases like NSF

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and p97 are believed to transfer hydrolysis energy by long ranging movements to its adjacent n-terminal domains aside of the rings, which contact the substrates [50, 51].

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AAA+-ATPases are organized in a modular manner and some combine many different AAA+modules within one polypeptide chain. For example, dynein combines six AAA+-segments on a single polypeptide chain forming a ring-shaped motor, together with domains for adaptor and cargo binding and a specialized stalk segment that facilitates the protein to walk along microtubule chains [52]. Beside this special example, there is a group of so called type II AAA+-ATPases that combine two consecutively arranged AAA+-domains (called D1 and D2) on one polypeptide chain (Clp/Hsp100, NSF, p97, Pex1p and Pex6p). In contrast to type I AAA+-proteins, which form one AAA+-ring upon oligomerisation, type II AAA+-ATPases arrange their AAA+-domains to a “double donut” structure. Here the D1 and D2 domains form two rings placed on top of each other, bringing together twelve AAA+-domains within one protein complex. However, usually only one of these type II AAA+-rings seems to

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constantly hydrolyze ATP, while the other one is reported to be involved in complex formation and to accomplish regulatory tasks (reviewed in [33]).

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To fulfill various cellular functions, the AAA+-modules are associated with different binding-,

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transmembrane- and/or catalytic- domains. For instance, mysterin was recently identified to

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be an ATPase, which combines its tandem AAA+-modules (type II) with an ubiquitin ligase segment [53]. Further examples are AAA+-proteases (FtsH, Lon, m-AAA+ and i-AAA+), which possess a protease-segment, covalently connected to their single AAA+-domain (type

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I). Upon oligomerisation, bi-functional proteases are formed, which recognize target proteins to thread them through the AAA+-ring and deliver them into the adjacent protease chamber,

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where they get degraded. Other AAA+-ATPases involved in quality control systems do not possess protease activity on their own (ClpX, ClpA, HslU, 19S proteasome regulatory

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particles) but can deliver unfolded substrates to separated proteases (ClpP, HslV, 20S proteasome core) (reviewed in [54]).

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All in all, it appears that during evolution different protein types evolved, which use the ringshaped organization of AAA+-modules with its intrinsic facility of concerted ATPase cycles

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to process different targets at various cellular loci. Associated to peroxisomes, so far four AAA+-proteins are identified, acting at the peroxisomal membrane (Msp1p/ATAD1), in the peroxisomal lumen (peroxisomal Lon) or participate in the import process of proteins into the peroxisomal matrix (Pex1p and Pex6p).

3. Msp1p/ATAD1 In a screen to identify yeast proteins involved in mitochondrial protein sorting, a protein was identified, which was termed along with its hypothetic function Msp1p [55]. The protein was classified as a type I AAA+-ATPase (Fig. 1B) exclusively located as an integral membrane protein in the outer mitochondrial membrane [55].

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The human protein ATAD1 (ATPase family AAA domain-containing protein 1) displays 50% identity and 70% similarity to Msp1p and thus very likely reflects the human counterpart of

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the yeast protein. Originally, ATAD1 was described as a neuroprotective gene (NPG6)

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involved in self-protection of the central nervous system from subsequent injuries in response

dissociate

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to sub-lethal insults [56]. Moreover, ATAD1 (also annotated as Thorase) was demonstrated to -amino-3-hydroxy-5-methylisoxazole-4-proprionate

(AMPA)

receptors

(AMPAR)-complexes. This process appears to play a crucial role in limiting endocytosis of

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AMPAR to adjust the level of AMPAR at the synaptic membrane. Thus, ATAD1 controls the

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endocytosis and removal of AMPAR from the postsynaptic membrane and thereby regulates synaptic plasticity and learning and memory [57].

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First hints that Msp1p/ATAD1 may also play a role for peroxisomes came from proteomic

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studies on mouse kidney peroxisomes. In these studies, ATAD1 (ATPase family AAA domain-containing protein 1) was identified as a peroxisome-associated component [58]. A

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more detailed analysis of the intracellular localization of Msp1p/ATAD1 revealed that the human as well as the yeast proteins display a dual localization to mitochondria and to

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peroxisomes [59, 60]. The affiliation of Msp1p/ATAD1 to the AAA+-superfamily of ATPases, whose members are involved in the unfolding of proteins or disassembly of protein complexes and aggregates, led to the assumption that both proteins are involved in some of these aspects in peroxisomal and mitochondria homeostasis [58]. In fact, it was demonstrated recently that cells lacking Msp1p display a dramatically altered localization of the peroxisomal tail-anchored protein Pex15p to the outer mitochondrial membrane [59, 60]. By the fact that Msp1p interacts with Pex15p and accelerates its turnover when inappropriately targeted to mitochondria [60], it was suggested that the Msp1p/ATAD1 protein family performs an evolutionarily conserved role in quality control of the mitochondrial outer membrane. Moreover, the dual localization of Msp1p/ATAD1 to both mitochondria and peroxisomes suggests a similar role in both organelles (Fig. 1C).

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4. LON-proteases

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The Lon protease was first discovered in Escherichia coli and functions in the degradation of mutated or abnormal proteins as well as short-lived regulatory proteins, in particular those produced under stress conditions [61]. Since its discovery, the protein was identified in many

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different organisms and subcellular locations. The Lon-protease is a highly conserved type I

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AAA+-ATPase that functions in the cytosol of prokaryotes, as well as eukaryotic organelles like mitochondria and peroxisomes [62, 63]. Lon-protease shares structural and functional features with other AAA+-proteases, like the bacterial FtsH [64], the mitochondrial i- and m-

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AAA proteases [65], which possess a single AAA+-domain and the protease domain on the same polypeptide chain (Fig. 1B). These proteases recognize their target proteins, which then

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are unfolded by their AAA+-domain and then degraded by the adjacent protease domain. Accordingly, Lon-proteases display a modular architecture of three distinct domains. Based

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on the domain composition and sequence characteristic of the domains, the Lon-proteases are subdivided into two classes, LonA and LonB [66]. The LonA subfamily are soluble enzymes, which function in the bacterial cytosol and the mitochondrial matrix, whereas LonB predominates in Archaea. However, in both classes, the Lon holoenzyme forms a homooligomer, in which each subunit carries a central domain for ATPase-activity and a Cterminal domain harboring the proteolytic activity. While LonA is defined by the presence of a large N-terminal domain, which facilitates substrate recognition and binding, LonB lacks this domain but possesses instead a membrane-spanning domain that anchors the protein to the cytoplasmic side of the membrane [67]. With the combination of ATPase and protease function within one polypeptide, Lon clearly differs from other ATP-dependent proteases that have separated ATPase and protease function in two different subunits [68].

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A peroxisome-specific Lon-isoform was first identified by proteomic study of rat liver peroxisomes [63, 69]. The peroxisomal isozyme exhibits a C-terminal Ser-Lys-Leu-motif,

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which represents a typical peroxisomal targeting signal type 1 (PTS1). In silico approaches on

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genome databases from Arabidopsis thaliana, Penicillium chrysogenum and Hansenula

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polymorpha also indicated the presence of peroxisomal Lon proteases in these organisms. In A. thaliana four Lon isoforms have been identified, which localize to mitochondria, plastids and peroxisomes. The peroxisome specific enzyme is Lon2, which carries a PTS1-signal and

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has also been identified in mammals [70, 71]. The peroxisomal P. chrysogenum Lon

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(designated Pln) was identified by the presence of a PTS1 and its peroxisomal localization was confirmed [72]. The genome of H. polymorpha indicates the presence of two Lon isoforms with one of them located in the peroxisomal matrix [73].

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Despite the increasing knowledge on Lon-proteases, the functional role of peroxisomal Lon is still not well defined. In P. chrysogenum, a deletion of peroxisomal Lon results in a specific

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growth defect on media containing oleic acid as a sole carbon source [72], conditions which require peroxisomal enzymes of the -oxidation pathway. Interestingly, the growth defect is

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accompanied by the formation of protein aggregates in the peroxisomal matrix. The authors demonstrate that the peroxisomal Lon-protease has chaperon-activity with damaged catalaseperoxidase as substrate [72]. The corresponding deletion strain of H. polymorpha did not display such a growth defect but a decreased viability of the cells. It is suggest that in this organism peroxisomal Lon and autophagy function together in peroxisomal quality control [73]. A similar function is discussed for Lon2 (APEM10) of plants where its absence leads to accumulation of enzymes in peroxisomes and results in an accelerated peroxisome degradation by pexophagy [74]. In plants, Lon2 is required for the elimination of unnecessary proteins during the functional transition of glyoxysomes to peroxisomes [74]. Altogether, the data indicate that Lon2 is part of a peroxisomal quality control system for the removal of unwanted and aggregated proteins (Fig. 1C).

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5. Molecular organization of the Pex1p-Pex6p complex

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Pex1p and Pex6p are categorized as classic type II AAA+-ATPases and both display a similar modular organization. They comprise two in tandem arranged AAA+-domains (D1 and D2) flanked by expanded individual N-terminal segments and short C-terminal regions [75]

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(Fig. 1B). Similar to other type II AAA+-ATPases, the tandem arranged AAA+-domains of Pex1p and Pex6p display different degrees of conservation. The second AAA+-domains (D2)

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of both AAA+-peroxins are highly conserved, including Walker A and Walker B motifs essential for ATP binding and hydrolysis, conserved aromatic amino acid residues positioned

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in the putative substrate binding loop region and two spaced out arginine fingers in the second region of homology [76]. Typical for classic AAA+-domains, the sensor 2 arginine is not

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present [44]. In general, mutations of either of these conserved D2 domain elements result in a loss of function of the AAA+ peroxins accompanied by peroxisomal matrix protein import

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defects [76-79]. However, an exception of this rule are particular Walker B motif point mutations in Pex1p D2. Corresponding yeast cells reveal no defects of peroxisomal functionality as judged by oleate utilization assays [76] and in human fibroblasts only a partially affected import of peroxisomal matrix proteins was detected due to Pex1p D2 Walker B mutations [79]. In contrast to the D2 domains, the D1 domains of both proteins are poorly conserved and harbor variations of the Walker consensus sequences. Generally for most species, nucleotide binding in Pex1p and Pex6p D1 seems to be possible, as these domains exhibit the GXXXXGK[T/S] Walker A consensus sequence [30, 43], with the exception of yeast Pex6p D1 featuring some amino acid substitutions. However, ATP hydrolysis in the D1 domains seems to play no or only a minor role. Particularly, the critical glutamate of the Walker B

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sequence hhhhDE (h, hydrophobic amino acid)[30, 43] of Pex1p in some species is functionally substituted to an aspartate [76], which might only keep some residual activity

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[79]. In other species, as in yeast, the glutamate is exchanged to an asparagine, which

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typically blocks ATP hydrolysis [33] and likewise, Pex6p D1 in all species possesses only

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non-functional Walker B motif remnants [76, 77, 79]. Without ATPase activity, the tasks of the D1 domains remain vague. At least for the human proteins, it was reported that functional D1 Walker A motifs are important for Pex1p-Pex6p interaction, indicating that nucleotide

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binding in the D1 domains is required for proper complex formation [79, 80].

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Pex1p and Pex6p are known to interact with each other in the presence of ATP [81-83] and in dependency of their nucleotide binding capability [78, 79, 84]. Early perceptions assumed that Pex1p and Pex6p form a dodecameric heteromer of two homo-hexameric complexes.

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Characterization of recombinant proteins revealed that yeast Pex1p and Pex6p associate to form a hetero-hexameric complex with 1:1 stoichometry [85]. Therefore, besides the six

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proteasomal Rpt proteins and the mitochondrial Yta10p/Yta12p (AFG3L2/paraplegin in human) m-AAA+-proteases, the Pex1p/Pex6p-complex represents one of the few examples of

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a hetero-hexameric AAA+-complex and is the only type II heteromeric AAA+-ATPase. Very recently, three groups independently analyzed the structure of the hexameric Pex1p-Pex6pcomplex from S. cerevisiae by negative stain and cyro electron microscopy. The studies showed that the complex consists of a trimer of Pex1p/Pex6p dimers, leading to an alternating organization of three Pex1p and three Pex6p molecules within the hexameric AAA+-complex [76, 86, 87]. Similar to p97 or NSF, the D1 and D2 AAA+-domains each form a ring positioned on top of the other to arrange in a double-tiered structure, whereby the conserved D2 ring harbors the main ATPase activity of the complex (Fig. 1B).

6. Dynamic Pex1p-Pex6p complex assembly at the peroxisomal membrane

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The Pex1p-Pex6p complex exhibits a dual localization, in the cytosol as well as attached to peroxisomal membranes [81, 88]. In contrast to these findings in yeast, where both proteins

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are more or less equally distributed between the cytosol and the membrane fraction,

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mammalian Pex1p was described as a predominantly cytosolic protein [82], which interact

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with predominantly peroxisome associated Pex6p [80, 89]. Since interaction of Pex1p and Pex6p strongly depends on accurate nucleotide binding [78-80, 85], hetero-hexameric complex formation might be a reversible process, auto-regulated by the ATPase cycle of the

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AAA+-peroxins. In this case, the human Pex1p-Pex6p complex might behave slightly

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different compared to the yeast proteins, leaving Pex6p at the peroxisomal membrane, whereas Pex1p is released to the cytosol, probably as homo-trimeric version [79, 90]. Interestingly, when the yeast Pex1p-Pex6p complex disassembled under ATP depleting

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conditions, Pex1p adopted a homo-trimeric conformation, while Pex6p was monomeric [85]. The tail-anchored protein Pex15p in yeast [91], its orthologue Pex26p in humans [92] and

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APM9 in plants [93] turned out to function as membrane anchors responsible for the recruitment of the Pex1p-Pex6p complex to the peroxisomal membrane [77, 88, 93, 94]. In

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particular, the N-terminal domain of Pex6p interacts with the cytosolic part of Pex15p/Pex26p and mediates the attachment of the Pex1p-Pex6p complex to the membrane [77, 80, 94]. Like the assembly of the hexameric Pex1p-Pex6p complex depends on nucleotides, also the interaction of Pex6p to Pex15p seems to be regulated by the nucleotide status of the AAA+ATPases. Particularly, inhibition of ATP hydrolysis by a Walker B mutation in the second AAA+-cassette (D2) of Pex6p stabilizes its interaction to Pex15p/Pex26p [77, 79, 80]. Binding of Pex15p to the Pex1p-Pex6p complex was shown to down regulate the ATPase activity of the AAA+-complex, possibly by influencing the D2 AAA+-domain of Pex1p [87]. These observations indicate that the interaction of all three proteins is correlated and regulated by the ATPase cycle. Accordingly, the ATP hydrolysis cycle of the AAA+-ATPases is supposed to regulate the assembly and disassembly of the Pex1p-Pex6p complex and its

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membrane association and release. It is tempting to speculate that also substrate binding might intervene with the ATPase cycle of Pex1p-Pex6p as proposed by the “Glutamate Switch”,

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which is supposed to regulate ATPase activity of AAA+-ATPases directly in response to

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handover of substrate between adjacent subunits [95, 96].

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substrate binding, or as observed for m-AAA proteases, which couple ATP hydrolysis and

7. Pex1p and Pex6p are crucial for peroxisome biogenesis

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The Pex1p-Pex6p complex is part of the peroxisomal matrix protein import machinery [88].

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This machinery can be divided into three physically and functionally associated subcomplexes, which fulfill concerted tasks in the translocation of proteins across the peroxisomal membrane into the matrix of the organelle: i) The docking complex consisting of

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the membrane proteins Pex13p and Pex14p (and Pex17p in yeast or Pex33p in filamentous fungi), ii) the RING-finger complex (really interesting new gene) comprising the E3-ligases

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Pex2p, Pex10p and Pex12p, and iii) the exportomer including Pex15p/Pex26p and the AAA+ATPases Pex1p and Pex6p (Fig. 2). While the docking complex and the RING-complex are

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supposed to be bridged by the intra-peroxisomally situated Pex8p to form the so called importomer [97], it is rather unclear how importomer and exportomer are interconnected, although they can be isolated together [88]. It appears that peroxisomal targeting of the AAA+-peroxins to Pex15p/Pex26p and the interaction of the membrane-bound complex to the importomer are dynamic processes, possibly regulated by the ATPase cycle of Pex1p-Pex6p [98]. Along this line, it was shown recently that human Pex26p directly interacts with the docking complex member Pex14p, whereby this binding was weakened in presence of the AAA+-ATPases Pex1p and Pex6p [99]. Other results implicate, that Pex26p might serve as a platform to arrange an active Pex1p-Pex6p conformation, but not necessarily needs to recruit the AAA+-peroxins to the import machinery at the peroxisomal membrane. In cells expressing a splicing variant of Pex26p without its transmembrane span, Pex1p-Pex6p function was not

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impaired, as judged by a functional import of matrix-proteins into peroxisomes. Same was true when Pex26p was mislocalized to the mitochondrial outer membrane. However, cells

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lacking Pex26p reveal a clear import defect [100].

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Although probably most peroxins involved in the import of matrix proteins are identified and many of their interconnections and functional tasks are known, the question of how protein transport across the membrane is mechanistically accomplished is only partially understood.

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One reason for this might be that peroxisomes import folded and even oligomerized proteins,

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a feature, which distinguishes this process from common import of proteins into mitochondria and chloroplasts. Folded proteins are also imported by the Twin-arginine-translocation (Tat) pathway of thylakoid, bacterial and some mitochondrial membranes (reviewed by [101, 102]).

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However, the components of the peroxisomal protein import machinery and the Tat-pathway share no sequence similarity and different to the Tat-pathway, peroxisomal import receptors

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switch between a soluble and a membrane bound state and shuttle between the cytosol and the peroxisomal import machinery [103, 104]. Comparable to Tat [105], binding of the receptors

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to the membrane and the formation of the import pore seems to be nucleotide independent [106-110]. However, the Tat-pathway is energetically driven by the transmembrane proton motive force, while the driving force for peroxisomal protein import seems to come from the hydrolysis of ATP, which is required to disassemble peroxisomal pore constituents and to extract the receptors for their release back into the cytosol. This receptor extraction step is performed by the exportomer, in particular by the AAA+-motor proteins Pex1p and Pex6p [84, 106].

8. Role of Pex1p/Pex6p in peroxisomal matrix protein import As other reviews in this compendium will focus on earlier steps in peroxisomal protein import, which do not require the AAA+-peroxins, they will only briefly described here.

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Peroxisomal proteins are synthesized on free ribosomes in the cytosol and subsequently transported into the organellar matrix in a post-translationally manner [111] (Fig. 2). In the

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cytosol, proteins destined for the peroxisomal lumen are recognized by soluble receptors via

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their peroxisomal targeting signals (PTS). Most matrix-proteins exhibit a C-terminal

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peroxisomal targeting signal 1 (PTS1) or an N-terminal PTS2, which are recognized and directed to the peroxisomal membrane by soluble import receptors Pex5p and Pex7p, respectively [112-117]. At the membrane, the receptor-cargo complex efficiently binds to the

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docking complex [118, 119] via interactions of the receptors to the membrane proteins

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Pex13p [120-122] and Pex14p [123, 124]. A characteristic feature of peroxisomes is the fact that they can import folded and even oligomeric protein complexes via a highly dynamic and transient pore, which assembles at the peroxisomal membrane and allows translocation of

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cargo in so far unknown manner (reference to appropriate reviews by this special issue). After cargo translocation across the peroxisomal membrane, the receptors shuttle back to the

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cytosol in an ATP dependent manner. Studies on yeast and human fibroblasts illustrated that the AAA+-ATPases Pex1p and Pex6p extract Pex5p from the membrane [84, 106].

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Accordingly, yeast cells lacking the AAA+-ATPases or its anchor Pex15p exhibit an accumulation of Pex5p at the membrane [125-127] and reveal an enhanced degradation of peroxisomes via pexophagy [128] (reference to appropriate reviews by this special issue). Interestingly, the accumulated Pex5p displays a poly-ubiquitin modification [125-127], which typically marks proteins for their proteasomal degradation. Ubiquitin is attached to Pex5p by the E3-ligases Pex2p, Pex10p and Pex12p of the RING-complex [129, 130] which function in concert with the ubiquitin-conjugating enzymes Pex4p and Ubc1/4/5p in yeast [125-127, 131, 132] or UbcH5a/b/c in human [133] (reference to appropriate reviews by this special issue). Hence, the extraction of ubiquitinated Pex5p from the peroxisomal membrane exhibit similarities to the endoplasmatic reticulum associated degradation (ERAD) pathway [134], which recognizes inaccurately folded proteins, expose them at the cytosolic side of the ER

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membrane where they are marked with ubiquitin chains and extracted by the AAA+-ATPase Cdc48p/p97 for final degradation in the proteasome (reviewed in [135, 136]. Interestingly,

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sequence analysis reveals Pex1p and Pex6p to be the closest relatives to Cdc48p/p97 and

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crystal structures of the extreme N-terminal part of murine Pex1p exposed a double-psi--

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barrel fold with similarities to adaptor binding domains of p97 and NSF [137]. NSF uses this domain to interact with SNAP adaptors [138, 139], while p97 is known to bind various adaptors comprising ubiquitin-like domains (reviewed in [140]). However, the murine Pex1p

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N-terminal domain lacks hydrophobic amino acids, which in case of p97 are crucially

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involved in binding of the Ubx adaptor p47 [141]. Very recent structural analysis of yeast Pex1p-Pex6p by cryo and negative stain electron microscopy revealed that the N-terminal part

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of both peroxins in fact possess two tandemly arranged domains (N1, N2), each forming a

highly flexible [86].

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double-psi--barrel fold, whereby especially the first N-terminal domain of Pex1p seems to be

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Although the exact mechanism of how the AAA+-peroxins interact with the ubiquitinated Pex5p is not known, ubiquitination of S. cerevisiae Pex5p was shown to be crucial for its

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Pex1p-Pex6p dependent extraction [131]. The human ubiquitin binding NF-B modulator AWP1 was identified to interact with Pex6p and to stimulate the export process of Pex5p, indicating an adaptor attended binding mechanism of the Pex1p-Pex6p AAA+-complex to the PTS1 receptor [142]. In PTS2 protein import, it was shown that the Pex7p-binding coreceptors Pex18p [143] and Pex20p [144] are ubiquitinated, suggesting a comparable export mechanism for these PTS2 receptors (reference to appropriate reviews by this special issue) . In wild-type cells, Pex5p is mono-ubiquitinated and showed a remarkable stability over time, indicating that its export is not necessarily associated with its degradation [127]. Interestingly, mono-ubiquitination of Pex5p [132, 133, 145], Pex18p [146] and Pex20p [147, 148] takes place at a conserved cysteine via a thioester bond. Usually, such thioester conjunctions are typical for E2 ubiquitin-conjugating enzymes and some E3 ubiquitin-ligases, which only bind

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ubiquitin temporally and allow to further transfer the ubiquitin downstream to its substrates. Likewise, the mono-ubiquitin modification of Pex5p has rather unstable features, as it easily

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can be removed by cytoplasmic concentrations of glutathione [149], or enzymatically by the

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action of the ubiquitin hydrolases Usp9x in humans [150] or Ubp15p in yeast [151]. Ubp15p

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is also part of the yeast AAA-complex and interacts with Pex6p [151]. It seems that attachment of ubiquitin marks the receptors for their export by the AAA+-peroxins, whereby the reversibility of the thioester bond protects the receptors for being recognized by the

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proteasomal degradation machinery [152]. Besides, it is discussed that the conserved cysteine

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of Pex5p might function in regulative import processes by sensing the redox status of the cytosol [153] or together with Pex8p might function in a redox-regulated cargo release at the import machinery [154]. De facto, the exact mechanism of cargo release at the import pore is

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not understood so far. Since the peroxisomal lumen has remarkable high protein density, proteins are supposed be imported against a concentration gradient. The only ATP-dependent

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steps within the import cycle are the activation of ubiquitin at the starting point of the ubiquitin-transducing cascade and the export of Pex5p by the Pex1p-Pex6p AAA+-complex.

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Therefore, the hypothesis of an “export driven import” process arose, suggesting that the ATP-dependent export of Pex5p is mechanistically coupled to cargo release into the peroxisomal lumen [134]. However, results obtained from in vitro assays point to a cargo translocation mechanism, which acts prior to the ATP dependent steps of ubiquitination and export of the receptors [108, 109].

9. Model of the Pex1p-Pex6p mode of action Recent studies revealed the molecular architecture of the yeast Pex1p-Pex6p-complex [76, 86, 87]. Trimers of Pex1p/Pex6p dimers assemble into a hexameric complex with double ring architecture and a central pore, traversing both rings (Fig. 3A). While the N-terminal domains (NTD) of Pex1p are positioned on top of the rings, the N-terminal domains (NTD) of Pex6p

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fold back and are situated aside of the double ring, forming the vertices of an atypically triangular geometry when the complex is viewed from top. By interactions of the Pex6p NTD

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to Pex15p [77], the Pex1p-Pex6p-complex is possibly anchored at its vertices to the

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peroxisomal membrane, whereby the affinity of the Pex1p NTD to phospholipids might

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support the attachment to the membrane [155]. Interestingly, D1 and D2 domains of the subunits are tilted, allowing the NTD of Pex6p to contact the D2 domain of the adjacent Pex1p subunit [76, 87]. Pex15p binding to the Pex1p-Pex6p complex was found to reduce its

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ATPase activity [87] which solely is attributed to the conserved and tightly structured D2

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ring, in particular to the Pex6p D2 domains [76, 86, 87]. It is plausible that Pex15p can intervene in the ATPase cycle upon binding to Pex6p NTD, which in turn contacts the D2 ring by its interaction to Pex1p.

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Using different nucleotides and Walker B mutants, snapshots of the Pex1p-Pex6p complex movements were taken by electron microscopy [76]. This study reveals that the conserved

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substrate binding loops within the D2 ring are oriented towards the central pore. Interestingly, these aromatic pore loop residues turned out to be essential for the function of the protein and

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during the ATPase cycle, they appear to move up and down along the central cavity (Fig. 3B). These observations strongly support a mechanism in which ATP hydrolysis within the D2 ring is transformed into a power stroke of the pore loops to thread the PTS receptors through the central channel of the Pex1p-Pex6p complex [76].

10. Final remarks Much progress has been made in the elucidation of the function of Pex1p, Pex6p, Msp1/ATAD1 and Lon, the four known peroxisomal AAA+-proteins. Pex1p was identified in 1991 as the first peroxisomal AAA+-protein and its molecular role in peroxisome biogenesis was disclosed in 2005. Now, in 2015, first structural data allow a glimpse on the architecture of the AAA+-complex of the peroxisomal protein import machinery, which in turn triggered

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our conception of how Pex1p/Pex6p facilitate the export of the import receptors from the peroxisomal membrane back to the cytosol. A multitude of questions remains, which may be

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addressed in future work. First, how does the Pex1p-Pex6p complex bind to the ubiquitinated

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Pex5p and does this binding regulate the ATPase cycle of the Pex1p-Pex6p complex. A

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further question is how pulling of the receptor and the attachment and detachment of the Pex1p-Pex6p complex at the import machinery are mechanistically linked, since both mechanisms seem to be regulated by the ATPase cycle. In contrast to Pex6p, ATP-hydrolysis

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activity of Pex1p was not essential for complex function in vivo [76]. Thus, the major physical

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work is supposed to be performed by Pex6p. Nevertheless, Pex1p is certainly required for the assembly and scaffolding of the complex and ATP-binding to Pex1p is supposed to regulate the export process. In addition, it needs to be addressed how the membrane anchor Pex15p

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and the substrate proteins contribute to the ATP-driven movements of the AAA+-complex. Finally, it would be interesting to find out, whether Pex5p is completely or only partially

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unfolded upon its threading through the central cavity of the AAA+-complex, since after its export the receptor is released in the cytosol and provided for further rounds of import. We

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have a clear conception about the role of ATAD1/Msp1p in the quality control of tailanchored proteins in mitochondria but whether the protein fulfills the same role for peroxisomes and how this task is mechanistically achieved is completely unknown. It is clear that peroxisomal Lon is involved in quality control of proteins but it remains to find out how the protease recognizes its substrates and whether generated peptides remain in the organelle until the organelle is disposed by pexophagy or whether the peptides are further degraded or exported for disposal.

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Acknowledgements

We apologize to all the scientists whose work could not be cited due to space limitations. We

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thank Dr. Petra Wendler and Dr. Susanne Ciniawsky for providing the structural image of Pex1p/Pex6p (Fig. 3A). This work was supported by the Deutsche Forschungsgemeinschaft

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(FOR1905, SFB642).

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Figure 1: Structural organization and localization of peroxisomal AAA+ ATPases A) AAA+ modules consist of an ASCE domain and a C-terminal attached C-domain. The ASCE domain harbors the Walker A (p-loop) and Walker B motifs as well as the Sensor 1 and arginine-fingers (Arg-finger) within the second region of homology (SRH). The Sensor 2 is located in the C-domain. All together are involved in ATP binding and hydrolysis events. Approximate location of nucleotide interacting key elements and the pore loop is depicted as linear diagram (upper left) and as 2D illustration of the folded AAA+ module (lower left). The principle of hexameric ring formation with ATP binding sides located between the interfaces of the AAA+ protomers is illustrated at the right.

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B) Organization of the peroxisomal AAA+-ATPases Msp1p, Lon and Pex1p/Pex6p. Msp1p and Lon form homo-hexamers with one AAA+ ring (type I). Msp1p is anchored to membranes

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by its N-terminal transmembrane domains (TM) and Lon combines its AAA+ ring with a C-

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terminal protease segment. Pex1p/Pex6p forms a type II hetero-hexameric complex with two

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AAA+ rings (D1 ring, D2 ring) and large N-terminal domains positioned on top and aside of the double ring structure.

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C) Topology of peroxisomal AAA+-proteins. Msp1p is anchored in the peroxisomal and mitochondrial outer membrane, the Lon protease is localized in the peroxisomal matrix and

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Pex1p/Pex6p is attached at the cytosolic side of the peroxisomal matrix protein import

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Figure 2: Protein import of PTS1 proteins into peroxisomes of S. cerevisiae (I) Newly synthesized peroxisomal matrix proteins are recognized by Pex5p via their

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targeting signal (PTS1). The PTS1-receptor Pex5p shuttles between the cytosol and the peroxisomal membrane, thereby guiding cargo proteins to the import machinery. (II) At the peroxisomal membrane, the cargo-loaded receptor is directed to the docking complex (Pex13p, Pex14p, Pex17p). (III) Interactions of the docking complex to the cargo loaded Pex5p is supposed to facilitate the formation of a transient import pore. The minimal pore is at least composed of Pex14p and Pex5p. Referring to the “export driven import” hypothesis cargo translocation might mechanistically be linked to the ATP-dependent export of Pex5p. (IV) Pex8p connects the docking complex with the RING-complex of RING-fingercontaining E3-ubiquitin-ligases (Pex2p, Pex10p, Pex12p). Pex12p functions in concert with the ubiquitin-conjugating Pex4p in transferring a single ubiquitin to Pex5p. (V) Mono-

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ubiquitination of Pex5p serves as signal for the ATP-dependent dislocation of the receptor from the peroxisomal membrane back to the cytosol. This step is accomplished by the Pex1p-

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Pex6p AAA+-complex, which is transiently anchored to the import machinery via reversible

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interactions of Pex6p to Pex15p. Finally, Pex5p is deubiquitinated by the ubiquitin-specific

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protease Ubp15p and released to the cytosol for new rounds of import.

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Figure 3: Model of Pex5p export by threading through the central Pex1p-Pex6p pore

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A) Structure of Pex1p-Pex6p complex (top-, side- and bottom view) revealed by negative stain electron microscopy in presence of ATPS. Pex1p (green) and Pex6p (purple) are

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alternatingly arranged. The D1- and D2 domains of Pex1p (light green, green) and Pex6p

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(light purple, purple) form a double-ring structure with a central pore. Large N-terminal domains of both peroxins (grey) are positioned on top (Pex1p NTD) and aside (Pex6p NTD)

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of the D1- and D2-rings, resulting in a triangular shaped overall appearance of the AAA+complex. Intermolecular contact sides appear between the NTD of Pex6p (grey) and the D2-

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domain of Pex1p (dark green, side view). B) Top view (upper left) and side views of Pex1p-Pex6p complex, reflecting the situation at the peroxisomal membrane. Alternating Pex1p (red) and Pex6p (yellow) molecules form a hexameric double ring structure with a central cavity and conserved pore loops (green) positioned in the lower D2 ring. Large N-terminal domains of Pex1p (red-grey) and Pex6p (yellow-grey) are positioned on top and aside of the double-tiered AAA+ ring structure. The N-terminal domain of Pex6p (yellow-grey) interacts with the tail anchored protein Pex15p (violet) and contacts the D2 domain of the adjacent Pex1p protomer (red). (I) Ubiquitinated Pex5p is recognized by the N-terminal domain of Pex1p (red-grey) and is pulled into the central cavity of the AAA+ complex. (II) Ubiquitin is cleaved off by Ubp15p, which is

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attached to the Pex6p D1 domain. (III) The Pex1p/Pex6p dimer in front and Ubp15p is removed to illustrate the action of two pore loops within the D2 ring. ATP binding elevates

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the pore loops to grab Pex5p. (IV) ATP hydrolysis results in a movement of the substrate-

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binding loop, which pulls the bound substrate downwards. The cycle of ATP binding and

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hydrolysis triggered movements create power strokes pulling Pex5p through the central channel, whereby Pex5p at least partially gets unfolded. (V) Finally, the AAA-complex together with its released cargo is dislocated from the membrane in an ATP-dependent

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Highlights   

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Four peroxisomal AAA+-proteins are known: Pex1p, Pex6p, ATAD1/Msp1p and Lon Pex1p/Pex6p form a hexameric complex required for peroxisomal protein import ATAD1/Msp1p plays a role in targeting of mitochondrial tail-anchored proteins, the function in peroxisomes is unknown  Lon is a AAA-protease required for the quality control of peroxisomal matrix proteins