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Jorge E Azevedo - One of the best experts on this subject based on the ideXlab platform.
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A Mechanistic Perspective on PEX1 and PEX6, Two AAA+ Proteins of the Peroxisomal Protein Import Machinery.
International Journal of Molecular Sciences, 2019Co-Authors: Ana G Pedrosa, Tânia Francisco, Tony A Rodrigues, Maria João Ferreira, Aurora Barros-barbosa, Jorge E AzevedoAbstract:In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to transport proteins across biological membranes, the peroxisomal matrix protein import machinery relies on a regulated self-assembly mechanism for this purpose and uses ATP hydrolysis only to reset its components. The ATP-dependent protein complex in charge of resetting this machinery—the Receptor Export Module (REM)—comprises two members of the “ATPases Associated with diverse cellular Activities” (AAA+) family, PEX1 and PEX6, and a membrane protein that anchors the ATPases to the organelle membrane. In recent years, a large amount of data on the structure/function of the REM complex has become available. Here, we discuss the main findings and their mechanistic implications.
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Chemically monoubiquitinated PEX5 binds to the components of the peroxisomal docking and export machinery
Scientific reports, 2018Co-Authors: Vera Hagmann, Jorge E Azevedo, Stefanie Sommer, Patrick Fabian, Jan Bierlmeier, Nadine D. Van Treel, Henning D. Mootz, Dirk Schwarzer, Gabriele DodtAbstract:Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. “Click” chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.
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peroxisomal monoubiquitinated pex5 interacts with the aaa atpases pex1 and pex6 and is unfolded during its dislocation into the cytosol
Journal of Biological Chemistry, 2018Co-Authors: Ana G Pedrosa, Gabriele Dodt, Tânia Francisco, Diana Bicho, Ana F Dias, Aurora Barrosbarbosa, Vera Hagmann, Tony A Rodrigues, Jorge E AzevedoAbstract:PEX1 and PEX6 are two members of the ATPases associated with diverse cellular activities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex that unfolds substrates by processive threading. However, whether the natural substrate of the PEX1–PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5–interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free in vitro system coupled with photoaffinity cross-linking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1–PEX6 complex.
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the cytosolic domain of PEX3 a protein involved in the biogenesis of peroxisomes binds membrane lipids
Biochimica et Biophysica Acta, 2009Co-Authors: Manuel P Pinto, Jorge E Azevedo, Claudia P Grou, Marc Fransen, Clara SamirandaAbstract:According to current models, most newly synthesized peroxisomal intrinsic membrane proteins are recognized in the cytosol and targeted to the peroxisomal membrane by PEX19. At the organelle membrane the PEX19-cargo protein complex interacts with PEX3, a protein believed to possess only one transmembrane domain and exposing the majority of its polypeptide chain into the cytosol. In agreement with this topological model, a recombinant protein comprising the cytosolic domain of PEX3 can be purified in a soluble and monomeric form in the absence of detergents or other solubilizing agents. Here, we show that this recombinant protein actually precipitates when incubated with mild detergents, suggesting that this domain of PEX3 interacts with amphipathic molecules. Following this observation, we tested this recombinant protein in lipid-binding assays and found that it interacts strongly with liposomes inducing their flocculation or even partial solubilization. The implications of these findings are discussed.
Andreas Martin - One of the best experts on this subject based on the ideXlab platform.
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The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading.
Nature communications, 2018Co-Authors: Brooke M. Gardner, Dominic T. Castanzo, Saikat Chowdhury, Goran Stjepanovic, Matthew S. Stefely, James H. Hurley, Gabriel C. Lander, Andreas MartinAbstract:Pex1 and Pex6 form a heterohexameric motor essential for peroxisome biogenesis and function, and mutations in these AAA-ATPases cause most peroxisome-biogenesis disorders in humans. The tail-anchored protein Pex15 recruits Pex1/Pex6 to the peroxisomal membrane, where it performs an unknown function required for matrix-protein import. Here we determine that Pex1/Pex6 from S. cerevisiae is a protein translocase that unfolds Pex15 in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Our structural studies of Pex15 in isolation and in complex with Pex1/Pex6 illustrate that Pex15 binds the N-terminal domains of Pex6, before its C-terminal disordered region engages with the pore loops of the motor, which then processively threads Pex15 through the central pore. Furthermore, Pex15 directly binds the cargo receptor Pex5, linking Pex1/Pex6 to other components of the peroxisomal import machinery. Our results thus support a role of Pex1/Pex6 in mechanical unfolding of peroxins or their extraction from the peroxisomal membrane during matrix-protein import.
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the pex1 pex6 complex is a heterohexameric aaa motor with alternating and highly coordinated subunits
Journal of Molecular Biology, 2015Co-Authors: Brooke M. Gardner, Saikat Chowdhury, Gabriel C. Lander, Andreas MartinAbstract:Abstract Pex1 and Pex6 are Type-2 AAA + ATPases required for the de novo biogenesis of peroxisomes. Mutations in Pex1 and Pex6 account for the majority of the most severe forms of peroxisome biogenesis disorders in humans. Here, we show that the ATP-dependent complex of Pex1 and Pex6 from Saccharomyces cerevisiae is a heterohexamer with alternating subunits. Within the Pex1/Pex6 complex, only the D2 ATPase ring hydrolyzes ATP, while nucleotide binding in the D1 ring promotes complex assembly. ATP hydrolysis by Pex1 is highly coordinated with that of Pex6. Furthermore, Pex15, the membrane anchor required for Pex1/Pex6 recruitment to peroxisomes, inhibits the ATP-hydrolysis activity of Pex1/Pex6.
Gabriele Dodt - One of the best experts on this subject based on the ideXlab platform.
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Chemically monoubiquitinated PEX5 binds to the components of the peroxisomal docking and export machinery
Scientific reports, 2018Co-Authors: Vera Hagmann, Jorge E Azevedo, Stefanie Sommer, Patrick Fabian, Jan Bierlmeier, Nadine D. Van Treel, Henning D. Mootz, Dirk Schwarzer, Gabriele DodtAbstract:Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. “Click” chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.
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peroxisomal monoubiquitinated pex5 interacts with the aaa atpases pex1 and pex6 and is unfolded during its dislocation into the cytosol
Journal of Biological Chemistry, 2018Co-Authors: Ana G Pedrosa, Gabriele Dodt, Tânia Francisco, Diana Bicho, Ana F Dias, Aurora Barrosbarbosa, Vera Hagmann, Tony A Rodrigues, Jorge E AzevedoAbstract:PEX1 and PEX6 are two members of the ATPases associated with diverse cellular activities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex that unfolds substrates by processive threading. However, whether the natural substrate of the PEX1–PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5–interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free in vitro system coupled with photoaffinity cross-linking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1–PEX6 complex.
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the role of conserved PEX3 regions in pex19 binding and peroxisome biogenesis
Traffic, 2012Co-Authors: Friederike Schmidt, Denise Dietrich, Roy Eylenstein, Yvonne Groemping, Thilo Stehle, Gabriele DodtAbstract:The human peroxins PEX3 and PEX19 are essential for peroxisome biogenesis. They mediate the import of membrane proteins as well as the de novo formation of peroxisomes. PEX19 binds newly synthesized peroxisomal membrane proteins post-translationally and directs them to peroxisomes by engaging PEX3, a protein anchored in the peroxisomal membrane. After protein insertion into the lipid bilayer, PEX19 is released back to the cytosol. Crystallographic analysis provided detailed insights into the PEX3-PEX19 interaction and identified three highly conserved regions, the PEX19-binding region, a hydrophobic groove and an acidic cluster, on the surface of PEX3. Here, we used site-directed mutagenesis and biochemical and functional assays to determine the role of these regions in PEX19-binding and peroxisome biogenesis. Mutations in the PEX19-binding region reduce the affinity for PEX19 and destabilize PEX3. Furthermore, we provide evidence for a crucial function of the PEX3-PEX19 complex during de novo formation of peroxisomes in peroxisome-deficient cells, pointing to a dual function of the PEX3-PEX19 interaction in peroxisome biogenesis. The maturation of preperoxisomes appears to require the hydrophobic groove near the base of PEX3, presumably by its involvement in peroxisomal membrane protein insertion, while the acidic cluster does not appear to be functionally relevant.
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insights into peroxisome function from the structure of PEX3 in complex with a soluble fragment of pex19
Journal of Biological Chemistry, 2010Co-Authors: Friederike Schmidt, Thilo Stehle, Nora Treiber, Georg Zocher, Sasa Bjelic, Michel O Steinmetz, Hubert Kalbacher, Gabriele DodtAbstract:The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.
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the interaction between human PEX3 and pex19 characterized by fluorescence resonance energy transfer fret analysis
European Journal of Cell Biology, 2003Co-Authors: Ania C Muntau, Adelbert A Roscher, Wolfh Kunau, Gabriele DodtAbstract:The process of peroxisome biogenesis involves several PEX genes that encode the machinery required to assemble the organelle. Among the corresponding peroxins the interaction between PEX3 and PEX19 is essential for early peroxisome biogenesis. However, the intracellular site of this protein interaction is still unclear. To address this question by fluorescence resonance energy transfer (FRET) analysis, we engineered the enhanced yellow fluorescent protein (EYFP) to the C-terminus of PEX3 and the enhanced cyan fluorescent protein (ECFP) to the N-terminus of PEX19. Functionality of the fusion proteins was shown by transfection of human PEX3- and PEX19-deficient fibroblasts from Zellweger patients with tagged versions of PEX3 and PEX19. This led to reformation of import-competent peroxisomes in both cell lines previously lacking detectable peroxisomal membrane structures. The interaction of PEX3-EYFP with ECFP-PEX19 in a PEX3-deficient cell line during peroxisome biogenesis was visualized by FRET imaging. Although PEX19 was predominantly localized to the cytoplasma, the peroxisome was identified to be the main intracellular site of the PEX3-PEX19 interaction. Results were confirmed and quantified by donor fluorescence photobleaching experiments. PEX3 deletion proteins lacking the N-terminal peroxisomal targeting sequence (PEX3 34-373-EYFP) or the PEX19-binding domain located in the C-terminal half of the protein (PEX3 1-140-EYFP) did not show the characteristic peroxisomal localization of PEX3, but were mislocalized to the cytoplasm (PEX3 34-373-EYFP) or to the mitochondria (PEX3 1-140-EYFP) and did not interact with ECFP-PEX19. We suggest that FRET is a suitable tool to gain quantitative spatial information about the interaction of peroxins during the process of peroxisome biogenesis in single cells. These findings complement and extend data from conventional in vitro protein interaction assays and support the hypothesis of PEX3 being an anchor for PEX19 at the peroxisomal membrane.
Ida J. Van Der Klei - One of the best experts on this subject based on the ideXlab platform.
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the peroxisome biogenesis factors PEX3 and pex19 multitasking proteins with disputed functions
FEBS Letters, 2019Co-Authors: Renate L M Jansen, Ida J. Van Der KleiAbstract:The peroxisomal membrane protein (PMP) PEX3 and its cytosolic interaction partner Pex19 have been implicated in peroxisomal membrane biogenesis. Although these peroxins have been extensively studied, no consensus has been reached yet on how they operate. Here, we discuss two major models of their function, namely, in direct insertion of proteins into the peroxisomal membrane or in formation of PMP-containing vesicles from the endoplasmic reticulum (ER). PEX3 can also recruit other proteins to the peroxisomal membrane (e.g., Inp1, Atg30, Atg36), thereby fulfilling roles in other processes such as autophagy and organelle retention. Recent studies indicate that PEX3 and Pex19 can also facilitate sorting of certain membrane proteins to other cellular organelles, including the ER, lipid droplets, and mitochondria.
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preperoxisomal vesicles can form in the absence of PEX3
Journal of Cell Biology, 2014Co-Authors: Kevin Knoops, Arjen M. Krikken, Selvambigai Manivannan, Malgorzata N Cepinska, Anita M Kram, Marten Veenhuis, Ida J. Van Der KleiAbstract:We demonstrate that the peroxin PEX3 is not required for the formation of peroxisomal membrane structures in yeast PEX3 mutant cells. Notably, PEX3 mutant cells already contain reticular and vesicular structures that harbor key proteins of the peroxisomal receptor docking complex—Pex13 and Pex14—as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). These reticular and vesicular structures are more abundant in cells of a PEX3 atg1 double deletion strain, as the absence of PEX3 may render them susceptible to autophagic degradation, which is blocked in this double mutant. Contrary to earlier suggestions, peroxisomes are not formed de novo from the endoplasmic reticulum when the PEX3 gene is reintroduced in PEX3 cells. Instead, we find that reintroduced PEX3 sorts to the preperoxisomal structures in PEX3 cells, after which these structures mature into normal peroxisomes.
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damaged peroxisomes are subject to rapid autophagic degradation in the yeast hansenula polymorpha
Autophagy, 2011Co-Authors: Tim Van Zutphen, Ida J. Van Der KleiAbstract:Evidence is accumulating that damaged components of eukaryotic cells are removed by autophagic degradation (e.g., mitophagy). Here we show that peroxisomes that are damaged by the abrupt removal of the membrane protein PEX3 are massively and rapidly degraded even when the cells are placed at peroxisome-inducing conditions and hence need the organelles for growth. PEX3 degradation was induced by a temperature shift using Hansenula polymorpha PEX3Δ cells producing a PEX3 fusion protein containing an N-terminal temperature sensitive degron sequence. The massive peroxisome degradation process, associated with PEX3 degradation, showed properties of both micro- and macropexophagy and was dependent on Atg1 and Ypt7. This mode of peroxisome degradation is of physiological significance as it was also observed at conditions that excessive ROS is formed from peroxisome metabolism, i.e., when methanol-grown wild-type cells are exposed to methanol excess conditions.
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overproduction of translation elongation factor 1 alpha eef1a suppresses the peroxisome biogenesis defect in a hansenula polymorpha PEX3 mutant via translational read through
Fems Yeast Research, 2007Co-Authors: Jan A K W Kiel, Ida J. Van Der Klei, Vladimir I Titorenko, Marten VeenhuisAbstract:In eukaryotes, elongation factor 1-alpha (eEF1A) is required during the elongation phase of translation. We observed that a portion of the cellular eEF1A colocalizes with purified peroxisomes from the methylotrophic yeast Hansenula polymorpha. We have isolated two genes (TEF1 and TEF2) that encode eEF1A, and which are constitutively expressed. We observed that overproduction of eEF1A suppressed the peroxisome deficient phenotype of an H. polymorpha PEX3-1 mutant, which was not observed in a strain deleted for PEX3. The PEX3-1 allele contains a UGG to UGA mutation, thereby truncating PEX3p after amino acid 242, suggesting that the suppression effect might be the result of translational read-through. Consistent with this hypothesis, overexpression of the PEX3-1 gene itself (including its now untranslated part) partly restored peroxisome biogenesis in a PEX3 null mutant. Subsequent co-overexpression of TEF2 in this strain fully restored its peroxisome biogenesis defect and resulted in the formation of major amounts of full-length PEX3p, presumably via translational read-through.
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reassembly of peroxisomes in hansenula polymorpha PEX3 cells on reintroduction of PEX3p involves the nuclear envelope
Fems Yeast Research, 2006Co-Authors: Gertjan Haan, Arjen M. Krikken, Marleen Otzen, Richard J S Baerends, Ida J. Van Der KleiAbstract:The reassembly of peroxisomes in Hansenula polymorpha PEX3 cells on reintroduction of PEX3p was examined. Using a PEX3-green fluorescent protein (PEX3-GFP) fusion protein, expressed under the control of an inducible promoter, it was observed that, initially on induction of PEX3-GFP synthesis, GFP fluorescence was localized to the endoplasmic reticulum and the nuclear envelope. Subsequently, a single organelle developed per cell that increased in size and multiplied by division. At these stages, GFP fluorescence was confined to peroxisomes. Fractionation experiments on homogenates of PEX3 cells, in which the endoplasmic reticulum and nuclear envelope were marked with GFP, identified a small amount of GFP in peroxisomes present in the initial stage of peroxisome reassembly. Our data suggest a crucial role for the endoplasmic reticulum/nuclear envelope in peroxisome reintroduction on complementation of PEX3 cells by the PEX3 gene.
Brooke M. Gardner - One of the best experts on this subject based on the ideXlab platform.
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The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading.
Nature communications, 2018Co-Authors: Brooke M. Gardner, Dominic T. Castanzo, Saikat Chowdhury, Goran Stjepanovic, Matthew S. Stefely, James H. Hurley, Gabriel C. Lander, Andreas MartinAbstract:Pex1 and Pex6 form a heterohexameric motor essential for peroxisome biogenesis and function, and mutations in these AAA-ATPases cause most peroxisome-biogenesis disorders in humans. The tail-anchored protein Pex15 recruits Pex1/Pex6 to the peroxisomal membrane, where it performs an unknown function required for matrix-protein import. Here we determine that Pex1/Pex6 from S. cerevisiae is a protein translocase that unfolds Pex15 in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Our structural studies of Pex15 in isolation and in complex with Pex1/Pex6 illustrate that Pex15 binds the N-terminal domains of Pex6, before its C-terminal disordered region engages with the pore loops of the motor, which then processively threads Pex15 through the central pore. Furthermore, Pex15 directly binds the cargo receptor Pex5, linking Pex1/Pex6 to other components of the peroxisomal import machinery. Our results thus support a role of Pex1/Pex6 in mechanical unfolding of peroxins or their extraction from the peroxisomal membrane during matrix-protein import.
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the peroxisomal aaa atpase pex1 pex6 unfolds substrates by processive threading
Nature Communications, 2018Co-Authors: Brooke M. Gardner, Dominic T. Castanzo, Saikat Chowdhury, Goran Stjepanovic, Matthew S. Stefely, James H. Hurley, Gabriel C. LanderAbstract:Pex1 and Pex6 form a heterohexameric motor essential for peroxisome biogenesis and function, and mutations in these AAA-ATPases cause most peroxisome-biogenesis disorders in humans. The tail-anchored protein Pex15 recruits Pex1/Pex6 to the peroxisomal membrane, where it performs an unknown function required for matrix-protein import. Here we determine that Pex1/Pex6 from S. cerevisiae is a protein translocase that unfolds Pex15 in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Our structural studies of Pex15 in isolation and in complex with Pex1/Pex6 illustrate that Pex15 binds the N-terminal domains of Pex6, before its C-terminal disordered region engages with the pore loops of the motor, which then processively threads Pex15 through the central pore. Furthermore, Pex15 directly binds the cargo receptor Pex5, linking Pex1/Pex6 to other components of the peroxisomal import machinery. Our results thus support a role of Pex1/Pex6 in mechanical unfolding of peroxins or their extraction from the peroxisomal membrane during matrix-protein import.
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the pex1 pex6 complex is a heterohexameric aaa motor with alternating and highly coordinated subunits
Journal of Molecular Biology, 2015Co-Authors: Brooke M. Gardner, Saikat Chowdhury, Gabriel C. Lander, Andreas MartinAbstract:Abstract Pex1 and Pex6 are Type-2 AAA + ATPases required for the de novo biogenesis of peroxisomes. Mutations in Pex1 and Pex6 account for the majority of the most severe forms of peroxisome biogenesis disorders in humans. Here, we show that the ATP-dependent complex of Pex1 and Pex6 from Saccharomyces cerevisiae is a heterohexamer with alternating subunits. Within the Pex1/Pex6 complex, only the D2 ATPase ring hydrolyzes ATP, while nucleotide binding in the D1 ring promotes complex assembly. ATP hydrolysis by Pex1 is highly coordinated with that of Pex6. Furthermore, Pex15, the membrane anchor required for Pex1/Pex6 recruitment to peroxisomes, inhibits the ATP-hydrolysis activity of Pex1/Pex6.