Peroxisomal membrane fusion requires two AAA family ATPases, Pex1p and Pex6p

Two AAA family ATPases, NSF and p97, have been implicated in membrane fusion during assembly and inheritance of organelles of the secretory pathway. We have now investigated the roles of AAA ATPases in membrane fusion during assembly of the peroxisome, an organelle outside the classical secretory system. Here, we show that peroxisomal membrane fusion in the yeast Yarrowia lipolytica requires two AAA ATPases, Pex1p and Pex6p. Release of membrane- associated Pex1p and Pex6p drives the asymmetric priming of two fusion partners. The next step, peroxisome docking, requires release of Pex1p from one partner. Subsequent fusion of the peroxisomal membranes is independent of both Pex1p and Pex6p.     

Pex19p-dependent targeting of Pex17p, a peripheral component of the peroxisomal protein import machinery

Pex19p is required for the topogenesis of peroxisomal membrane proteins (PMPs). Here we have demonstrated that Pex19p is also required for the peroxisomal targeting and stability of Pex17p, a peripheral component of the docking complex of the peroxisomal protein import machinery. We have demonstrated that Pex17p is associated with the peroxisomal Pex13p-Pex14p complex as well as with Pex19p. We have identified the corresponding binding sites for Pex14p and Pex19p and demonstrated that a specific loss of the Pex19p interaction resulted in mistargeting of Pex17p. We have shown that a construct consisting only of the Pex19p- and Pex14p-binding sites of Pex17p is sufficient to direct an otherwise cytosolic reporter protein to the peroxisomal membrane in a Pex19p-dependent manner. Our data show that the function of Pex19p as chaperone or import receptor is not restricted to integral membrane proteins but may also include peripheral PMPs. As a consequence of our data, the previous definition of a targeting signal for PMPs (mPTS) as a Pex19p-binding motif in conjunction with a transmembrane segment should be extended to regions comprising a Pex19p-binding motif and a peroxisomal anchor sequence.     

Peroxisomal matrix protein import

In the past decade, much progress has been made in understanding the mechanisms that govern sorting of proteins to the peroxisomal lumen. This article summarizes the principal features of how peroxisomal matrix enzymes are thought to reach the peroxisome. In addition, it describes recent data that indicate that, in specific pex mutants of the methylotrophic yeast Hansenula polymorpha, defects in matrix protein import can be (partly) rescued by overproduction of the receptor essential for import of these proteins. The implication of these results on the mechanisms of matrix protein import is discussed.     

Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import

Two isoforms of the peroxisomal targeting signal type 1 (PTS1) receptor, termed Pex5pS and (37-amino-acid-longer) Pex5pL, are expressed in mammals. Pex5pL transports PTS1 proteins and Pex7p-PTS2 cargo complexes to the initial Pex5p-docking site, Pex14p, on peroxisome membranes, while Pex5pS translocates only PTS1 cargoes. Here we report functional Pex5p domains responsible for interaction with peroxins Pex7p, Pex13p, and Pex14p. An N-terminal half, such as Pex5pL(1-243), comprising amino acid residues 1 to 243, bound to Pex7p, Pex13p, and Pex14p and was sufficient for restoring the impaired PTS2 import of pex5 cell mutants, while the C-terminal tetratricopeptide repeat motifs were required for PTS1 binding. N-terminal Pex5p possessed multiple Pex14p-binding sites. Alanine-scanning analysis of the highly conserved seven (six in Pex5pS) pentapeptide WXXXF/Y motifs residing at the N-terminal region indicated that these motifs were essential for the interaction of Pex5p with Pex14p and Pex13p. Moreover, mutation of several WXXXF/Y motifs did not affect the PTS import-restoring activity of Pex5p, implying that the binding of Pex14p to all of the WXXXF/Y sites was not a prerequisite for the translocation of Pex5p-cargo complexes. Pex5p bound to Pex13p at the N-terminal part, not to the C-terminal SH3 region, via WXXXF/Y motifs 2 to 4. PTS1 and PTS2 import required the interaction of Pex5p with Pex14p but not with Pex13p, while Pex5p binding to Pex13p was essential for import of catalase with PTS1-like signal KANL. Pex5p recruited PTS1 proteins to Pex14p but not to Pex13p. Pex14p and Pex13p formed a complex with PTS1-loaded Pex5p but dissociated in the presence of cargo-unloaded Pex5p, implying that PTS cargoes are released from Pex5p at a step downstream of Pex14p and upstream of Pex13p. Thus, Pex14p and Pex13p very likely form mutually and temporally distinct subcomplexes involved in peroxisomal matrix protein import.     

The peroxisomal membrane protein import receptor Pex3p is directly transported to peroxisomes by a novel Pex19p- and Pex16p-dependent pathway

Two distinct pathways have recently been proposed for the import of peroxisomal membrane proteins (PMPs): a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex3p-independent class II pathway. We show here that Pex19p plays an essential role as the chaperone for full-length Pex3p in the cytosol. Pex19p forms a soluble complex with newly synthesized Pex3p in the cytosol and directly translocates it to peroxisomes. Knockdown of Pex19p inhibits peroxisomal targeting of newly synthesized full-length Pex3p and results in failure of the peroxisomal localization of Pex3p. Moreover, we demonstrate that Pex16p functions as the Pex3p-docking site and serves as the peroxisomal membrane receptor that is specific to the Pex3p–Pex19p complexes. Based on these novel findings, we suggest a model for the import of PMPs that provides new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p.     

Characterization of the mammalian peroxisomal import machinery: Pex2p, Pex5p, Pex12p, and Pex14p are subunits of the same protein assembly

Although many of the proteins involved in the biogenesis of the mammalian peroxisome have already been identified, our knowledge of the architecture of all this machinery is still very limited. In this work we used native gel electrophoresis and sucrose gradient sedimentation analysis in combination with immunoprecipitation experiments to address this issue. After solubilization of rat liver peroxisomes with the mild detergent digitonin, comigration of Pex5p, Pex14p, and a fraction of Pex12p was observed upon native electrophoresis and sucrose gradient sedimentation. The existence of a complex comprising Pex2p, Pex5p, Pex12p, and Pex14p was demonstrated by preparative coimmunoprecipitation experiments using an antibody directed to Pex14p. No stoichiometric amounts of Pex13p were detected in the Pex2p-Pex5p-Pex12p-Pex14p complex, although the presence of a small fraction of Pex13p in this complex could be demonstrated by Western blot analysis. Pex13p is also a component of a high molecular mass complex. Strikingly, partial purification of this Pex13p-containing complex revealed Pex13p as the major (if not the only) component. Taken together, our data indicate that Pex2p, Pex5p, Pex12p, and Pex14p, on one side, and Pex13p, on the other, are subunits of two stable protein complexes that probably interact with each other in the peroxisomal membrane.     

The Peroxisomal Matrix Import of Pex8p Requires Only PTS Receptors and Pex14p

Pichia pastoris (Pp) Pex8p, the only known intraperoxisomal peroxin at steady state, is targeted to peroxisomes by either the peroxisomal targeting signal (PTS) type 1 or PTS2 pathway. Until recently, all cargoes entering the peroxisome matrix were believed to require the docking and really interesting new gene (RING) subcomplexes, proteins that bridge these two subcomplexes and the PTS receptor-recycling machinery. However, we reported recently that the import of PpPex8p into peroxisomes via the PTS2 pathway is Pex14p dependent but independent of the RING subcomplex (Zhang et al., 2006 ). In further characterizing the peroxisome membrane-associated translocon, we show that two other components of the docking subcomplex, Pex13p and Pex17p, are dispensable for the import of Pex8p. Moreover, we demonstrate that the import of Pex8p via the PTS1 pathway also does not require the RING subcomplex or intraperoxisomal Pex8p. In receptor-recycling mutants (Δpex1, Δpex6, and Δpex4), Pex8p is largely cytosolic because Pex5p and Pex20p are unstable. However, upon overexpression of the degradation-resistant Pex20p mutant, hemagglutinin (HA)-Pex20p(K19R), in Δpex4 and Δpex6 cells, Pex8p enters peroxisome remnants. Our data support the idea that PpPex8p is a special cargo whose translocation into peroxisomes depends only on the PTS receptors and Pex14p and not on intraperoxisomal Pex8p, the RING subcomplex, or the receptor-recycling machinery.     

Pex7p and Pex20p of Neurospora crassa function together in PTS2-dependent protein import into peroxisomes

Recruiting matrix proteins with a peroxisomal targeting signal type 2 (PTS2) to the peroxisomal membrane requires species-specific factors. In Saccharomyces cerevisiae, the PTS2 receptor Pex7p acts in concert with the redundant Pex18p/Pex21p, whereas in Yarrowia lipolytica, Pex20p might unite the function of both S. cerevisiae peroxins. Herein, the genome of the filamentous fungus Neurospora crassa was analyzed for peroxin-encoding genes. We identified a set of 18 peroxins that resembles that of Y. lipolytica rather than that of S. cerevisiae. Interestingly, proteins homologous to both S. cerevisiae Pex7p and Y. lipolytica Pex20p exist in N. crassa. We report on the isolation of these PTS2-specific peroxins and demonstrate that NcPex20p can substitute for S. cerevisiae Pex18p/Pex21p, but not for ScPex7p. Like Pex18p, NcPex20p did not bind PTS2 protein or the docking proteins in the absence of ScPex7p. Rather, NcPex20p was required before docking to form an import-competent complex of cargo-loaded PTS2 receptors. NcPex7p did not functionally replace yeast Pex7p, probably because the N. crassa PTS2 receptor failed to associate with Pex18p/Pex21p. However, once NcPex7p and NcPex20p had been coexpressed, it proved possible to replace yeast Pex7p. Pex20p and Pex18p/Pex21p are therefore true orthologues, both of which are in need of Pex7p for PTS2 protein import.     

The import competence of a peroxisomal membrane protein is determined by Pex19p before the docking step

Biogenesis of the mammalian peroxisomal membrane requires the action of Pex3p and Pex16p, two proteins present in the organelle membrane, and Pex19p, a protein that displays a dual subcellular distribution (peroxisomal and cytosolic). Pex19p interacts with most peroxisomal intrinsic membrane proteins, but whether this property reflects its role as an import receptor for this class of proteins or a chaperone-like function in the assembly/disassembly of peroxisomal membrane proteins has been the subject of much controversy. Here, we describe an in vitro system particularly suited to address this issue. It is shown that insertion of a reporter protein into the peroxisomal membrane is a Pex3p-dependent process that does not require ATP/GTP hydrolysis. The system can be programmed with recombinant versions of Pex19p, allowing us to demonstrate that Pex19p-cargo protein complexes formed in the absence of peroxisomes are the substrates for the peroxisomal docking/insertion machinery. Data suggesting that cargo-loaded Pex19p displays a much higher affinity for Pex3p than Pex19p alone are also provided. These results suggest that soluble Pex19p participates in the targeting of newly synthesized peroxisomal membrane proteins to the organelle membrane and support the existence of a cargo-induced peroxisomal targeting mechanism for Pex19p.     

Involvement of Pex13p in Pex14p localization and peroxisomal targeting signal 2-dependent protein import into peroxisomes

Pex13p is the putative docking protein for peroxisomal targeting signal 1 (PTS1)-dependent protein import into peroxisomes. Pex14p interacts with both the PTS1- and PTS2-receptor and may represent the point of convergence of the PTS1- and PTS2-dependent protein import pathways. We report the involvement of Pex13p in peroxisomal import of PTS2-containing proteins. Like Pex14p, Pex13p not only interacts with the PTS1-receptor Pex5p, but also with the PTS2-receptor Pex7p; however, this association may be direct or indirect. In support of distinct peroxisomal binding sites for Pex7p, the Pex7p/Pex13p and Pex7p/ Pex14p complexes can form independently. Genetic evidence for the interaction of Pex7p and Pex13p is provided by the observation that overexpression of Pex13p suppresses a loss of function mutant of Pex7p. Accordingly, we conclude that Pex7p and Pex13p functionally interact during PTS2-dependent protein import into peroxisomes. NH2-terminal regions of Pex13p are required for its interaction with the PTS2-receptor while the COOH-terminal SH3 domain alone is sufficient to mediate its interaction with the PTS1-receptor. Reinvestigation of the topology revealed both termini of Pex13p to be oriented towards the cytosol. We also found Pex13p to be required for peroxisomal association of Pex14p, yet the SH3 domain of Pex13p may not provide the only binding site for Pex14p at the peroxisomal membrane.     

Yeast thermotolerance does not require protein synthesis.

Heat shock at 37 degrees C induces synthesis of stress (heat shock) proteins in Saccharomyces cerevisiae and also induces thermotolerance. Amino acid analogs that are powerful inducers of stress protein synthesis failed to induce thermotolerance, suggesting that the stress proteins do not play a causal role in acquired thermotolerance at 37 degrees C. This suggestion was confirmed by the observation that protein synthesis was not required for the induction of thermotolerance at 37 degrees C.     

Pex17p is required for import of both peroxisome membrane and lumenal proteins and interacts with Pex19p and the peroxisome targeting signal-receptor docking complex in Pichia pastoris

Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Delta mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Delta mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal-receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.     

The AAA-type ATPases Pex1p and Pex6p and their role in peroxisomal matrix protein import in Saccharomyces cerevisiae

The recognition of the conserved ATP-binding domains of Pex1p, p97 and NSF led to the discovery of the family of AAA-type ATPases. The biogenesis of peroxisomes critically depends on the function of two AAA-type ATPases, namely Pex1p and Pex6p, which provide the energy for import of peroxisomal matrix proteins. Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and guided to the peroxisomal membrane by specific soluble receptors. At the membrane, the cargo-loaded receptors bind to a docking complex and the receptor-docking complex assembly is thought to form a dynamic pore which enables the transition of the cargo into the organellar lumen. The import cycle is completed by ubiquitination- and ATP-dependent dislocation of the receptor from the membrane to the cytosol, which is performed by the AAA-peroxins. Receptor ubiquitination and dislocation are the only energy-dependent steps in peroxisomal protein import. The export-driven import model suggests that the AAA-peroxins might function as motor proteins in peroxisomal import by coupling ATP-dependent removal of the peroxisomal import receptor and cargo translocation into the organelle.     

Interactions of Pex7p and Pex18p/Pex21p with the peroxisomal docking machinery: implications for the first steps in PTS2 protein import

Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13delta mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p- and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event.     

Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p

Peroxisomes require peroxin (Pex) proteins for their biogenesis. The interaction between Pex3p, which resides on the peroxisomal membrane, and Pex19p, which resides in the cytosol, is crucial for peroxisome formation and the post-translational targeting of peroxisomal membrane proteins (PMPs). It is not known how Pex3p promotes the specific interaction with Pex19p for the purpose of PMP translocation. Here, we present the three-dimensional structure of the complex between a cytosolic domain of Pex3p and the binding-region peptide of Pex19p. The overall shape of Pex3p is a prolate spheroid with a novel fold, the 'twisted six-helix bundle.' The Pex19p-binding site is at an apex of the Pex3p spheroid. A 16-residue region of the Pex19p peptide forms an α-helix and makes a contact with Pex3p; this helix is disordered in the unbound state. The Pex19p peptide contains a characteristic motif, consisting of the leucine triad (Leu18, Leu21, Leu22), and Phe29, which are critical for the Pex3p binding and peroxisome biogenesis.     

PUMA-mediated apoptosis in fibroblast-like synoviocytes does not require p53

PUMA (p53-upregulated modulator of apoptosis) is a pro-apoptotic gene that can induce rapid cell death through a p53-dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53-/- murine FLS were also examined to evaluate whether p53 is required. p53-deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were then determined by Western blot analysis. Apoptosis was assayed by ELISA to measure histone release and caspase-3 activation, or by trypan blue dye exclusion to measure cell viability. Initial studies showed that p53 siRNA decreased p53 expression by more than 98% in human FLS. Loss of p53 increased the growth rate of cells and suppressed p21 expression. However, PUMA still induced apoptosis in control and p53-deficient FLS after PUMA cDNA transfection. Similar results were observed in p53-/- murine FLS or in human FLS transfected with a dominant-negative mutant p53 gene. These data suggest that PUMA-induced apoptosis in FLS does not require p53. Therefore, approaches to gene therapy that involve increasing PUMA expression could be an effective inducer of synoviocyte cell death in rheumatoid arthritis regardless of the p53 status in the synovium.     

PUMA-mediated apoptosis in fibroblast-like synoviocytes does not require p53

PUMA (p53-upregulated modulator of apoptosis) is a pro-apoptotic gene that can induce rapid cell death through a p53-dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53^-/- murine FLS were also examined to evaluate whether p53 is required. p53-deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were then determined by Western blot analysis. Apoptosis was assayed by ELISA to measure histone release and caspase-3 activation, or by trypan blue dye exclusion to measure cell viability. Initial studies showed that p53 siRNA decreased p53 expression by more than 98% in human FLS. Loss of p53 increased the growth rate of cells and suppressed p21 expression. However, PUMA still induced apoptosis in control and p53-deficient FLS after PUMA cDNA transfection. Similar results were observed in p53^-/- murine FLS or in human FLS transfected with a dominant-negative mutant p53 gene. These data suggest that PUMA-induced apoptosis in FLS does not require p53. Therefore, approaches to gene therapy that involve increasing PUMA expression could be an effective inducer of synoviocyte cell death in rheumatoid arthritis regardless of the p53 status in the synovium.     

Pex17p of Saccharomyces cerevisiae Is a Novel Peroxin and Component of the Peroxisomal Protein Translocation Machinery

The Saccharomyces cerevisiae pex17-1 mutant was isolated from a screen to identify mutants defective in peroxisome biogenesis. pex17-1 and pex17 null mutants fail to import matrix proteins into peroxisomes via both PTS1- and PTS2-dependent pathways. The PEX17 gene (formerly PAS9; Albertini, M., P. Rehling, R. Erdmann, W. Girzalsky, J.A.K.W. Kiel, M. Veenhuis, and W.-H Kunau. 1997. Cell. 89:83–92) encodes a polypeptide of 199 amino acids with one predicted membrane spanning region and two putative coiled-coil structures. However, localization studies demonstrate that Pex17p is a peripheral membrane protein located at the surface of peroxisomes. Particulate structures containing the peroxisomal integral membrane proteins Pex3p and Pex11p are evident in pex17 mutant cells, indicating the existence of peroxisomal remnants (“ghosts”). This finding suggests that pex17 null mutant cells are not impaired in peroxisomal membrane biogenesis. Two-hybrid studies showed that Pex17p directly binds to Pex14p, the recently proposed point of convergence for the two peroxisomal targeting signal (PTS)-dependent import pathways, and indirectly to Pex5p, the PTS1 receptor. The latter interaction requires Pex14p, indicating the potential of these three peroxins to form a trimeric complex. This conclusion is supported by immunoprecipitation experiments showing that Pex14p and Pex17p coprecipitate with both PTS receptors in the absence of Pex13p. From these and other studies we conclude that Pex17p, in addition to Pex13p and Pex14p, is the third identified component of the peroxisomal translocation machinery.     

Saccharomyces cerevisiae Pex14p contains two independent Pex5p binding sites, which are both essential for PTS1 protein import

Pex14p is a peroxisomal membrane-associated protein involved in docking of both Pex5p and Pex7p to the peroxisomal membrane. Previous studies have shown that, in humans, the N-terminal region of Pex14p interacts with WxxxF/Y motifs in Pex5p. Here, we report that Saccharomyces cerevisiae Pex14p contains two independent Pex5p binding sites, one in the N- and one in the C-terminus. Using deletion analysis we show that, in vivo, both of these interactions are needed for PTS1 import. Furthermore, we show that the characterized WxxxF/Y motifs of Pex5p are not essential for binding to the N-terminus of Pex14p but do play a role in the interaction with the Pex14 C-terminus. Thus, the data suggest that the mechanism of the Pex14p-Pex5p interaction in yeast is different from that previously reported for humans.