Human pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences

The molecular machinery underlying peroxisomal membrane biogenesis is not well understood. The observation that cells deficient in the peroxins Pex3p, Pex16p, and Pex19p lack peroxisomal membrane structures suggests that these molecules are involved in the initial stages of peroxisomal membrane formation. Pex19p, a predominantly cytosolic protein that can be farnesylated, binds multiple peroxisomal integral membrane proteins, and it has been suggested that it functions as a soluble receptor for the targeting of peroxisomal membrane proteins (PMPs) to the peroxisome. An alternative view proposes that Pex19p functions as a chaperone at the peroxisomal membrane. Here, we show that the peroxisomal sorting determinants and the Pex19p-binding domains of a number of PMPs are distinct entities. In addition, we extend the list of peroxins with which human Pex19p interacts to include the PMP Pex16p and show that Pex19p's CaaX prenylation motif is an important determinant in the affinity of Pex19p for Pex10p, Pex11pbeta, Pex12p, and Pex13p.     

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.     

Pex30p, Pex31p, and Pex32p form a family of peroxisomal integral membrane proteins regulating peroxisome size and number in Saccharomyces cerevisiae

The peroxin Pex23p of the yeast Yarrowia lipolytica exhibits high sequence similarity to the hypothetical proteins Ylr324p, Ygr004p, and Ybr168p encoded by the Saccharomyces cerevisiae genome. Ylr324p, Ygr004p, and Ybr168p are integral to the peroxisomal membrane and act to control peroxisome number and size. Synthesis of Ylr324p and Ybr168p, but not of Ygr004p, is induced during incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for YLR324w exhibit increased numbers of peroxisomes, whereas cells deleted for YGR004w or YBR168w exhibit enlarged peroxisomes. Ylr324p and Ybr168p cannot functionally substitute for one another or for Ygr004p, whereas Ygr004p shows partial functional redundancy with Ylr324p and Ybr168p. Ylr324p, Ygr004p, and Ybr168p interact within themselves and with Pex28p and Pex29p, which have been shown also to regulate peroxisome size and number. Systematic deletion of genes demonstrated that PEX28 and PEX29 function upstream of YLR324w, YGR004w, and YBR168w in the regulation of peroxisome proliferation. Our data suggest a role for Ylr324p, Ygr004p, and Ybr168p--now designated Pex30p, Pex31p, and Pex32p, respectively--together with Pex28p and Pex29p in controlling peroxisome size and proliferation in Saccharomyces cerevisiae.     

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.     

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.     

Multiple targeting modules on peroxisomal proteins are not redundant: discrete functions of targeting signals within Pmp47 and Pex8p

Several peroxisomal proteins have two nonoverlapping targeting signals. These signals have been termed “redundant” because targeting can still occur with only one signal. We now report that separate targeting motifs within both Pmp47 and Pex8 provide complementary function. Pmp47 is an ATP translocator that contains six transmembrane domains (TMDs). We had previously shown that the TMD2 region (termed TMD2R, consisting of TMD2 and a short adjacent segment of cytosolic loop) was required for targeting to proliferated peroxisomes in Saccharomyces cerevisiae. We now report that the analogous TMD4R, which cannot target to proliferated peroxisomes, targets at least as well, or much better (depending on strain and growth conditions) in cells containing only basal (i.e., nonproliferated) peroxisomes. These data suggest differences in the targeting pathway among peroxisome populations. Pex8p, a peripheral protein facing the matrix, contains a typical carboxy terminal targeting sequence (PTS1) that has been shown to be nonessential for targeting, indicating the existence of a second targeting domain (not yet defined in S. cerevisiae); thus, its function was unknown. We show that targeting to basal peroxisomes, but not to proliferated peroxisomes, is more efficient with the PTS1 than without it. Our results indicate that multiple targeting signals within peroxisomal proteins extend coverage among heterogeneous populations of peroxisomes and increase efficiency of targeting in some metabolic states.     

Interaction of Pex5p, the type 1 peroxisome targeting signal receptor, with the peroxisomal membrane proteins Pex14p and Pex13p

Pex5p, a receptor for peroxisomal matrix proteins with a type 1 peroxisome targeting signal (PTS1), has been proposed to cycle from the cytoplasm to the peroxisomal membrane where it docks with Pex14p and Pex13p, the latter an SH3 domain-containing protein. Using in vitro binding assays we have demonstrated that binding of Pex5p to Pex14p is enhanced when Pex5p is loaded with a PTS1-containing peptide. In contrast, Pex5p binding to Pex13p, which involves only the SH3 domain, occurs at 20-40-fold lower levels and is reduced when Pex5p is preloaded with a PTS1 peptide. Pex14p was also shown to bind weakly to the Pex13p SH3 domain. Site-directed mutagenesis of the Pex13p SH3 domain attenuated binding to Pex5p and Pex14p, consistent with both of these proteins being binding partners for this domain. The SH3 binding site in Pex5p was determined to lie within a 114-residue peptide (Trp(100)-Glu(213)) in the amino-terminal region of the protein. The interaction between this peptide and the SH3 domain was competitively inhibited by Pex14p. We interpret these data as suggesting that docking of the Pex5p-PTS1 protein complex at the peroxisome membrane occurs at Pex14p and that the Pex13p SH3 domain functions as an associated component possibly involved in sequestering Pex5p after relinquishment of the PTS1 protein cargo to components of the translocation machinery.     

Ubiquitination of the peroxisomal import receptor Pex5p is required for its recycling

Pex5p, which is the import receptor for peroxisomal matrix proteins harboring a type I signal sequence (PTS1), is mono- and polyubiquitinated in Saccharomyces cerevisiae. We identified Pex5p as a molecular target for Pex4p-dependent monoubiquitination and demonstrated that either poly- or monoubiquitination of the receptor is required for the ATP-dependent release of the protein from the peroxisomal membrane to the cytosol as part of the receptor cycle. Therefore, the energy requirement of the peroxisomal import pathway has to be extended by a second ATP-dependent step, namely receptor monoubiquitination.     

The peroxin Pex19p interacts with multiple, integral membrane proteins at the peroxisomal membrane

Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome.     

Hansenula polymorpha Pex19p is essential for the formation of functional peroxisomal membranes

We have cloned and characterized the Hansenula polymorpha PEX19 gene. In cells of a pex19 disruption strain (Hppex19), induced on methanol, peroxisome structures were not detectable; peroxisomal matrix proteins accumulated in the cytosol, whereas peroxisomal membrane proteins (PMPs) were mislocalized to the cytosol (Pex3p) and mitochondria (Pex14p) or strongly reduced to undetectable levels (Pex10p). The defect in peroxisome formation in Hppex19 cells was largely suppressed upon overproduction of HpPex3p or a fusion protein that consisted of the first 50 N-terminal amino acids of Pex3p and GFP. In these cells PMPs were again correctly sorted to peroxisomal structures, which also harbored peroxisomal matrix proteins. In Saccharomyces cerevisiae pex19 cells overproduction of ScPex3p led to the formation of numerous vesicles that contained PMPs but lacked the major matrix protein thiolase. Taken together, our data are consistent with a function of Pex19p in membrane protein assembly and function.     

Saccharomyces cerevisiae pex3p and pex19p are required for proper localization and stability of peroxisomal membrane proteins

The mechanisms by which peroxisomal membrane proteins (PMPs) are targeted to and inserted into membranes are unknown, as are the required components. We show that among a collection of 16 Saccharomyces cerevisiae peroxisome biogenesis (pex) mutants, two mutants, pex3Delta and pex19Delta, completely lack detectable peroxisomal membrane structures and mislocalize their PMPs to the cytosol where they are rapidly degraded. The other pexDelta mutants contain membrane structures that are properly inherited during vegetative growth and that house multiple PMPs. Even Pex15p requires Pex3p and Pex19p for localization to peroxisomal membranes. This PMP was previously hypothesized to travel via the endoplasmic reticulum (ER) to peroxisomes. We provide evidence that ER-accumulated Pex15p is not a sorting intermediate on its way to peroxisomes. Our results show that Pex3p and Pex19p are required for the proper localization of all PMPs tested, including Pex15p, whereas the other Pex proteins might only be required for targeting/import of matrix proteins.     

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.     

Quantitative analysis of peroxisomal targeting signal type-1 binding to wild-type and pathogenic mutants of Pex5p supports an affinity threshold for peroxisomal protein targeting

Peroxisomal biogenesis disorders (PBDs) are caused by mutations in 12 distinct genes that encode the components of the peroxisome assembly machinery. Three mutations in the gene encoding Pex5p, the peroxisomal targeting signal type-1 (PTS1) receptor, have been reported, each associated with a disorder of the Zellweger spectrum of different severity. Here, we report studies of the affinities of mutated forms of Pex5p for a series of PTS1 peptides and conclude that PTS1-affinity reductions are correlated with disease severity and cell biological phenotype. A quantitative model has been developed that allows estimation of the dissociation constants for complexes with a wide range of PTS1 sequences bound to wild-type and mutant Pex5p. In the context of this model, the binding measurements suggest that no PTS1-containing proteins are targeted by Pex5p(N489K) and only a relatively small subset of PTS1-containing proteins with the highest affinity for Pex5p are targeted to peroxisomes by Pex5p(S563W). Furthermore, the results of the analysis are consistent with an approximate dissociation constant threshold near 500 nM required for efficient protein targeting to peroxisomes.     

Pex19p Interacts with Pex3p and Pex10p and Is Essential for Peroxisome Biogenesis in Pichia pastoris

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Delta cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.     

Peroxisomal membrane proteins contain common Pex19p-binding sites that are an integral part of their targeting signals

Targeting of peroxisomal membrane proteins (PMPs) is a multistep process that requires not only recognition of PMPs in the cytosol but also their insertion into the peroxisomal membrane. As a consequence, targeting signals of PMPs (mPTS) are rather complex. A candidate protein for the PMP recognition event is Pex19p, which interacts with most PMPs. However, the respective Pex19p-binding sites are ill-defined and it is currently disputed whether these sites are contained within mPTS. By using synthetic peptide scans and yeast two-hybrid analyses, we determined and characterized Pex19p-binding sites in Pex11p and Pex13p, two PMPs from Saccharomyces cerevisiae. The sites turned out to be composed of a short helical motif with a minimal length of 11 amino acids. With the acquired data, it proved possible to predict and experimentally verify Pex19p-binding sites in several other PMPs by applying a pattern search and a prediction matrix. A peroxisomally targeted Pex13p fragment became mislocalized to the endoplasmic reticulum in the absence of its Pex19p-binding site. By adding the heterologous binding site of Pex11p, peroxisomal targeting of the Pex13p fragment was restored. We conclude that Pex19p-binding sites are well-defined entities that represent an essential part of the mPTS.     

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.     

Hansenula polymorpha and Saccharomyces cerevisiae Pex5p's recognize different, independent peroxisomal targeting signals in alcohol oxidase

Peroxisomal alcohol oxidase (AO) from Hansenula polymorpha is inactive and partially mislocalized to the cytosol upon synthesis in Saccharomyces cerevisiae. Co-production with H. polymorpha pyruvate carboxylase (HpPyc1p) resulted in AO activation, but did not improve import into peroxisomes. We show that import of AO mediated by S. cerevisiae Pex5p is strictly dependent on the peroxisomal targeting signal 1 (PTS1) of AO and independent of HpPyc1p. In contrast, HpPex5p-mediated sorting of AO into S. cerevisiae peroxisomes is independent of the PTS1, but requires an alternative PTS that is only formed when HpPyc1p is co-produced and most likely involves folding and co-factor binding to AO.     

Mutations in the peroxin Pex26p responsible for peroxisome biogenesis disorders of complementation group 8 impair its stability, peroxisomal localization, and interaction with the Pex1p x Pex6p complex

Peroxisome biogenesis disorders (PBDs) are fatal autosomal recessive diseases and are caused by impaired peroxisome biogenesis. PBDs are genetically heterogeneous and classified into 13 complementation groups (CGs). CG8 is one of the most common groups and has three clinical phenotypes, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD). We recently isolated PEX26 as the pathogenic gene for PBD of CG8. Pex26p functions in recruiting to peroxisomes the complexes of the AAA ATPase peroxins, Pex1p and Pex6p. In the present work, we identified four distinct mutations in PEX26 from five patients of CG8 PBD including 2 with ZS and 3 with IRD, in addition to 7 mutant alleles in 8 patients in the first report describing the pathogenic PEX26 gene for CG8 PBD. Phenotype-genotype analyses revealed that temperature-sensitive (ts) peroxisome assembly gave rise to a milder IRD in contrast to the non-ts phenotype of the cells from ZS patients. Furthermore, we present several lines of evidence that show that the instability, insufficient binding to Pex1p x Pex6p complexes, or mislocalization of patient-derived Pex26p mutants is most likely responsible for the CG8 PBDs.     

The human peroxisomal targeting signal receptor, Pex5p, is translocated into the peroxisomal matrix and recycled to the cytosol

Peroxisomal targeting signals (PTSs) are recognized by predominantly cytosolic receptors, Pex5p and Pex7p. The fate of these PTS receptors following their interactions on the peroxisomal membrane with components of docking and putative translocation complexes is unknown. Using both novel and multiple experimental approaches, we show that human Pex5p does not just bind cargo and deliver it to the peroxisome membrane, but participates in multiple rounds of entry into the peroxisome matrix and export to the cytosol independent of the PTS2 import pathway. This unusual shuttling mechanism for the PTS1 receptor distinguishes protein import into peroxisomes from that into most other organelles, with the exception of the nucleus.