Peroxisome reintroduction in Hansenula polymorpha requires Pex25 and Rho1

We identified two proteins, Pex25 and Rho1, which are involved in reintroduction of peroxisomes in peroxisome-deficient yeast cells. These are, together with Pex3, the first proteins identified as essential for this process. Of the three members of the Hansenula polymorpha Pex11 protein family-Pex11, Pex25, and Pex11C-only Pex25 was required for reintroduction of peroxisomes into a peroxisome-deficient mutant strain. In peroxisome-deficient pex3 cells, Pex25 localized to structures adjacent to the ER, whereas in wild-type cells it localized to peroxisomes. Pex25 cells were not themselves peroxisome deficient but instead contained a slightly increased number of peroxisomes. Interestingly, pex11 pex25 double deletion cells, in which both peroxisome fission (due to the deletion of PEX11) and reintroduction (due to deletion of PEX25) was blocked, did display a peroxisome-deficient phenotype. Peroxisomes reappeared in pex11 pex25 cells upon synthesis of Pex25, but not of Pex11. Reintroduction in the presence of Pex25 required the function of the GTPase Rho1. These data therefore provide new and detailed insight into factors important for de novo peroxisome formation in yeast.     

Depletion of GIM5 causes cellular fragility,a decreased glycosome number,and reduced levels of ether-linked phospholipids in trypanosomes

Microbody division in mammalian cells, trypanosomes, and yeast depends on the PEX11 microbody membrane proteins. The function of PEX11 is not understood, and the suggestion that it affects microbody (peroxisome) numbers in mammals and yeast, because it plays a role in beta-oxidation of fatty acids, is controversial. PEX11 and two PEX11-related proteins, GIM5A and GIM5B, are the predominant membrane proteins of the microbodies (glycosomes) of Trypanosoma brucei. The compartmentation of glycosomal enzymes is essential in trypanosomes. Deletion of the GIM5A gene from the form of the parasite that lives in the mammalian blood has no effect on trypanosome growth, but depletion of GIM5B on a gim5a null background causes death. We show here that procyclic trypanosomes, adapted for life in the Tsetse fly vector, survive without GIM5A and with very low levels of GIM5B. The depleted cells have fewer glycosomes than usual and are osmotically fragile, which is a novel observation for a microbody defect. Thus trypanosomes require both GIM5B and PEX11 for the maintenance of normal glycosome numbers. Procyclic cells lacking GIM5A, like mouse cells partially defective in PEX11, have fewer ether-linked phospholipids, even when GIM5B levels are not reduced. Metabolite measurements on GIM5A/B-depleted bloodstream form trypanosomes suggested a change in the flux through the glycolytic pathway. We conclude that PEX11 family proteins play important roles in determining microbody membrane structure, with secondary effects on a subset of microbody metabolic pathways.     

Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p

PEX5 functions as an import receptor for proteins with the type-1 peroxisomal targeting signal (PTS1). Although PEX5 is not involved in the import of PTS2-targeted proteins in yeast, it is essential for PTS2 protein import in mammalian cells. Human cells generate two isoforms of PEX5 through alternative splicing, PEX5S and PEX5L, and PEX5L contains an additional insert 37 amino acids long. Only one isoform, PEX5L, is involved in PTS2 protein import, and PEX5L physically interacts with PEX7, the import receptor for PTS2-containing proteins. In this report we map the regions of human PEX5L involved in PTS2 protein import, PEX7 interaction, and targeting to peroxisomes. These studies revealed that amino acids 1-230 of PEX5L are required for PTS2 protein import, amino acids 191-222 are sufficient for PEX7 interaction, and amino acids 1-214 are sufficient for targeting to peroxisomes. We also identified a 21-amino acid-long peptide motif of PEX5L, amino acids 209-229, that overlaps the regions sufficient for full PTS2 rescue activity and PEX7 interaction and is shared by Saccharomyces cerevisiae Pex18p and Pex21p, two yeast peroxins that act only in PTS2 protein import in yeast. A mutation in PEX5 that changes a conserved serine of this motif abrogates PTS2 protein import in mammalian cells and reduces the interaction of PEX5L and PEX7 in vitro. This peptide motif also lies within regions of Pex18p and Pex21p that interact with yeast PEX7. Based on these and other results, we propose that mammalian PEX5L may have acquired some of the functions that yeast Pex18p and/or Pex21p perform in PTS2 protein import. This hypothesis may explain the essential role of PEX5L in PTS2 protein import in mammalian cells and its lack of importance for PTS2 protein import in yeast.     

Molecular anatomy of the peroxin Pex12p: ring finger domain is essential for Pex12p function and interacts with the peroxisome-targeting signal type 1-receptor Pex5p and a ring peroxin, Pex10p

The three peroxin genes, PEX12, PEX2, and PEX10, encode peroxisomal integral membrane proteins with RING finger at the C-terminal part and are responsible for human peroxisome biogenesis disorders. Mutation analysis in PEX12 of Chinese hamster ovary cell mutants revealed a homozygous nonsense mutation at residue Trp263Ter in ZP104 cells and a pair of heterozygous nonsense mutations, Trp170Ter and Trp114Ter, in ZP109. This result and domain mapping of Pex12p showed that RING finger is essential for peroxisome-restoring activity of Pex12p but not necessary for targeting to peroxisomes. The N-terminal region of Pex12p, including amino acid residues at positions 17-76, was required for localization to peroxisomes, while the sequence 17-76 was not sufficient for peroxisomal targeting. Peroxins interacting with RING finger of Pex2p, Pex10p, and Pex12p were investigated by yeast two-hybrid as well as in vitro binding assays. The RING finger of Pex12p bound to Pex10p and the PTS1-receptor Pex5p. Pex10p also interacted with Pex2p and Pex5p in vitro. Moreover, Pex12p was co-immunoprecipitated with Pex10p from CHO-K1 cells, where Pex5p was not associated with the Pex12p-Pex10p complex. This observation suggested that Pex5p does not bind to, or only transiently interacts with, Pex10p and Pex12p when Pex10p and Pex12p are in the oligomeric complex in peroxisome membranes. Hence, the RING finger peroxins are most likely to be involved in Pex5p-mediated matrix protein import into peroxisomes.     

The tetratricopeptide repeat domains of human, tobacco, and nematode PEX5 proteins are functionally interchangeable with the analogous native domain for peroxisomal import of PTS1-terminated proteins in yeast

In the yeast Saccharomyces cerevisiae, beta-oxidation of fatty acids is compartmentalised in peroxisomes. Most yeast peroxisomal matrix proteins contain a type 1C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof. PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain. Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids. Here, the PEX5-TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras. These hybrid proteins were examined for functional complementation of the pex5delta mutant phenotype. Expression of the Pex5p chimaeras in pex5delta mutant cells restored peroxisomal import of PTS1-terminated proteins. Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source. We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s. Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5-TPR domain in the two-hybrid system. HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids. Although mutant pex5delta cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, they were able to grow on oleic acid. We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL.     

ARF- and coatomer-mediated peroxisomal vesiculation

The authors characterized on a molecular level the clofibrate-inducible 26-kDa integral peroxisomal membrane protein (Pmp26p, Pex11-1p) of rat liver. By screening cDNA databases with the obtained Pex11-1p-cDNA, a second homologous cDNA was identified that codes for a polypeptide with slightly larger molecular mass than Pex11-1p. The authors call this polypeptide Pex11-2p. Studies on the topology of Pex11-1p revealed two transmembrane domains with the N- and C-terminus facing the cytoplasm. The C-terminal tail of Pex11-1p ends in a consensus dilysine motif of the type-KXKXX-COOH, which is known to be involved in the ADP-ribosylation factor (ARF)1-coat protein (COP) I coat (ARF)1-dependent membrane recruitment to Golgi membranes. Studies with isolated peroxisomes incubated in the presence of cytosol, adenosine triphosphate and GTPγS, indeed, provided evidence for specific binding of ARF and coatomer to peroxisomes. Expression of Pex11-1p in Chinese hamster ovary (CHO) wild-type cells led to a twofold increase in the number of peroxisomes, but expression in a temperature-sensitive CHO mutant, defective in coatomer, induced elongation and tubulation of peroxisomal structures, rather than numerical proliferation. The obtained results for the first time offer a mechanism explaining Pex11-1p-, as well as ARF- and coatomer-mediated peroxisomal vesiculation. Two models are presented that may explain how these observations fit in with peroxisome biogenesis.     

PEX genes in fungal genomes: common, rare or redundant

PEX genes encode proteins, termed peroxins, that are required for the biogenesis and proliferation of microbodies (peroxisomes). We have screened the available protein and DNA databases to identify putative peroxin orthologs in 17 fungal species (yeast and filamentous fungi) and in humans. This analysis demonstrated that most peroxins are present in all fungi under study. Only Pex16p is absent in most yeast species, with the exception of Yarrowia lipolytica, but this peroxin is present in all filamentous fungi. Furthermore, we found that the Y. lipolytica PEX9 gene, a putative orphan gene, might encode a Pex26p ortholog. In addition, in the genomes of Saccharomyces cerevisiae and Candida glabrata, several PEX genes appear to have been duplicated, exemplified by the presence of paralogs of the peroxins Pex5p and Pex21p, which were absent in other organisms. In all organisms, we observed multiple paralogs of the peroxins involved in organelle proliferation. These proteins belong to two groups of peroxins that we propose to designate the Pex11p and Pex23p families. This redundancy may complicate future studies on peroxisome biogenesis and proliferation in fungal species.     

Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p.

Kinetoplastid protozoa confine large parts of glycolysis within glycosomes, which are microbodies related to peroxisomes. We cloned the gene encoding the second most abundant integral membrane protein of Trypanosoma brucei glycosomes. The 24 kDa protein is very basic and hydrophobic, with two predicted transmembrane domains. It is targeted to peroxisomes when expressed in mammalian cells and yeast. The protein is a functional homologue of Pex11p from Saccharomyces cerevisiae: pex11Delta mutants, which are defective in peroxisome proliferation, can be complemented by the trypanosome gene. Sequence conservation is significant in the N- and C-terminal domains of all putative Pex11p homologues known, from trypanosomes, yeasts and mammals. Several lines of evidence indicate that these domains are oriented towards the cytosol. TbPex11p can form homodimers, like its yeast counterpart. The TbPEX11 gene is essential in trypanosomes. Inducible overexpression of the protein in T.brucei bloodstream forms causes growth arrest, the globular glycosomes being transformed to clusters of long tubules filling significant proportions of the cytoplasm. Reduced expression results in trypanosomes with fewer, but larger, organelles.     

A role for the peroxin Pex8p in Pex20p-dependent thiolase import into peroxisomes of the yeast Yarrowia lipolytica

Peroxins are proteins required for peroxisome assembly. The cytosolic peroxin Pex20p binds directly to the beta-oxidation enzyme thiolase and is necessary for its dimerization and peroxisomal targeting. The intraperoxisomal peroxin Pex8p has a role in the import of peroxisomal matrix proteins, including thiolase. We report the results of yeast two-hybrid analyses with various peroxins of the yeast Yarrowia lipolytica and characterize more fully the interaction between Pex8p and Pex20p. Coimmunoprecipitation showed that Pex8p and Pex20p form a complex, while in vitro binding studies demonstrated that the interaction between Pex8p and Pex20p is specific, direct, and autonomous. Pex8p fractionates with peroxisomes in cells of a PEX20 disruption strain, indicating that Pex20p is not necessary for the targeting of Pex8p to peroxisomes. In cells of a PEX8 disruption strain, thiolase is mostly cytosolic, while Pex20p and a small amount of thiolase associate with peroxisomes, suggesting the involvement of Pex8p in the import of thiolase after docking of the Pex20p-thiolase complex to the membrane. In the absence of Pex8p, peroxisomal thiolase and Pex20p are protected from the action of externally added protease. This finding, together with the fact that Pex8p is intraperoxisomal, suggests that Pex20p may accompany thiolase into peroxisomes during import.     

Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum.     

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.     

Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes

Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (Saccharomyces cerevisiae and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane. In this report, we show that the specificity of peroxisomal membrane protein binding for Trypanosoma brucei PEX19 is very similar to those previously identified for human and yeast PEX19. Our studies show that trafficking is conserved across these distant phyla and that both a PEX19 binding site and a transmembrane domain are required for the insertion of two test proteins into the glycosomal membrane. However, in contrast to T. brucei PEX10 and PEX12, T. brucei PEX14 does not traffic to human peroxisomes, indicating that it is not recognized by the human PEX14 import mechanism.     

Ubiquitination of the peroxisomal targeting signal type 1 receptor, Pex5p, suggests the presence of a quality control mechanism during peroxisomal matrix protein import

PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.     

Function of the PEX19-binding site of human adrenoleukodystrophy protein as targeting motif in man and yeast. PMP targeting is evolutionarily conserved

We predicted in human peroxisomal membrane proteins (PMPs) the binding sites for PEX19, a key player in the topogenesis of PMPs, by virtue of an algorithm developed for yeast PMPs. The best scoring PEX19-binding site was found in the adrenoleukodystrophy protein (ALDP). The identified site was indeed bound by human PEX19 and was also recognized by the orthologous yeast PEX19 protein. Likewise, both human and yeast PEX19 bound with comparable affinities to the PEX19-binding site of the yeast PMP Pex13p. Interestingly, the identified PEX19-binding site of ALDP coincided with its previously determined targeting motif. We corroborated the requirement of the ALDP PEX19-binding site for peroxisomal targeting in human fibroblasts and showed that the minimal ALDP fragment targets correctly also in yeast, again in a PEX19-binding site-dependent manner. Furthermore, the human PEX19-binding site of ALDP proved interchangeable with that of yeast Pex13p in an in vivo targeting assay. Finally, we showed in vitro that most of the predicted binding sequences of human PMPs represent true binding sites for human PEX19, indicating that human PMPs harbor common PEX19-binding sites that do resemble those of yeast. Our data clearly revealed a role for PEX19-binding sites as PMP-targeting motifs across species, thereby demonstrating the evolutionary conservation of PMP signal sequences from yeast to man.     

Pex11p plays a primary role in medium-chain fatty acid oxidation, a process that affects peroxisome number and size in Saccharomyces cerevisiae

The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p has previously been implicated in peroxisome proliferation based on morphological observations of PEX11 mutant cells. Pex11p-deficient cells fail to increase peroxisome number in response to growth on fatty acids and instead accumulate a few giant peroxisomes. We report that mutants deficient in genes required for medium-chain fatty acid (MCFA) beta-oxidation display the same phenotype as Pex11p-deficient cells. Upon closer inspection, we found that Pex11p is required for MCFA beta-oxidation. Disruption of the PEX11 gene results in impaired formation of MCFA-CoA esters as measured in intact cells, whereas their formation is normal in cell lysates. The sole S. cerevisiae MCFA-CoA synthetase (Faa2p) remains properly localized to the inner leaflet of the peroxisomal membrane in PEX11 mutant cells. Therefore, the in vivo latency of MCFA activation observed in Pex11p-deficient cells suggests that Pex11p provides Faa2p with substrate. When PEX11 mutant cells are shifted from glucose to oleate-containing medium, we observed an immediate deficiency in beta-oxidation of MCFAs whereas giant peroxisomes and a failure to increase peroxisome abundance only became apparent much later. Our observations suggest that the MCFA oxidation pathway regulates the level of a signaling molecule that modulates the number of peroxisomal structures in a cell.     

New insights into dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p in shuttling of PTS1-receptor Pex5p during peroxisome biogenesis

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders such as Zellweger syndrome. Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for PBDs of complementation groups 1 and 4, respectively. PEX26 responsible for peroxisome biogenesis disorders of complementation group 8 codes for C-tail-anchored type-II membrane peroxin Pex26p, the recruiter of Pex1p-Pex6p complexes to peroxisomes. Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by ATPase cycle. Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome targeting signal type-1 and shuttles between the cytosol and peroxisomes. AAA peroxins are involved in the export from peroxisomes of Pex5p. Pex5p is ubiquitinated at the conserved cysteine11 in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p-C11A, abrogates peroxisomal import of proteins harboring peroxisome targeting signals 1 and 2 in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, hence suggesting an essential role of the cysteine residue in the export of Pex5p.     

Pexophagy is responsible for 65% of cases of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) is a group of diseases caused by mutations in one of the peroxins, proteins responsible for biogenesis of the peroxisomes. In recent years, it became clear that many peroxins (e.g., PEX3 and PEX14) play additional roles in peroxisome homeostasis (such as promoting autophagic degradation of peroxisomes or pexophagy), which are often opposite to their originally established functions in peroxisome formation and maintenance. Even more interesting, the peroxins that make up the peroxisomal AAA ATPase complex (AAA-complex) in yeast (Pex1, Pex6 and Pex15) or mammals (PEX1, PEX6, PEX26) are responsible for the downregulation of pexophagy. Moreover, this might be even their primary role in human: to prevent pexophagy by removing from the peroxisomal membrane the ubiquitinated peroxisomal matrix protein import receptor, Ub-PEX5, which is also a signal for the Ub-binding pexophagy receptor, NBR1. Remarkably, the peroxisomes rescued from pexophagy by autophagic inhibitors in PEX1^G843D (the most common PBD mutation) cells are able to import matrix proteins and improve their biochemical function suggesting that the AAA-complex per se is not essential for the protein import function in human. This paradigm-shifting discovery published in the current issue of Autophagy has raised hope for up to 65% of all PBD patients with various deficiencies in the AAA-complex. Recognizing PEX1, PEX6 and PEX26 as pexophagy suppressors will allow treating these patients with a new range of tools designed to target mammalian pexophagy.