Pex14p is Not Required for Microautophagy and in Catalytic Amounts for Macropexophagy in Hansenula polymorpha

We showed before that the two oppositely directed processes of peroxisome biogenesis and selective peroxisome degradation (macropexophagy) converge at the peroxisomal membrane protein Pex14p. Here we show that this protein is not required for peroxisomal degradation during nitrogen starvation-induced general autophagy, thereby limiting its function to the selective degradation process. Pex14p is present in two forms, namely an unmodified (Pex14p) and a phosphorylated form (Pex14p^Pi) that are differently induced during peroxisome proliferation. The data suggest that Pex14p is required for peroxisome biogenesis during organelle proliferation and Pex14p^Pi in macropexophagy. Finally, we show that macropexophagy is not coupled to normal peroxisome assembly and is required in only catalytic amounts to allow initiation of the selective peroxisome degradation process.     

Removal of Pex3p is an important initial stage in selective peroxisome degradation in Hansenula polymorpha

Selective degradation of peroxisomes (macropexophagy) in Hansenula polymorpha involves the sequestration of individual organelles to be degraded by membranes prior to the fusion of this compartment with the vacuole and subsequent degradation of the whole organelle by vacuolar hydrolases. Here we show that Pex3p, a peroxisomal membrane protein essential for peroxisome biogenesis, escapes this autophagic process. Upon induction of macropexophagy, Pex3p is removed from the organelle tagged for degradation prior to its sequestration. Our data indicate that Pex3p degradation is essential to allow the initiation of the organellar degradation process. Also, in a specific peroxisome degradation-deficient (pdd) mutant in which sequestration still occurs but the vacuolar fusion event is disturbed, the turnover of Pex3p is still observed. Taken together, our data suggest that degradation of Pex3p is part of the initial degradation machinery of individual peroxisomes.     

Pex14 is the sole component of the peroxisomal translocon that is required for pexophagy

Pex14 was initially identified as a peroxisomal membrane protein that is involved in docking of the soluble receptor proteins Pex5 and Pex7, which are required for import of PTS1- or PTS2-containing peroxisomal matrix proteins. However, Hansenula polymorpha Pex14 is also required for selective degradation of peroxisomes (pexophagy). Previously we showed that Pex1, Pex4, Pex6 and Pex8 are not required for this process. Here we show that also in the absence of various other peroxins, namely Pex2, Pex10, Pex12, Pex13 and Pex17, pexophagy can normally occur. These peroxins are, like Pex14, components of the peroxisomal translocon. Our data confirm that Pex14 is the sole peroxin that has a unique dual function in two apparent opposite processes, namely peroxisome formation and selective degradation.     

Peroxisome biogenesis and selective degradation converge at Pex14p

We have analyzed the function of Hansenula polymorpha Pex14p in selective peroxisome degradation. Previously, we showed that Pex14p was involved in peroxisome biogenesis and functions in peroxisome matrix protein import. Evidence for the additional function of HpPex14p in selective peroxisome degradation (pexophagy) came from cells defective in HpPex14p synthesis. The suggestion that the absence of HpPex14p interfered with pexophagy was further analyzed by mutational analysis. These studies indicated that deletions at the C terminus of up to 124 amino acids of HpPex14p did not affect peroxisome degradation. Conversely, short deletions of the N terminus (31 and 64 amino acids, respectively) of the protein fully impaired pexophagy. Peroxisomes present in these cells remained intact for at least 6 h of incubation in the presence of excess glucose, conditions that led to the rapid turnover of the organelles in wild-type control cells. We conclude that the N terminus of HpPex14p contains essential information to control pexophagy in H. polymorpha and thus, that organelle development and turnover converge at Pex14p.     

Peroxisome fission is associated with reorganization of specific membrane proteins

Membrane remodeling is an important aspect in organelle biogenesis. We show that different peroxisome membrane proteins that play a role in organelle biogenesis and proliferation (Pex8, Pex10, Pex14, Pex25 and Pex11) are subject to spatiotemporal behavior during organelle development. Using fluorescence microscopy analysis of Hansenula polymorpha dnm1 cells that are blocked in the normal fission process, we show that green fluorescent protein (GFP) fusions of Pex8, Pex10, Pex14 and Pex25 show enhanced fluorescence at the organelle extensions that are formed in budding cells. In contrast, Pex11 fluorescence is enriched at the base of this extension on the mother organelle. A fusion protein of GFP with the transporter Pmp47, used as a control, did not show enhanced fluorescence at any specific region of the organelle. The concentration of specific peroxins at the peroxisome surface was lost upon deletion of PEX11 or the N-terminal domain of Pex11 that is involved in membrane remodeling. Comparable distribution patterns as in dnm1 cells were observed in wild-type cells where Pex8, Pex10, Pex14 and Pex25, but not Pex11, were especially present at newly formed organelles that migrated to the bud. We speculate that peroxin reorganization events result in enhanced levels of peroxins involved in peroxisome biogenesis in nascent organelles.     

Damaged peroxisomes are subject to rapid autophagic degradation in the yeast Hansenula polymorpha

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.     

Damaged peroxisomes are subject to rapid autophagic degradation in the yeast Hansenula polymorpha

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.     

PpAtg30 tags peroxisomes for turnover by selective autophagy

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.     

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.     

A subtle interplay between three Pex11 proteins shapes de novo formation and fission of peroxisomes

The organization of eukaryotic cells into membrane-bound compartments must be faithfully sustained for survival of the cell. A subtle equilibrium exists between the degradation and the proliferation of organelles. Commonly, proliferation is initiated by a membrane remodeling process. Here, we dissect the function of proteins driving organelle proliferation in the particular case of peroxisomes. These organelles are formed either through a growth and division process from existing peroxisomes or de novo from the endoplasmic reticulum (ER). Among the proteins involved in the biogenesis of peroxisomes, peroxins, members of the Pex11 protein family participate in peroxisomal membrane alterations. In the yeast Saccharomyces cerevisiae, the Pex11 family consists of three proteins, Pex11p, Pex25p and Pex27p. Here we demonstrate that yeast mutants lacking peroxisomes require the presence of Pex25p to regenerate this organelle de novo. We also provide evidence showing that Pex27p inhibits peroxisomal function and illustrate that Pex25p initiates elongation of the peroxisomal membrane. Our data establish that although structurally conserved each of the three Pex11 protein family members plays a distinct role. While ScPex11p promotes the proliferation of peroxisomes already present in the cell, ScPex25p initiates remodeling at the peroxisomal membrane and ScPex27p acts to counter this activity. In addition, we reveal that ScPex25p acts in concert with Pex3p in the initiation of de novo peroxisome biogenesis from the ER.     

Hansenula polymorpha Vam7p is required for macropexophagy

We have analyzed the functions of two vacuolar t-SNAREs, Vam3p and Vam7p, in peroxisome degradation in the methylotrophic yeast Hansenula polymorpha. A Hp-vam7 mutant was strongly affected in peroxisome degradation by selective macropexophagy as well as non-selective microautophagy. Deletion of Hp-Vam3p function had only a minor effect on peroxisome degradation processes. Both proteins were located at the vacuolar membrane, with Hp-Vam7p also having a partially cytosolic location. Previously, in baker's yeast Vam3p and Vam7p have been demonstrated to be components of a t-SNARE complex essential for vacuole biogenesis. We speculate that the function of this complex in macropexophagy includes a role in membrane fusion processes between the outer membrane layer of sequestered peroxisomes and the vacuolar membrane. Our data suggest that Hp-Vam3p may be functionally redundant in peroxisome degradation. Remarkably, deletion of Hp-VAM7 also significantly affected peroxisome biogenesis and resulted in organelles with multiple, membrane-enclosed compartments. These morphological defects became first visible in cells that were in the mid-exponential growth phase of cultivation on methanol, and were correlated with accumulation of electron-dense extensions that were connected to mitochondria.     

The Saccharomyces cerevisiae peroxisomal import receptor Pex5p is monoubiquitinated in wild type cells

Pex5p is a mobile receptor for peroxisomal targeting signal type I-containing proteins that cycles between the cytoplasm and the peroxisome. Here we show that Pex5p is a stable protein that is monoubiquitinated in wild type cells. By making use of mutants defective in vacuolar or proteasomal degradation we demonstrate that monoubiquitinated Pex5p is not a breakdown intermediate of either system. Monoubiquitinated Pex5p is localized to peroxisomes, and ubiquitination requires the presence of functional docking and RING finger complexes, which suggests that it is a late event in peroxisomal matrix protein import. In pex1, pex4, pex6, pex15, and pex22 mutants, all of which are blocked in the terminal steps of peroxisomal matrix protein import, polyubiquitinated forms of Pex5p accumulate, ubiquitination being dependent on the ubiquitin-conjugating enzyme Ubc4p. However, Ubc4p is not required for Pex5p ubiquitination in wild type cells, and cells lacking Ubc4p are not affected in peroxisome biogenesis. These results indicate that Pex5p monoubiquitination in wild type cells serves to regulate rather than to degrade Pex5p, which is supported by the observed stability of Pex5p. We propose that Pex5p monoubiquitination in wild type cells is required for the recycling of Pex5p from the peroxisome, whereas Ubc4p-mediated polyubiquitination of Pex5p in mutants blocked in the terminal steps of peroxisomal matrix protein import may function as a disposal mechanism for Pex5p when it gets stuck in the import pathway.     

Peroxisome biogenesis occurs in an unsynchronized manner in close association with the endoplasmic reticulum in temperature-sensitive Yarrowia lipolytica Pex3p mutants

Pex3p is a peroxisomal integral membrane protein required early in peroxisome biogenesis, and Pex3p-deficient cells lack identifiable peroxisomes. Two temperature-sensitive pex3 mutant strains of the yeast Yarrowia lipolytica were made to investigate the role of Pex3p in the early stages of peroxisome biogenesis. In glucose medium at 16 degrees C, these mutants underwent de novo peroxisome biogenesis and exhibited early matrix protein sequestration into peroxisome-like structures found at the endoplasmic reticulum-rich periphery of cells or sometimes associated with nuclei. The de novo peroxisome biogenesis seemed unsynchronized, with peroxisomes occurring at different stages of development both within cells and between cells. Cells with peripheral nascent peroxisomes and cells with structures morphologically distinct from peroxisomes, such as semi/circular tubular structures that immunostained with antibodies to peroxisomal matrix proteins and to the endoplasmic reticulum-resident protein Kar2p, and that surrounded lipid droplets, were observed during up-regulation of peroxisome biogenesis in cells incubated in oleic acid medium at 16 degrees C. These structures were not detected in wild-type or Pex3p-deficient cells. Their role in peroxisome biogenesis remains unclear. Targeting of peroxisomal matrix proteins to these structures suggests that Pex3p directly or indirectly sequesters components of the peroxisome biogenesis machinery. Such a role is consistent with Pex3p overexpression producing cells with fewer, larger, and clustered peroxisomes.     

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.     

Obstruction of polyubiquitination affects PTS1 peroxisomal matrix protein import

Pex4p is an ubiquitin-conjugating enzyme that functions at a late stage of peroxisomal matrix protein import. Here we show that in the methylotrophic yeast Hansenula polymorpha production of a mutant form of ubiquitin (Ub(K48R)) has a dramatic effect on PTS1 matrix protein import. This effect was not observed in cells lacking Pex4p, in which the peroxisome biogenesis defect was largely suppressed. These findings provide the first indication that the function of Pex4p in matrix protein import involves polyubiquitination. We also demonstrate that the production of Ub(K48R) in H. polymorpha results in enhanced Pex5p degradation. A similar observation was made in cells in which the PEX4 gene was deleted. We demonstrate that in both strains Pex5p degradation was due to ubiquitination and subsequent degradation by the proteasome. This process appeared to be dependent on a conserved lysine residue in the N-terminus of Pex5p (Lys21) and was prevented in a Pex5p(K21R) mutant. We speculate that the degradation of Pex5p by the proteasome is important to remove receptor molecules that are stuck at a late stage of the Pex5p-mediated protein import pathway.     

Peroxisome biogenesis disorders: Molecular basis for impaired peroxisomal membrane assembly: In metabolic functions and biogenesis of peroxisomes in health and disease

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS). Gene defects of peroxins required for both membrane assembly and matrix protein import are identified: ten mammalian pathogenic peroxins for ten complementation groups of PBDs, are required for matrix protein import; three, Pex3p, Pex16p and Pex19p, are shown to be essential for peroxisome membrane assembly and responsible for the most severe ZS in PBDs of three complementation groups 12, 9, and 14, respectively. Patients with severe ZS with defects of PEX3, PEX16, and PEX19 tend to carry severe mutation such as nonsense mutations, frameshifts and deletions. With respect to the function of these three peroxins in membrane biogenesis, two distinct pathways have been proposed for the import of peroxisomal membrane proteins in mammalian cells: a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex16p-dependent class II pathway. In class II pathway, Pex19p also forms a soluble complex with newly synthesized Pex3p as the chaperone for Pex3p in the cytosol and directly translocates it to peroxisomes. Pex16p functions as the peroxisomal membrane receptor that is specific to the Pex3p-Pex19p complexes. A model for the import of peroxisomal membrane proteins is suggested, providing new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p. Another model suggests that in Saccharomyces cerevisiae peroxisomes likely emerge from the endoplasmic reticulum. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease.     

Pex15p of Saccharomyces cerevisiae provides a molecular basis for recruitment of the AAA peroxin Pex6p to peroxisomal membranes

The gene products (peroxins) of at least 29 PEX genes are known to be necessary for peroxisome biogenesis but for most of them their precise function remains to be established. Here we show that Pex15p, an integral peroxisomal membrane protein, in vivo and in vitro binds the AAA peroxin Pex6p. This interaction functionally interconnects these two hitherto unrelated peroxins. Pex15p provides the mechanistic basis for the reversible targeting of Pex6p to peroxisomal membranes. We could demonstrate that the N-terminal part of Pex6p contains the binding site for Pex15p and that the two AAA cassettes D1 and D2 of Pex6p have opposite effects on this interaction. A point mutation in the Walker A motif of D1 (K489A) decreased the binding of Pex6p to Pex15p indicating that the interaction of Pex6p with Pex15p required binding of ATP. Mutations in Walker A (K778A) and B (D831Q) motifs of D2 abolished growth on oleate and led to a considerable larger fraction of peroxisome bound Pex6p. The nature of these mutations suggested that ATP-hydrolysis is required to disconnect Pex6p from Pex15p. On the basis of these results, we propose that Pex6p exerts at least part of its function by an ATP-dependent cycle of recruitment and release to and from Pex15p.     

Pex18p is constitutively degraded during peroxisome biogenesis

Pex18p and Pex21p are structurally related yeast peroxins (proteins required for peroxisome biogenesis) that are partially redundant in function. One or the other is essential for the import into peroxisomes of proteins with type 2 peroxisomal targeting sequences (PTS2). These sequences bind to the soluble PTS2 receptor, Pex7p, which in turn binds to Pex18p (or Pex21p or possibly both). Here we show that Pex18p is constitutively degraded with a half-time of less than 10 min in wild-type Saccharomyces cerevisiae. This degradation probably occurs in proteasomes, because it requires the related ubiquitin-conjugating enzymes Ubc4p and Ubc5p and occurs normally in a mutant lacking the Pep4p vacuolar protease. The turnover of Pex18p stops, and Pex18p accumulates to a much higher than normal abundance in pex mutants in which the import of all peroxisomal matrix proteins is blocked. This includes mutants that lack peroxins involved in receptor docking at the membrane (Deltapex13 or Deltapex14), a mutant that lacks the peroxisomal member of the E2 family of ubiquitin-conjugating enzymes (Deltapex4), and others (Deltapex1). This stabilization in a variety of pex mutants indicates that Pex18p turnover is associated with its normal function. A Pex18p-Pex7p complex is detected by immunoprecipitation in wild type cells, and its abundance increases considerably in the Deltapex14 peroxisome biogenesis mutant. Cells that lack Pex7p fail to stabilize and accumulate Pex18p, indicating an important role for complex formation in the stabilization. Mono- and diubiquitinated forms of Pex18p are detected in wild-type cells, and there is no Pex18p turnover in a yeast doa4 mutant in which ubiquitin homeostasis is defective. These data represent, to the best of our knowledge, the first instance of an organelle biogenesis factor that is degraded constitutively and rapidly.     

Two independent pathways traffic the intraperoxisomal peroxin PpPex8p into peroxisomes: mechanism and evolutionary implications

Among peroxins involved in peroxisome biogenesis, only Pex8p is predominantly intraperoxisomal at steady state. Pex8p is necessary for peroxisomal matrix protein import via the PTS1 and PTS2 pathways. It is proposed to bridge two peroxisomal membrane subcomplexes comprised of the docking (Pex13p, Pex14p, Pex17p) and RING (Pex2p, Pex10p, Pex12p) peroxins and is also implicated in cargo release of PTS1 proteins in the matrix. We show that Pichia pastoris Pex8p (PpPex8p) enters the peroxisome matrix using two redundant pathways in a Pex14p-dependent, but Pex2p-independent, manner, showing that the intact importomer and RING subcomplex are not required for its import. One pathway depends on the TPR motifs in Pex5p, the C-terminal PTS1 sequence (AKL) in PpPex8p, and the intraperoxisomal presence of this peroxin. The alternative pathway uses the PTS2 receptor, Pex7p, its accessory protein, Pex20p, and a putative PTS2 motif in PpPex8p, but does not require intraperoxisomal PpPex8p. Pex20p interaction with PpPex8p is independent of Pex7p, but the interaction of PpPex8p with Pex7p requires Pex20p. These data suggest a direct interaction between PpPex8p and Pex20p. Our studies shed light on the mechanism and evolution of the dual import pathways for PpPex8p.     

AAA Peroxins and Their Recruiter Pex26p Modulate the Interactions of Peroxins Involved in Peroxisomal Protein Import

Pex1p and Pex6p are required for the relocation of the import receptor Pex5p from the peroxisomal membrane to the cytosol. We herein show that mammalian Pex26p directly binds to Pex14p, the initial docking receptor of Pex5p, and interacts with Pex5p via Pex14p. The binding affinity of Pex26p to Pex14p is altered by Pex5p. Further evidence suggests that the N-terminal region in Pex26p acts as a scaffold protein to recruit Pex14p·Pex5p complex together with Pex1p·Pex6p complexes on peroxisomes. Pex26p binding to Pex14p was suppressed by overexpression of Pex1p and Pex6p in an ATP-dependent manner, whereas Pex14p was not competed out by Pex1p and Pex6p from Pex26p mutant defective in peroxisomal matrix protein import. These results suggested that peroxisome biogenesis requires Pex1p- and Pex6p-regulated dissociation of Pex14p from Pex26p. Pex1p homo-oligomer directly binds to Pex5p as assessed by a surface plasmon resonance-based assay. Moreover, cytosolic Pex1p is likely to maintain the functional oligomer of Pex5p. Taken together, in the peroxisomal protein import, AAA peroxins modulate the interaction between Pex26p and Pex14p on peroxisome membrane as well as Pex5p oligomer in the cytosol.