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.     

Atg21p is essential for macropexophagy and microautophagy in the yeast Hansenula polymorpha

ATG genes are required for autophagy-related processes that transport proteins/organelles destined for proteolytic degradation to the vacuole. Here, we describe the identification and characterisation of the Hansenula polymorpha ATG21 gene. Its gene product Hp-Atg21p, fused to eGFP, had a dual location in the cytosol and in peri-vacuolar dots. We demonstrate that Hp-Atg21p is essential for two separate modes of peroxisome degradation, namely glucose-induced macropexophagy and nitrogen limitation-induced microautophagy. In atg21 cells subjected to macropexophagy conditions, sequestration of peroxisomes tagged for degradation is initiated but fails to complete.     

The Hansenula polymorpha PDD7 gene is essential for macropexophagy and microautophagy

Hansenula polymorpha PDD genes are involved in the selective degradation of peroxisomes via macropexophagy. We have isolated various novel pdd mutants by a gene-tagging method. Here we describe the isolation and characterisation of PDD7, which encodes a protein with high sequence similarity (40% identity) to Saccharomyces cerevisiae Apg1p/Aut3p, previously described to be involved in random autophagy and the cytoplasm-to-vacuole targeting pathway. Our data indicate that HpPdd7p is essential for two processes that degrade peroxisomes, namely the highly selective process of macropexophagy and microautophagy, which occurs in H. polymorpha upon nitrogen starvation.     

Hansenula polymorpha Pex14p phosphorylated in vivo

Hansenula polymorpha Pex14p is a novel peroxisomal membrane protein essential for peroxisome biogenesis. In vivo labeling experiment of wild-type cells with ³²P-orthophosphate and alkaline phosphatase treatment of labeled Pex14p indicate that Pex14p is phosphorylated in vivo. Analysis of the phosphoamino acid in the phosphorylated Pex14p suggested that the major phosphoamino acid was acid labile. Using expression system of several truncated Pex14ps in a PEX14-deletion strain it is suggested that the phosphorylation site of Pex14p resides in the C-terminal 58 residues.     

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.     

The peroxisomal membrane protein Pex14p of Hansenula polymorpha is phosphorylated in vivo.

Hansenula polymorpha Pex14p (HpPex14p) is a component of the peroxisomal membrane essential for peroxisome biogenesis. Here, we show that HpPex14p is phosphorylated in vivo. In wild-type H. polymorpha cells, grown in the presence of [32P]orthophosphate, the 32P label was incorporated into HpPex14p. Labelled HpPex14p was induced after a shift of cells to methanol-containing media and rapidly disappeared after a shift to glucose medium, which induces specific peroxisome degradation. Alkaline phosphatase treatment of labelled HpPex14p resulted in the release of 32P and a minor shift of the HpPex14p band on Western blots. Phosphoamino acid analysis by two dimensional silica gel thin layer chromatography suggested that the major phosphoamino acid in phosphorylated HpPex14p was acid-labile.     

The Hansenula polymorpha ATG25 gene encodes a novel coiled-coil protein that is required for macropexophagy

We have isolated the Hansenula polymorpha ATG25 gene, which is required for glucose-induced selective peroxisome degradation by macropexophagy. ATG25 represents a novel gene that encodes a 45 kDa coiled-coil protein. We show that this protein colocalizes with Atg11 on a small structure, which most likely represents the pre-autophagosomal structure (PAS). In cells of a constructed ATG25 deletion strain (atg25) peroxisomes are constitutively degraded by nonselective microautophagy, a process that in WT H. polymorpha is only observed at nitrogen limitation conditions. This suggests that nonselective microautophagy is deregulated in H. polymorpha atg25 cells.     

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.     

A conserved alpha helical domain at the N-terminus of Pex14p is required for PTS1 and PTS2 protein import in Hansenula polymorpha

We have analyzed the highly conserved N-terminus of Hansenula polymorpha Pex14p for its function in peroxisomal matrix protein import. The region comprising aa 10-54 of HpPex14p is predicted to contain three alpha-helices. Its alpha-helical structure was confirmed by CD analysis of a synthetic peptide, corresponding to residues 8-58. Deletion of aa 1-21 of HpPex14p, but not of aa 1-9, completely abolished PTS1 and PTS2 matrix protein import. An extensive mutational analysis of the first alpha-helix (aa 10-21) demonstrated that its secondary structure, as well as residues Phe20 and Leu21, are essential for PTS1 and PTS2 matrix protein import.     

Hansenula polymorpha Pex3p is a peripheral component of the peroxisomal membrane

Hansenula polymorpha Pex3p plays an essential role in the biogenesis and maintenance of the peroxisomal membrane. In the initial report, bakers' yeast Pex3p was suggested to represent an integral component of the peroxisomal membrane, containing one membrane-spanning region that exposes the N terminus of the protein into the organellar matrix. Biochemically, HpPex3p behaved like an integral membrane protein as it was resistant toward high salt and carbonate treatment. However, urea fully removed Pex3p from the membrane under conditions in which the integral membrane protein Pex10p was resistant to this treatment. Additional experiments, including protease protection assays and pre-embedding labeling experiments on purified organellar fractions from cells that produced Pex3ps carrying Myc epitopes at various selected locations in the protein, revealed that invariably all Myc tags were accessible for externally added proteases and antibodies, independent of the presence of detergents. Also, overproduction of Pex3p failed to demonstrate the typical integral membrane protein structures in fracture faces of freeze-fractured peroxisomes. Taken together, our data suggest that HpPex3p does not span the peroxisomal membrane but instead is tightly associated to the cytosolic face of the organelle where it may be present in focal protein clusters.     

Microautophagy and macropexophagy may occur simultaneously in Hansenula polymorpha

We subjected methanol-grown cells of wild type Hansenula polymorpha simultaneously to nitrogen depletion and excess glucose conditions. Both treatments induce the degradation of peroxisomes, either selective (via excess glucose) or non-selective (via nitrogen limitation). Our combined data strongly suggest that both processes occur simultaneously under these conditions. The implications of these findings on studies of autophagy and related transport pathways to the vacuole in yeast are discussed.     

Normal peroxisome development from vesicles induced by truncated Hansenula polymorpha Pex3p.

We show that the synthesis of the N-terminal 50 amino acids of Pex3p (Pex3p(1-50)) in Hansenula polymorpha pex3 cells is associated with the formation of vesicular membrane structures. Biochemical and ultrastructural findings suggest that the nuclear membrane is the donor membrane compartment of these vesicles. These structures also contain Pex14p and can develop into functional peroxisomes after subsequent reintroduction of the full-length Pex3p protein. We discuss the significance of this finding in relation to peroxisome reintroduction, e.g. in case peroxisomes are lost due to failure in inheritance.     

Overproduction of Pex5p stimulates import of alcohol oxidase and dihydroxyacetone synthase in a Hansenula polymorpha Pex14 null mutant

Hansenula polymorpha Deltapex14 cells are affected in peroxisomal matrix protein import and lack normal peroxisomes. Instead, they contain peroxisomal membrane remnants, which harbor a very small amount of the major peroxisomal matrix enzymes alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS). The bulk of these proteins is, however, mislocated in the cytosol. Here, we show that in Deltapex14 cells overproduction of the PTS1 receptor, Pex5p, leads to enhanced import of the PTS1 proteins AO and DHAS but not of the PTS2 protein amine oxidase. The import of the PTS1 protein catalase (CAT) was not stimulated by Pex5p overproduction. The difference in import behavior of AO and CAT was not related to their PTS1, since green fluorescent protein fused to the PTS1 of either AO or CAT were both not imported in Deltapex14 cells overproducing Pex5p. When produced in a wild type control strain, both proteins were normally imported into peroxisomes. In Deltapex14 cells overproducing Pex5p, Pex5p had a dual location and was localized in the cytosol and bound to the outer surface of the peroxisomal membrane. Our results indicate that binding of Pex5p to the peroxisomal membrane and import of certain PTS1 proteins can proceed in the absence of Pex14p.     

The ubiquitin-conjugating enzyme Pex4p of Hansenula polymorpha is required for efficient functioning of the PTS1 import machinery.

We have cloned the Hansenula polymorpha PEX4 gene by functional complementation of a peroxisome-deficient mutant. The PEX4 translation product, Pex4p, is a member of the ubiquitin-conjugating enzyme family. In H.polymorpha, Pex4p is a constitutive, low abundance protein. Both the original mutant and the pex4 deletion strain (Deltapex4) showed a specific defect in import of peroxisomal matrix proteins containing a C-terminal targeting signal (PTS1) and of malate synthase, whose targeting signal is not yet known. Import of the PTS2 protein amine oxidase and the insertion of the peroxisomal membrane proteins Pex3p and Pex14p was not disturbed in Deltapex4 cells. The PTS1 protein import defect in Deltapex4 cells could be suppressed by overproduction of the PTS1 receptor, Pex5p, in a dose-response related manner. In such cells, Pex5p is localized in the cytosol and in peroxisomes. The peroxisome-bound Pex5p specifically accumulated at the inner surface of the peroxisomal membrane and thus differed from Pex5p in wild-type peroxisomes, which is localized throughout the matrix. We hypothesize that in H. polymorpha Pex4p plays an essential role for normal functioning of Pex5p, possibly in mediating recycling of Pex5p from the peroxisome to the cytosol.     

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.     

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.     

Pex14p is a member of the protein linkage map of Pex5p.

To identify members of the translocation machinery for peroxisomal proteins, we made use of the two-hybrid system to establish a protein linkage map centered around Pex5p from Saccharomyces cerevisiae, the receptor for the C-terminal peroxisomal targeting signal (PTS1). Among the five interaction partners identified, Pex14p was found to be induced under conditions allowing peroxisome proliferation. Deletion of the corresponding gene resulted in the inability of yeast cells to grow on oleate as well as the absence of peroxisomal structures. The PEX14 gene product of approximately 38 kDa was biochemically and ultrastructurally demonstrated to be a peroxisomal membrane protein, despite the lack of a membrane-spanning domain. This protein was shown to interact with itself, with Pex13p and with both PTS receptors, Pex5p and Pex7p, indicating a central function for the import of peroxisomal matrix proteins, either as a docking protein or as a releasing factor at the organellar membrane.     

Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction

Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.