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.     

The Hansenula polymorpha ATG25 Gene Encodes a Novel Coiled-Coil Protein that is Required for Macropexophagy

We have isolated the Hansenula polymorpha ATG11 and ATG25 genes, which are both required for glucose-induced selective peroxisome degradation (macropexophagy). ATG11 was identified before in other yeast species and shown to be involved in the Cvt pathway in Saccharomyces cerevisiae and glucose-induced micropexophagy in Pichia pastoris. Our data indicate that HpATG11 is required for macropexophagy. ATG25 represents a novel gene that encodes a 45 kDa coiled-coil protein. We show that this protein co-localizes with Atg11 on a small structure, which most likely represents the pre-autophagosomal structure (PAS). Cells of a constructed ATG25 deletion strain (atg25) displayed relatively slow, continuous degradation of peroxisomes by microautophagy during growth on methanol in the presence of excess nitrogen that also continued after induction of selective peroxisome degradation. This suggests that the processes of selective and non-selective autophagy are dysregulated in atg25 cells.     

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.     

The requirement of sterol glucoside for pexophagy in yeast is dependent on the species and nature of peroxisome inducers

Sterol glucosyltransferase, Ugt51/Atg26, is essential for both micropexophagy and macropexophagy of methanol-induced peroxisomes in Pichia pastoris. However, the role of this protein in pexophagy in other yeast remained unclear. We show that oleate- and amine-induced peroxisomes in Yarrowia lipolytica are degraded by Atg26-independent macropexophagy. Surprisingly, Atg26 was also not essential for macropexophagy of oleate- and amine-induced peroxisomes in P. pastoris, suggesting that the function of sterol glucoside (SG) in pexophagy is both species and peroxisome inducer specific. However, the rates of degradation of oleate- and amine-induced peroxisomes in P. pastoris were reduced in the absence of SG, indicating that P. pastoris specifically uses sterol conversion by Atg26 to enhance selective degradation of peroxisomes. However, methanol-induced peroxisomes apparently have lost the redundant ability to be degraded without SG. We also show that the P. pastoris Vac8 armadillo repeat protein is not essential for macropexophagy of methanol-, oleate-, or amine-induced peroxisomes, which makes PpVac8 the first known protein required for the micropexophagy, but not for the macropexophagy, machinery. The uniqueness of Atg26 and Vac8 functions under different pexophagy conditions demonstrates that not only pexophagy inducers, such as glucose or ethanol, but also the inducers of peroxisomes, such as methanol, oleate, or primary amines, determine the requirements for subsequent pexophagy in yeast.     

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.     

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.     

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.     

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.     

Pex14p is not required for N-starvation induced 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 peroxisome 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, because Pex14p is required in only catalytic amounts to allow initiation of the selective peroxisome degradation process.     

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.     

Cold-inducible selective degradation of peroxisomes in Hansenula polymorpha

Exposure of Hansenula polymorpha cells, grown in batch cultures on methanol at 37 degrees C, to a cold treatment (18 degrees C) is paralleled by a rapid degradation of peroxisomes present in these cells. Remarkably, the events accompanying organelle degradation at 18 degrees C are similar to those of selective glucose-induced peroxisome degradation in wild-type cells, described before. This observation was strengthened by the finding that cold-induced peroxisome degradation was not observed in mutants impaired in selective peroxisome degradation (Atg(-) mutants). Biochemical data indicated that the onset of peroxisome degradation was not triggered by the inactivation of peroxisome function due to the fall in temperature. We show that our findings have implications in case of fluorescence microscopy studies that are generally not conducted at physiological temperatures and thus may lead to strong morphological alterations unless proper precautions are taken.     

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.     

The membrane dynamics of pexophagy are influenced by Sar1p in Pichia pastoris

Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy.     

Tagging Hansenula polymorpha genes by random integration of linear DNA fragments (RALF)

We have investigated the feasibility of using gene tagging by restriction enzyme-mediated integration (REMI) to isolate mutants in Hansenula polymorpha. A plasmid that cannot replicate in H. polymorpha and contains a dominant zeocin resistance cassette, pREMI-Z, was used as the integrative/mutagenic plasmid. We observed that high transformation efficiency was primarily dependent on the use of linearised pREMI-Z, and that the addition of restriction endonuclease to linearised pREMI-Z prior to transformation increased the transformation frequency only slightly. Integration of linearised pREMI-Z occurred at random in the H. polymorpha genome. Therefore, we termed this method Random integration of Linear DNA Fragments (RALF). To explore the potential of RALF in H. polymorpha, we screened a collection of pREMI-Z transformants for mutants affected in peroxisome biogenesis (pex) or selective peroxisome degradation (pdd). Many previously described PEX genes were obtained from the mutant collection, as well as a number of new genes, including H. polymorpha PEX12 and genes whose function in peroxisome biogenesis is still unclear. These results demonstrate that RALF is a powerful tool for tagging genes in H. polymorpha that should make it possible to carry out genome-wide mutagenesis screens.     

Isolation and characterization of mutants impaired in the selective degradation of peroxisomes in the yeast Hansenula polymorpha.

We have isolated a collection of peroxisome degradation-deficient (Pdd-) mutants of the yeast Hansenula polymorpha which are impaired in the selective autophagy of alcohol oxidase-containing peroxisomes. Two genes, designated PDD1 and PDD2, have been identified by complementation and linkage analyses. In both mutant strains, the glucose-induced proteolytic turnover of peroxisomes is fully prevented. The pdd1 and pdd2 mutant phenotypes were caused by recessive monogenic mutations. Mutations mapped in the PDD1 gene appeared to affect the initial step of peroxisome degradation, namely, sequestration of the organelle to be degraded by membrane multilayers. Thus, Pdd1p may be involved in the initial signalling events which determine which peroxisome will be degraded. The product of the PDD2 gene appeared to be essential for mediating the second step in selective peroxisome degradation, namely, fusion and subsequent uptake of the sequestered organelles into the vacuole. pdd1 and pdd2 mutations showed genetic interactions which suggested that the corresponding gene products may physically or functionally interact with each other.     

Glucose-induced and nitrogen-starvation-induced peroxisome degradation are distinct processes in Hansenula polymorpha that involve both common and unique genes

In the methylotrophic yeast Hansenula polymorpha non-selective autophagy, induced by nitrogen starvation, results in the turnover of cytoplasmic components, including peroxisomes. We show that the uptake of these components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast. A selective mode of autophagy in H. polymorpha, namely glucose-induced peroxisome degradation, involves sequestration of individual peroxisomes tagged for degradation by membrane layers that subsequently fuse with the vacuole where the organelle is digested. H. polymorpha pdd mutants are blocked in selective peroxisome degradation. We observed that pdd1-201 is also impaired in non-selective autophagy, whereas this process still normally functions in pdd2-4. These findings suggest that mechanistically distinct processes as selective and non-selective autophagy involve common but also unique genes.     

Excess peroxisomes are degraded by autophagic machinery in mammals

Peroxisomes are degraded by autophagic machinery termed "pexophagy" in yeast; however, whether this is essential for peroxisome degradation in mammals remains unknown. Here we have shown that Atg7, an essential gene for autophagy, plays a pivotal role in the degradation of excess peroxisomes in mammals. Following induction of peroxisomes by a 2-week treatment with phthalate esters in control and Atg7-deficient livers, peroxisomal degradation was monitored within 1 week after discontinuation of phthalate esters. Although most of the excess peroxisomes in the control liver were selectively degraded within 1 week, this rapid removal was exclusively impaired in the mutant liver. Furthermore, morphological analysis revealed that surplus peroxisomes, but not mutant hepatocytes, were surrounded by autophagosomes in the control. Our results indicated that the autophagic machinery is essential for the selective clearance of excess peroxisomes in mammals. This is the first direct evidence for the contribution of autophagic machinery in peroxisomal degradation in mammals.     

Atg26 is not involved in autophagy-related pathways in Saccharomyces cerevisiae

Autophagy is a degradative pathway conserved among eukaryotes. It is a major route for degradation of long-lived proteins and entire organelles, such as peroxisomes. Atg26, a sterol glucosyltransferase, is specifically required for micro- and macropexophagy, but not for starvation-induced bulk autophagy in Pichia pastoris. Here we study the requirement of Saccharomyces cerevisiae Atg26 in the Cvt pathway, nonspecific autophagy and pexophagy. Our results show that the S. cerevisiae atg26Delta strain is not defective in prApe1 maturation, macroautophagy or peroxisome degradation, in contrast to the situation seen in Pichia pastoris. These studies highlight the importance of examining mutants in multiple organisms.