Selective Degradation Of Peroxisomes Research Articles

Mycobacterium tuberculosis Rv3034c regulates mTORC1 and PPAR-γ dependant pexophagy mechanism to control redox levels in macrophages.

Mycobacterium tuberculosis survives inside the macrophages by employing several host immune evasion strategies. Here, we reported a novel mechanism in which M. tuberculosis acetyltransferase, encoded by Rv3034c, induces peroxisome homeostasis to regulate host oxidative stress levels to facilitate intracellular mycobacterial infection. Presence of M. tuberculosis Rv3034c induces the expression of peroxisome biogenesis and proliferation factors such as Pex3, Pex5, Pex19, Pex11b, Fis-1 and DLP-1; while depletion of Rv3034c decreased the expression of these molecules, thereby selective degradation of peroxisomes via pexophagy. Further studies revealed that M. tuberculosis Rv3034c inhibit induction of pexophagy mechanism by down-regulating the expression of pexophagy associated proteins (p-AMPKα, p-ULK-1, Atg5, Atg7, Beclin-1, LC3-II, TFEB and Keap-1) and adaptor molecules (NBR1 and p62). Inhibition was found to be dependent on the phosphorylation of mTORC1 and activation of peroxisome proliferator activated receptor-γ. In order to maintain intracellular homeostasis during oxidative stress, M. tuberculosis Rv3034c was found to induce degradation of dysfunctional and damaged peroxisomes through activation of Pex14 in infected macrophages. In conclusion, this is the first report which demonstrated that M. tuberculosis acetyltransferase regulate peroxisome homeostasis in response to intracellular redox levels to favour mycobacterial infection in macrophage.

Autophagic Machinery of Plant Peroxisomes.

Peroxisomes are cell organelles that play an important role in plants in many physiological and developmental processes. The plant peroxisomes harbor enzymes of the β-oxidation of fatty acids and the glyoxylate cycle; photorespiration; detoxification of reactive oxygen and nitrogen species; as well as biosynthesis of hormones and signal molecules. The function of peroxisomes in plant cells changes during plant growth and development. They are transformed from organelles involved in storage lipid breakdown during seed germination and seedling growth into leaf peroxisomes involved in photorespiration in green parts of the plant. Additionally, intensive oxidative metabolism of peroxisomes causes damage to their components. Therefore, unnecessary or damaged peroxisomes are degraded by selective autophagy, called pexophagy. This is an important element of the quality control system of peroxisomes in plant cells. Despite the fact that the mechanism of pexophagy has already been described for yeasts and mammals, the molecular mechanisms by which plant cells recognize peroxisomes that will be degraded via pexophagy still remain unclear. It seems that a plant-specific mechanism exists for the selective degradation of peroxisomes. In this review, we describe the physiological role of pexophagy in plant cells and the current hypotheses concerning the mechanism of plant pexophagy.

Catalase inhibition induces pexophagy through ROS accumulation

Peroxisomes are dynamic and multifunctional organelles involved in various cellular metabolic processes, and their numbers are tightly regulated by pexophagy, a selective degradation of peroxisomes through autophagy to maintain peroxisome homeostasis in cells. Catalase, a major peroxisome protein, plays a critical role in removing peroxisome-generated reactive oxygen species (ROS) produced by peroxisome enzymes, but the contribution of catalase to pexophagy has not been reported. Here, we investigated the role of catalase in peroxisome degradation during nutrient deprivation. Both short interfering RNA-mediated silencing of catalase and pharmacological inhibition by 3-aminotriazole (3AT) decreased the number of peroxisomes and resulted in the downregulation of peroxisomal proteins, such as PMP70 and PEX14 under serum starvation. In addition, treatment with 3AT induced NBR1-dependent autophagy and PEX5 ubiquitination in the absence of serum, which was accompanied by accumulation of ROS. Co-treatment with antioxidant agent N-acetyl-l-cysteine (NAC) prevented ROS accumulation and pexophagy by modulating peroxisome protein levels and the association of NBR1, a pexophagy receptor with peroxisomes. Taken together, these findings demonstrate that catalase plays an important role in pexophagy during nutrient deprivation.

Pex3 and Atg37 compete to regulate the interaction between the pexophagy receptor, Atg30, and the Hrr25 kinase

ABSTRACTMacroautophagy/autophagy is a highly conserved process in which subcellular components destined for degradation are sequestered within autophagosomes. The selectivity of autophagy is determined by autophagy receptors, such as Pichia pastoris Atg30 (autophagy-related 30), which controls the selective degradation of peroxisomes (pexophagy) through the assembly of a receptor-protein complex (RPC). Previously, we proved that the peroxisomal acyl-CoA-binding protein, Atg37, and the highly conserved peroxin, Pex3, are required for RPC formation and efficient pexophagy. Here, we describe how Atg37 and Pex3 regulate the assembly and activation of the pexophagic RPC. We demonstrate that Atg30 requires both Atg37 and Pex3 to recruit Atg8 and Atg11 to the pexophagic RPC, because Atg37 depends on Pex3 for its localization at the peroxisomal membrane. We establish that due to close proximity of Atg37- and Pex3-binding sites in the middle domain of Atg30, the binding of these proteins to Atg30 is mutually exclusive within this region. We also show that direct binding of Pex3 and Atg37 to Atg30 regulates its phosphorylation by the Hrr25 kinase, negatively and positively, respectively. Based on these results we present a model that clarifies the assembly and activation of the pexophagic RPC through the phosphoregulation of Atg30.

2,2′-dipyridyl induces pexophagy

Pexophagy is the selective degradation of peroxisomes for maintaining peroxisome homeostasis within cells. Peroxisome dynamics and pexophagy are important events required to maintain the quality control of peroxisomes, thereby preventing peroxisome-associated diseases. To identify novel pexophagy modulators, we developed a cell-based screening system and selected 2,2′-dipyridyl (2,2-DP) as a candidate molecule. 2,2-DP treatment induced peroxisome degradation as evidenced by an increased number of low-pH autolysosomes originating from peroxisomes and a decrease in the expression of peroxisomal proteins such as catalase, Pex14, and PMP70. The phenotype was defined as pexophagy, because 2,2-DP induced autophagy and inhibition of autophagy significantly reduced the degree of peroxisome degradation. Mechanistically, 2,2-DP-dependent pexophagy seemed to be mediated by iron chelation, since another iron chelator displayed a similar effect on pexophagy, but a copper chelator did not. Notably, iron replenishment prevented 2,2-DP-mediated pexophagy. Taken together, our results suggest that 2,2-DP treatment disrupts peroxisome dynamics and promotes pexophagy through iron depletion.

Peroxisome homeostasis: Mechanisms of division and selective degradation of peroxisomes in mammals

Peroxisome number and quality are maintained by its biogenesis and turnover and are important for the homeostasis of peroxisomes. Peroxisomes are increased in number by division with dynamic morphological changes including elongation, constriction, and fission. In the course of peroxisomal division, peroxisomal morphogenesis is orchestrated by Pex11β, dynamin-like protein 1 (DLP1), and mitochondrial fission factor (Mff). Conversely, peroxisome number is reduced by its degradation. Peroxisomes are mainly degraded by pexophagy, a type of autophagy specific for peroxisomes. Upon pexophagy, an adaptor protein translocates on peroxisomal membrane and connects peroxisomes to autophagic machineries. Molecular mechanisms of pexophagy are well studied in yeast systems where several specific adaptor proteins are identified. Pexophagy in mammals also proceeds in a manner dependent on adaptor proteins. In this review, we address the recent progress in studies on peroxisome morphogenesis and pexophagy. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann.

Autophagy in plants and algae.

Autophagy in plants and algae.

The peroxisomal receptor dislocation pathway: To the exportomer and beyond

The biogenesis of peroxisomes is an ubiquitin-dependent process. In particular, the import of matrix proteins into the peroxisomal lumen requires the modification of import receptors with ubiquitin. The matrix proteins are synthesized on free polyribosomes in the cytosol and are recognized by import receptors via a peroxisomal targeting sequence (PTS). Subsequent to the transport of the receptor/cargo-complex to the peroxisomal membrane and the release of the cargo into the peroxisomal lumen, the PTS-receptors are exported back to the cytosol for further rounds of matrix protein import. The exportomer represents the molecular machinery required for the retrotranslocation of the PTS-receptors. It comprises enzymes for the ubiquitination as well as for the ATP-dependent extraction of the PTS-receptors from the peroxisomal membrane. Furthermore, recent evidence indicates a mechanistic interconnection of the ATP-dependent removal of the PTS-receptors with the translocation of the matrix protein into the organellar lumen. Interestingly, the components of the peroxisomal exportomer seem also to be involved in cellular tasks that are distinct from the ubiquitination and dislocation of the peroxisomal PTS-receptors. This includes work that indicates a central function of this machinery in the export of peroxisomal matrix proteins in plants, while a subset of exportomer components is involved in the meiocyte formation in some fungi, the peroxisome-chloroplast contact during photorespiration in plants and possibly even the selective degradation of peroxisomes via pexophagy. In this review, we want to discuss the central role of the exportomer during matrix protein import, but also highlight distinct roles of exportomer constituents in additional cellular processes. This article is part of a Special Issue entitled: Peroxisomes: biogenesis, functions and diseases.

Molecular mechanisms of selective autophagy

Autophagy is the catabolic process by which the cell degrades cytoplasmic components within lysosomes.1 The balance between the biosynthesis of cellular components and their eventual degradation is of cardinal importance for normal cellular homeostasis and health.2 Autophagy has long been thought to be an essential but non-selective bulk degradation pathway. However, recently accumulating evidence has highlighted the selective elimination by autophagy of unwanted components such as aberrant protein aggregates, lipid droplets, dysfunctional organelles and invading pathogens.3 Some of the molecular components involved in selective autophagy have been identified, implying that we are beginning to understand how selectivity is achieved in this process. Specific autophagy receptors are responsible for selective autophagy by tethering cargo to the site of autophagosomal engulfment.4 In addition, it is becoming clear that post-translational modifications have an important role in ensuring substrate recognition and selectivity in vertebrates.5 For example, protein ubiquitination, besides its role in proteasome-mediated degradation, can constitute a modification that targets proteins to turnover by autophagy.5 The recognition of ubiquitinated substrates is provided by molecular adaptors including p62, NBR1, NDP52, VCP and optineurin, which bind on one side to ubiquitin and, on the other end, to autophagosome-specific proteins, such as the members of the LC3/GABARAP/Gate16 family.4 So far, several autophagy-‘specific' receptors and adaptor proteins that regulate the selective degradation of damaged organelles, protein aggregate and pathogens have been identified and partially characterized.4, 6 However, the physiological roles of selective autophagy are not yet fully understood. In this thematic issue, five reviews summarize the mechanistic and structural basis of receptor-mediated selective autophagy. Shaid et al.7 focus on the role of ubiquitinylation signals and selective autophagy receptors in selecting cargo for autophagic degradation. Suzuki8 provides an overview of selective autophagy in yeast, concentrating on Atg11-dependent pathways. In particular, Atg11 appears to be important for organelle-selective autophagy, such as mitophagy (selective degradation of mitochondria), pexophagy (selective degradation of peroxisomes) and piecemeal microautophagy of the nucleus. In contrast, ribophagy (ribosomal degradation) could represent a non-selective and Atg-11-independent autophagic pathway.8 Ashrafi and Schwarz9 describe the state of the art in the process of mitophagy. The selective autophagy of mitochondria is probably the only quality control mechanism that allows for eliminating these organelles upon irreversible damage. Mitophagy also mediates the removal of superfluous mitochondria from differentiating erythrocytes, and contributes to the exclusively maternal inheritance of mitochondrial DNA via the elimination of sperm-derived mitochondria.9 Liu and Czaja10 analyse ‘lipophagy', a recently identified alternative pathway of lipid degradation mediated by lysosomal hydrolases. In this very specific form of selective autophagy, triglycerides and cholesterol contained in lipid droplet are taken up by autophagosomes and delivered to lysosomes for degradation by acidic hydrolases. Intracellular lipids can regulate levels of autophagy by yet unknown mechanisms. Interestingly, impaired lipophagy can promote excessive tissue lipid accumulation such as hepatic steatosis induced by viral infections,11 alter hypothalamic neuropeptide release to affect body mass,12 block cellular transdifferentiation and sensitize cells to death stimuli.13 Finally, Isakson et al.14 discuss the current knowledge of the most prominent mammalian adaptor protein identified thus far, autophagy-linked FYVE protein (ALFY). ALFY is a large, scaffolding, multidomain protein implicated in the selective degradation of ubiquitinated protein aggregates by autophagy.14 The avid reader of this collection of excellent reviews will appreciate that several major problems remain to be solved. Which are the early upstream signals triggering different types of selective autophagy? Do different stress/damage agents activate selective autophagy through common or distinct receptor proteins? How can a balanced crosstalk be achieved between selective autophagy and programmed cell death pathways? We anticipate that answering these questions will yield clinically useful information because autophagy has an essential role in removing potentially harmful intracellular components. Thus, impaired or disabled autophagy has been linked to many human pathologies including cancer, neurodegeneration, maladaptive immune responses and premature ageing.15 In keeping with this, we hope that this CDD review set will contribute to stimulating the interest of the scientific community in this ever more intriguing research field.

Identification of phosphorylation sites in Hansenula polymorpha Pex14p by mass spectrometry

Pex14p is a peroxisomal membrane protein that is involved in both peroxisome biogenesis and selective peroxisome degradation. Previously, we showed that Hansenula polymorpha Pex14p was phosphorylated in vivo. In this study, we identified its phosphorylation site by mass spectrometry. Recombinant His-tagged Pex14p (H6-Pex14p) was overexpressed and purified from the yeast. The protein band corresponding to H6-Pex14p was in-gel digested with trypsin and subjected to LC/MS. As a result of LC/MS, Thr248 and Ser258 were identified as the phosphorylated sites. To confirm the phosphorylation sites and explore its functions, we made Ala mutants of the candidate amino acids. In the western blot analysis with anti-Pex14p, S258A mutant gave doublet bands while wild type (WT) and T248A mutants gave triplet bands. Moreover, the double mutant (T248A/S258A) gave a single band. WT and all mutant Pex14p labeled with [32P] orthophosphate were immunoprecipitated and analyzed by autoradiography. The phosphorylation of Pex14p was suppressed in S258A mutant, but enhanced in T248A mutant compared to WT. Moreover, the phosphorylated Pex14p was not detected in the T248A/S258A double mutant. All mutants were able to grow on methanol and their matrix proteins (alcohol oxidase and amine oxidase) were mostly localized in peroxisomes. Furthermore all mutants showed selective degradation of peroxisome like WT during the glucose-induced macropexophagy.

Selective Types of Autophagy

The focus of this special issue of the International Journal of Cell Biology is to underscore the recent developments in the field of macroautophagy and how this degradative pathway intersects with cellular metabolism, complex physiological functions, and human diseases. During the last decade, autophagy has become an expanding field in biomedical life sciences due to its involvement with numerous intracellular processes. Autophagy also plays a role in pathology, and it has the therapeutic potential to be the target for the treatment of specific human diseases. Early studies suggested that autophagy was a nonselective process in which cytoplasmic structures were randomly sequestered into autophagosomes before being delivered to the mammalian lysosome or the plant and yeast vacuole for degradation. Now there is growing evidence that unwanted cellular structures can be selectively recognized and exclusively eliminated within cells (F. Reggiori et al., “Selective types of autophagy”). This is achieved through the action of specific autophagy receptors, as reviewed by C. Behrends and S. Fulda in “Receptor proteins in selective autophagy”) and studied by K. Marchbank et al. “MAP1B interaction with the FW domain of the autophagic receptor Nbr1 facilitates its association to the microtubule network”. Thus excess or damaged organelles including mitochondria (A. May et al., “The many faces of mitochondrial autophagy: making sense of contrasting observations in recent research”; Y. Hirota et al., “The physiological role of mitophagy: new insights into phosphorylation events”), peroxisomes (A. Till et al., “Pexophagy: the selective degradation of peroxisomes”), lipid droplets (R. Singh and A. Cuervo, “Lipophagy: connecting autophagy and lipid metabolism”), endoplasmic reticulum and ribosomes (E. Cebollero et al., “Reticulophagy and ribophagy: regulated degradation of protein production factories”) can be specifically sequestered by autophagosomes and targeted to the lysosome for degradation. Importantly, there is growing evidence that selective autophagy subtypes also have a wide range of physiological functions. In yeast, the cytosol-to-vacuole (Cvt) pathway transports hydrolases into the vacuole, which is reviewed by M. Umekawa and D. Klionsky in “The cytoplasm-to-vacuole targeting pathway: a historical perspective”. In eukaryotes, autophagy plays a central role in both innate and acquired immunity. Further sequestration and elimination of invading pathogens such as Salmonella and Staphylococcus aureus have been exploited to study autophagosome biogenesis (T. Noda et al., “Three-axis model for Atg recruitment in autophagy against Salmonella”; M. Mauthe et al., “WIPI-1 positive autophagosome-like vesicles entrap pathogenic Staphylococcus aureus for lysosomal degradation”). In pancreas cells, autophagy has recently been shown to specifically turn over secretory granules, as described by M. Vaccaro in “Zymophagy: selective autophagy of secretory granules”. Dysregulation of autophagic function has been implicated in a growing list of disease processes and has underscored the selective or substrate-specific versions of the pathway. Examples in this special issue include the clearance of aggregates associated with neurological diseases, as reviewed by T. Lamark and T. Johansen in “Aggrephagy: selective disposal of protein aggregates by macroautophagy” and by I. Nezis in “Selective autophagy in Drosophila”. In terms of cancer biology, autophagy has been viewed as having dual roles in both tumor suppression and progression. K. Hughson et al. in “Implications of therapy-induced selective autophagy on tumor metabolism and survival” review how activation of autophagy selective forms can be used as a potential therapeutic approach for the treatment of specific cancers. Adding to the complexity of autophagic function and regulation, the article by K. Juenemann and E. Reits “Alternative macroautophagic pathways” explores alternative macroautophagic pathways that are independent of key core autophagy components such as Beclin-1 or Atg5. We expect future research on the mechanism and regulation of selective autophagy, and the physiological importance of this pathway in human disease will be very exciting and expand on the findings highlighted in this issue of IJCB. Fulvio Reggiori Maasaki Komatsu Kim Finley Anne Simonsen

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.

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 (Pex14pPi) that are differently induced during peroxisome proliferation. The data suggest that Pex14p is required for peroxisome biogenesis during organelle proliferation and Pex14pPi 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.

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.

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.

Cold-inducible selective degradation of peroxisomes in

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.

Atg8 is Essential for Macropexophagy in Hansenula polymorpha

We have isolated a peroxisome-degradation-deficient (pdd) mutant of the methylotrophic yeast Hansenula polymorpha via gene tagging mutagenesis. Sequencing revealed that the mutant was affected in the HpATG8 gene. HpAtg8 is a protein with high sequence similarity to both Pichia pastoris and Saccharomyces cerevisiae Atg8 and appeared to be essential for selective peroxisome degradation (macropexophagy) and nitrogen-limitation induced microautophagy. Fluorescence microscopy revealed that a GFP.Atg8 fusion protein was located close to the vacuole. After induction of macropexophagy, the GFP.Atg8 containing spot extended to engulf an individual peroxisome. In cells of a constructed deletion strain, sequestration of individual organelles was never completed; analysis of series of serial sections revealed that invariably a minor diaphragm-like opening remained. We hypothesize that H. polymorpha Atg8 facilitates sealing of the sequestering membranes during selective peroxisome degradation.

Hansenula polymorpha Tup1p is important for peroxisome degradation.

In the yeast Hansenula polymorpha peroxisomes are selectively degraded upon a shift of cells from methanol to glucose-containing media. We identified the H. polymorpha TUP1 gene by functional complementation of the peroxisome degradation deficient mutant pdd2-4. Tup1 proteins function in transcriptional repression of specific sets of genes involved in various cellular processes. Our combined data indicate that H. polymorpha TUP1 is involved in regulation of the switch between peroxisome biogenesis and selective degradation. The initial DNA fragment that complemented H. polymorpha pdd2-4 contained a second gene, encoding H. polymorpha Vps4p. Deletion of the VPS4 gene did not affect selective peroxisome degradation.

Transcriptional Down-regulation of Peroxisome Numbers Affects Selective Peroxisome Degradation in Hansenula polymorpha

We have isolated and characterized a novel transcription factor of Hansenula polymorpha that is involved in the regulation of peroxisomal protein levels. This protein, designated Mpp1p, belongs to the family of Zn(II)2Cys6 proteins. In cells deleted for the function of Mpp1p the levels of various proteins involved in peroxisome biogenesis (peroxins) and function (enzymes) are reduced compared with wild type or, in the case of the matrix protein dihydroxyacetone synthase, fully absent. Also, upon induction of mpp1 cells on methanol, the number of peroxisomes was strongly reduced relative to wild type cells and generally amounted to one organelle per cell. Remarkably, this single organelle was not susceptible to selective peroxisome degradation (pexophagy) and remained unaffected during exposure of methanol-induced cells to excess glucose conditions. We show that this mechanism is a general phenomenon in H. polymorpha in the case of cells that contain only a single peroxisome.

Selective degradation of peroxisomes in yeasts.

In the last two decades, much progress has been made in understanding the process of induction and biogenesis of peroxisomes, essential organelles in all eukaryotes. Only relatively recently, the first molecular studies on the selective degradation of this important organelle-a process known as pexophagy, which occurs when the organelles have become redundant-have been performed, especially using methylotrophic yeasts. The finding that pexophagy and other transport pathways to the vacuole (vacuolar protein sorting, autophagy, cytoplasm-to-vacuole-targeting and endocytosis) utilize common but also unique genes has placed pexophagy in the heart of the machinery that recycles cellular material. The quest is now on to understand how peroxisome degradation has become such a highly selective process and what the signals are that trigger it. In addition, because the prime determinant of pexophagy is located on the peroxisome itself, it has become essential to study the role of peroxisomal membrane proteins in the degradation process in detail. This review highlights the main achievements of the last years.