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 stability of the lactose and citrate plasmids in Lactococcus lactis subsp. lactis biovar. diacetylactis

We have studied the intraperoxisomal location of catalase in peroxisomes of methanol-grown Hansenula polymorpha by (immuno)cytochemical means. In completely crystalline peroxisomes, in which the crystalline matrix is composed of octameric alcohol oxidase (AO) molecules, most of the catalase protein is located in a narrow zone between the crystalloid and the peroxisomal membrane. In non-crystalline organelles the enzyme was present throughout the peroxisomal matrix. Other peroxisomal matrix enzymes studied for comparison, namely dihydroxyacetone synthase, amine oxidase and malate synthase, all were present throughout the AO crystalloid. The advantage of location of catalase at the edges of the AO crystalloids for growth of the organism on methanol is discussed.     

Pexophagy: autophagic degradation of peroxisomes

The abundance of peroxisomes within a cell can rapidly decrease by selective autophagic degradation (also designated pexophagy). Studies in yeast species have shown that at least two modes of peroxisome degradation are employed, namely macropexophagy and micropexophagy. During macropexophagy, peroxisomes are individually sequestered by membranes, thus forming a pexophagosome. This structure fuses with the vacuolar membrane, resulting in exposure of the incorporated peroxisome to vacuolar hydrolases. During micropexophagy, a cluster of peroxisomes is enclosed by vacuolar membrane protrusions and/or segmented vacuoles as well as a newly formed membrane structure, the micropexophagy-specific membrane apparatus (MIPA), which mediates the enclosement of the vacuolar membrane. Subsequently, the engulfed peroxisome cluster is degraded. This review discusses the current state of knowledge of pexophagy with emphasis on studies on methylotrophic yeast species.     

The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes

In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regulated. Isocitrate lyase is induced by growth on C2 compounds and long-chain fatty acids and repressed by glucose. In addition, activity of isocitrate lyase is subject to a second mechanism of catabolite control, glucose-induced inactivation. Here, we demonstrate that the catabolite inactivation of A. nidulans isocitrate lyase, a process that takes place during glucose adaptation of cells grown under gluconeogenic conditions, occurs by proteolysis of the enzyme. Ultrastructural analyses were carried out in order to investigate the cellular processes that govern the catabolite inactivation of this peroxisomal enzyme. Addition of glucose to oleate-induced cells triggered the specific engulfment and sequestration of peroxisomes by the vacuoles. Sequestration of various peroxisomes by a single vacuole was a frequently observed phenomenon. Results obtained by immunoelectron microscopy using antibodies against A. nidulans isocitrate lyase showed that degradation of this peroxisomal enzyme occurred inside the vacuole. In addition, ultrastructural studies demonstrated that microautophagy was the autophagic pathway involved in degradation of redundant peroxisomes during glucose adaptation of oleate-induced cells of A. nidulans.     

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.     

Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver

We isolated membranes from leupeptin-induced autophagic vacuoles and compared them with lysosomal membranes purified from dextran-administered rats. In protein composition, autophagic vacuole membranes prepared from long term-starved (36 h) rats bear marked resemblance to lysosomal membranes, whereas vacuole membranes prepared from short term-starved (12 h) animals differ significantly from lysosomal membranes. Immunoblotting analyses showed that only autophagic vacuole membranes from short term-starved rats possess endoplasmic reticulum markers such as cytochrome P450 and NADPH-cytochrome c reductase. None of the membranes contain sialyltransferase, a Golgi membrane marker. In experiments in which rats were starved after feeding to induce autophagy, the appearance of the endoplasmic reticulum markers occurred during 6-12 h of starvation, concomitantly with increases in vacuolar proteins and sequestered cytosolic aldolase. The endoplasmic reticulum membrane markers and sequestered aldolase declined gradually after 20-36 h of starvation, suggesting that prolonged starvation causes no further increase in the formation of autophagic vacuoles but an increase in the population of matured autophagic vacuoles. Thus, the prominent markers of endoplasmic reticulum from which autophagosomes originate are well preserved in autophagic vacuole membranes, and retention of these markers is highly dependent on the formation and subsequent maturation process of autophagic vacuoles.     

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.     

Early and late molecular events of glucose-induced pexophagy in Pichia pastoris require Vac8

We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like "segmented" extensions that almost completely surrounded the adjacent peroxisomes. Vac8-GFP was found at the vacuolar membrane and concentrated at the base of the arm-like protrusions that extend from the vacuole to sequester the peroxisomes. The localization of Vac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8 resulted in decreased protein stability, impaired vacuolar association and reduced degradation of peroxisomal alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a non-functional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.     

Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(DeltaATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein. This conjugate was not observed in cells expressing Gsa7(DeltaATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.     

Alcohol oxidase expressed under nonmethylotrophic conditions is imported, assembled, and enzymatically active in peroxisomes of Hansenula polymorpha

We have introduced into Hansenula polymorpha an extra copy of its alcohol oxidase gene. This gene which is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter is integrated in a chromosome different from the one containing the endogenous gene. Cells with the extra alcohol oxidase gene, grown on glucose or ethanol as the sole carbon source, express enzymatically active alcohol oxidase. However, other enzymes characteristic for methylotrophic growth conditions are absent or present at low levels. Most of the alcohol oxidase occurs in the octameric state and immuno- and cytochemical evidence shows that it is located in a single enlarged peroxisome per cell. Such peroxisomes show crystalloid inclusions which are lacking in the peroxisomes present in glucose grown control cells. Our results suggest that import into peroxisomes of H. polymorpha, assembly and activation of alcohol oxidase is not conditionally dependent on adaptation to methylotrophic growth conditions and that proliferation of peroxisomes is a well-programmed process that is not triggered solely by overproduction of a peroxisomal protein.     

Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.     

Monitoring autophagy in wheat living cells by visualization of fluorescence protein-tagged ATG8

Autophagy is a highly conserved eukaryotic degradation process during which bulk cytoplasmic materials are transported by double-membrane autophagosomes into the vacuole for degradation. Methods of monitoring autophagy are indispensable in studying the mechanism and functions of autophagy. AuTophaGy-related protein 8 (ATG8) functions in autophagosome assembly by decorating on autophagic membranes, and the inner membrane-bound ATG8 proteins enter the vacuole via active autophagy flux. Fluorescence protein (FP)-tagged forms of ATG8 have been explored as visual markers to monitor autophagy in animals and several plant species. Here, we evaluated and modified this FP-ATG8-based autophagy monitoring method in wheat (Triticum aestivum L.) by fluorescence observation of green fluorescence protein (GFP)-tagged and Discosoma red fluorescent protein (DsRED)-tagged forms of one wheat ATG8, TaATG8h, in wheat mesophyll protoplasts. Under a nutrient-starvation condition, punctate GFP/DsRED- TaATG8h fluorescence representing autophagosomes was clearly observed in the cytoplasm. The accumulation of GFP-TaATG8h-labeled autophagosomes was impaired by the autophagosome biogenesis inhibitor 3-methyladenine but enhanced by the vacuolar degradation inhibitor concanamycin A. In addition, accumulated spreading fluorescence was observed in the vacuole, indicating active autophagy fluxes which led to continuous degradation of GFP/DsRED-TaATG8h fusions and release of protease-tolerant free GFP/DsRED proteins in the vacuole. To observe FP-tagged TaATG8h in other types of wheat cell, we also expressed GFP-TaATG8h in leaf epidermal cells. Consistent with its performance in protoplasts, GFP-TaATG8h showed punctate fluorescence representing autophagosomes in leaf epidermal cells. Taken together, our results proved the feasibility of using FP-tagged ATG8 to monitor both autophagosome accumulation and autophagy flux in living wheat cells.     

Degradative inactivation of the peroxisomal enzyme, alcohol oxidase, during adaptation of methanol-grown Candida boidinii to ethanol.

Adaptation of methanol-grown C. boidinii to ethanol-utilization in non-growing cells resulted in decreased activity of the peroxisomal enzyme alcohol oxidase. Re-appearance of alcohol oxidase activity was dependent on protein synthesis de novo. Degradation of alcohol oxidase protein was shown to parallel the decrease in activity. Adaptation of methanol-grown cells to ethanol-utilization resulted in increased absorbance due to cytochromes and decreased absorbance due to flavoprotein. Decrease in alcohol oxidase activity was associated with loss of the flavin coenzyme, FAD, from the organisms and the appearance of flavins (FAD, FMN, riboflavin) in the surrounding medium. Electron microscopic observations showed that general degradation of whole peroxisomes rather than specific loss of crystalline cores (alcohol oxidase protein) occurred during the adaptation.     

Peroxisome degradation in mammals

This review summarizes the historical aspects of the study of peroxisome degradation in mammalian cells. Peroxisomes have diverse metabolic roles in response to environmental changes and are degraded in a preferential manner, by comparison with cytosolic proteins. This review introduces three hypotheses on the degradation mechanisms: (a) the action of the peroxisome-specific Lon protease; (b) the membrane disruption effect of 15-lipoxygenase; and (c) autophagy that sequesters and degrades the organelles by lysosomal enzymes. Among these hypotheses, autophagy is now recognized as the most important mechanism for excess peroxisome degradation. One of the most striking characteristics of peroxisomes is that they are markedly proliferated in the liver by the administration of hypolipidemic drugs and industrial plasticizers. The effects of these substances were fully reversed after withdrawal of the substances, and most of the excess peroxisomes were selectively degraded and recovered to a normal number and size. Autophagic degradation of peroxisomes has been examined using this characteristic phenomenon. Excessive peroxisome degradation that occurs after cessation of hypolipidemic drugs has been extensively investigated biochemically and morphologically. The evidence shows that the degradation of excess peroxisomes and peroxisomal enzymes is inhibited by 3-methyladenine (3-MA), a specific inhibitor of autophagy. Furthermore, in liver-specific autophagy-deficient mice, rapid removal of peroxisomes was exclusively impaired, and degradation of peroxisomal enzymes was not detected. Thus, the significant contribution of autophagic machinery to peroxisomal degradation in mammals was confirmed. However, the important question of the mechanism for the selective recognition of peroxisomes by autophagosomes remains to be fully elucidated.     

Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha

The subcellular localization of dihydroxyacetone synthase (DHAS) in the methylotrophic yeast Hansenula polymorpha was studied by various biochemical and immunocytochemical methods. After cell fractionation involving differential and sucrose gradient centrifugation of protoplast homogenates prepared from methanol-grown cells, DHAS cosedimented with the peroxisomal enzymes alcohol oxidase and catalase. Electron microscopy of this fraction showed that it contained mainly intact peroxisomes, whereas SDS-polyacrylamide gel electrophoresis revealed two major protein bands (75 and 78 kDa) which were identified as alcohol oxidase and DHAS, respectively. The localization of DHAS in peroxisomes was further established by immunocytochemistry. After immuno-gold staining carried out on ultrathin sections of methanol-grown H. polymorpha using DHAS-specific antibodies, labelling was confined to the peroxisomal matrix.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid - DTT dithiothreitol - SDS sodium dodecyl sulphate - TPP thiamin pyrophosphate - DHAS dihydroxyacetone synthase - GSH reduced glutathione     

Import of alcohol oxidase into peroxisomes of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is unable to grow on methanol because it lacks the enzymes required for its metabolism. To study the possibility of whether or not the methanol oxidation pathway of Hansenula polymorpha can be transferred to S. cerevisiae, the gene coding for alcohol oxidase, a peroxisomal homo-octameric flavoprotein, was introduced into S. cerevisiae. Transformed cells contain varying amounts of alcohol oxidase depending on the plasmid used. Immunocytochemical experiments indicate that the protein is imported into peroxisomes, whether organelle proliferation is induced or not. Cells lack alcohol oxidase activity however, and only the monomeric, non-functional, form of the protein is found. These findings indicate that the H. polymorpha peroxisomal targeting signal of alcohol oxidase is recognized in S. cerevisiae and protein monomers are imported.     

Role of Vac8 in Cellular Degradation Pathways in Pichia pastoris

We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy and mitophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity and GFP-labeled mitochondria in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like “segmented” extensions that almost completely surrounded the adjacent peroxisomes. PpVac8-GFP was found at the vacuolar membrane and concentrated at the base of the sequestering membranes that extend from the vacuole to engulf the peroxisomes. The localization of PpVac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8, resulted in an impaired vacuolar association and decreased degradation of alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a nonfunctional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.     

Autophagy in organelle homeostasis: peroxisome turnover

When cells are confronted with an insufficient supply of nutrients in their extracellular fluid, they may begin to cannibalize some of their internal proteins as well as whole organelles for reuse in the synthesis of new components. This process is termed autophagy and it involves the formation of a double-membrane structure within the cell, which encloses the material to be degraded into a vesicle called an autophagosome. The autophagosome subsequently fuses with a lysosome/vacuole whose hydrolytic enzymes degrade the sequestered organelle. Degradation of peroxisomes is a specific type of autophagy, which occurs in a selective manner and has been mostly studied in yeast. Recently, it was reported that a similar selective process of autophagy occurs in mammalian cells with proliferated peroxisomes. Here we discuss characteristics of the autophagy of peroxisomes in mammalian cells and present a comprehensive model of their likely mechanism of degradation on the basis of known and common elements from other systems.     

Changes in proteinase activities and subcellular distribution during inactivation of alcohol oxidase in Candida boidinii.

Adaptation of methanol-grown Candida boidinii to ethanol utilization was accompanied by an increase in proteolytic activities, which behaved like known vacuolar enzymes. Degradation of alcohol oxidase protein was partially prevented by the serine proteinase inhibitor phenylmethanesulphonyl fluoride, but not by the carboxyl proteinase inhibitor pepstatin. Fractionation of cell-free extracts, by high-speed zonal centrifugation, of methanol-grown C. boidinii showed non-sedimentable and sedimentable proteolytic activities. Naturally occurring inhibitors of vacuolar proteinases were non-sedimentable. Fractionation of extracts prepared from methanol-grown cells which had been adapted to ethanol utilization for 5 h revealed significant changes in the sedimentability and distribution of proteolytic and acid phosphatase activities. These results suggest the possible involvement of a vacuolar process during alcohol oxidase degradation.