Atg36: The Saccharomyces cerevisiae receptor for pexophagy

Eukaryotic cells adapt their organelle composition and abundance according to environmental conditions. Analysis of the peroxisomal membrane protein Pex3 has revealed that this protein plays a crucial role in peroxisome maintenance as it is required for peroxisome formation, segregation and breakdown. Although its function in peroxisome formation and segregation was known to involve its recruitment to the peroxisomal membrane of factors specific for these processes, the role of Pex3 in peroxisome breakdown was unclear until our recent identification of Atg36 as a novel Saccharomyces cerevisiae Pex3-interacting protein. Atg36 is recruited to peroxisomes by Pex3 and is required specifically for pexophagy. Atg36 is distinct from Atg30, the pexophagy receptor identified in Pichia pastoris. Atg36 interacts with Atg11 in vivo, and to a lesser extent with Atg8. These latter proteins link autophagic cargo receptors to the core autophagy machinery. Like other autophagic cargo receptors, Atg36 is a suicide receptor and is broken down in the vacuole together with its cargo. Unlike other cargo receptors, the interaction between Atg36 and Atg8 does not seem to be direct. Our recent findings suggest that Atg36 is a novel pexophagy receptor that may target peroxisomes for degradation via a noncanonical mechanism.     

Atg36

Eukaryotic cells adapt their organelle composition and abundance according to environmental conditions. Analysis of the peroxisomal membrane protein Pex3 has revealed that this protein plays a crucial role in peroxisome maintenance as it is required for peroxisome formation, segregation and breakdown. Although its function in peroxisome formation and segregation was known to involve its recruitment to the peroxisomal membrane of factors specific for these processes, the role of Pex3 in peroxisome breakdown was unclear until our recent identification of Atg36 as a novel Saccharomyces cerevisiae Pex3-interacting protein. Atg36 is recruited to peroxisomes by Pex3 and is required specifically for pexophagy. Atg36 is distinct from Atg30, the pexophagy receptor identified in Pichia pastoris. Atg36 interacts with Atg11 in vivo, and to a lesser extent with Atg8. These latter proteins link autophagic cargo receptors to the core autophagy machinery. Like other autophagic cargo receptors, Atg36 is a suicide receptor and is broken down in the vacuole together with its cargo. Unlike other cargo receptors, the interaction between Atg36 and Atg8 does not seem to be direct. Our recent findings suggest that Atg36 is a novel pexophagy receptor that may target peroxisomes for degradation via a noncanonical mechanism.     

Pex3-anchored Atg36 tags peroxisomes for degradation in Saccharomyces cerevisiae

Peroxisomes undergo rapid, selective autophagic degradation (pexophagy) when the metabolic pathways they contain are no longer required for cellular metabolism. Pex3 is central to the formation of peroxisomes and their segregation because it recruits factors specific for these functions. Here, we describe a novel Saccharomyces cerevisiae protein that interacts with Pex3 at the peroxisomal membrane. We name this protein Atg36 as its absence blocks pexophagy, and its overexpression induces pexophagy. We have isolated pex3 alleles blocked specifically in pexophagy that cannot recruit Atg36 to peroxisomes. Atg36 is recruited to mitochondria if Pex3 is redirected there, where it restores mitophagy in cells lacking the mitophagy receptor Atg32. Furthermore, Atg36 binds Atg8 and the adaptor Atg11 that links receptors for selective types of autophagy to the core autophagy machinery. Atg36 delivers peroxisomes to the preautophagosomal structure before being internalised into the vacuole with peroxisomes. We conclude that Pex3 recruits the pexophagy receptor Atg36. This reinforces the pivotal role played by Pex3 in coordinating the size of the peroxisome pool, and establishes its role in pexophagy in S. cerevisiae.     

Atg37 regulates the assembly of the pexophagic receptor protein complex

Like other selective autophagy pathways, the selective autophagy of peroxisomes, pexophagy, is controlled by receptor protein complexes (RPCs). The pexophagic RPC in Pichia pastoris consists of several proteins: Pex3 and Pex14 ligands in the peroxisomal membrane, Atg30 receptor, Atg11, and Atg17 scaffolds, and the phagophore protein Atg8. Recently, we identified a new component of the pexophagic RPC, Atg37, which is involved in the assembly of this complex. Atg37 is an integral peroxisomal membrane protein (PMP) that binds Pex3 and Atg30, but not Pex14 or Atg8. In the absence of Atg37, the recognition of Pex3 and recruitment of Atg17 by Atg30 are normal. However, the recruitment of Atg11 is severely affected suggesting that the role of Atg37 is to facilitate the Atg30-Atg11 interaction. Palmitoyl-CoA competes with Atg30 for the acyl-CoA binding domain of Atg37 in vitro and might regulate the dynamics of the pexophagic RPC in vivo. The human counterpart of Atg37, ACBD5, also localizes to peroxisomes and is specifically required for pexophagy. Therefore, it is tempting to speculate that ACBD5/ATG37 regulates the assembly of the pexophagic RPC in mammalian cells.     

Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36–Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36–Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.     

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

Macroautophagy/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.     

Up for grabs; trashing peroxisomes

EMBO J 31 13, 2852–2868 (2012); published online May292012Together with the proteasome, autophagy is one of the major catabolic pathways of the cell. In response to cellular needs or environmental cues, this transport route targets specific structures for degradation into the mammalian lysosomes or the yeast and plant vacuoles. The mechanisms allowing exclusive autophagic elimination of unwanted structures are currently the object of intensive investigations. The emerging picture is that there is a series of autophagy receptors that determines the specificity of the different selective types of autophagy. How cargo binding and recognition is regulated by these receptors, however, is largely unknown. In their study, Motley et al (2012) have shed light into the molecular principles underlying the turnover of excess peroxisomes in the budding yeast Saccharomyces cerevisiae.Peroxisomes perform a series of crucial functions and their number is regulated in response to the metabolic demands of the cell. After proliferation and when no more required, a selective type of autophagy called pexophagy degrades superfluous peroxisomes (Manjithaya et al, 2010). This turnover allows the cell to save the energy required for the maintenance of excess organelles and to generate metabolites that can be used to carry out other functions. Like all selective types of autophagy, pexophagy relies on the conserved core of the autophagy-related (Atg) machinery, but also requires additional proteins that confer specificity of the pathway such as cargo selection and membrane dynamics (Manjithaya et al, 2010). It is still unclear, however, which peroxisomal protein allows the recognition of peroxisomes by the autophagosomes. Although Pex3 and Pex14 have previously been indicated as possible suspects (Bellu et al, 2001, 2002; Farre et al, 2008), their specific contribution to pexophagy was difficult to establish. Deletion of either PEX3 or PEX14, as well as most other PEX genes, leads to defects in peroxisome biogenesis, which makes the dissection of their contribution to peroxisome degradation very difficult to assess. Motley et al (2012) have elegantly exploited S. cerevisiae genetics to isolate pex3 alleles specifically impaired in pexophagy and could thus demonstrate that Pex3 (and not Pex14) mediates the selective engulfment of peroxisomes by autophagosomes. In support to this result, the authors have also identified a new protein, Atg36, which binds Pex3 (Figure 1). Importantly, Atg36 interacts with Atg11, an autophagy adaptor involved in numerous selective types of autophagy in yeast, thereby bringing peroxisomes to the site where autophagosomes will be generated and coordinating the activation of the Atg machinery at this location (Kim et al, 2001; Reggiori et al, 2005; Monastyrska et al, 2008). Atg36, however, is only present in S. cerevisiae and related yeasts. Methylotrophic yeasts, in contrast, appear to have a different protein with the same properties, Atg30 (Farre et al, 2008). It is unclear, however, whether Atg30 is the functional counterpart of Atg36 because these two proteins do not display similarities in their amino-acid sequence.Open in a separate windowFigure 1Schematic representation for a putative Pex3 checkpoint. The peroxisomal integral membrane protein Pex3 acts as a master regulator to determine peroxisome fate. Organelle abundance is regulated by formation of new organelles, and their subsequent segregation (inheritance) and degradation. A new paradigm has been uncovered, whereby Pex3 controls peroxisome abundance through the regulated binding to specific co-factors. At the endoplasmic reticulum (ER), together with Pex19, it initiates biogenesis of new peroxisomes. At the peroxisomal membrane, it ensures that both mother and daughter cells obtain the correct number of peroxisomes, whereas when the organelles become dispensable, Pex3 can initiate their selective degradation. To keep peroxisomes in the mother cell during cell division, Pex3 associates with Inp1 and tether peroxisomes to cortical actin patches. Under pexophagy-inducing conditions, Pex3 binds the newly identified pexophagy factor Atg36 and delivers peroxisomes to the site of autophagosome formation for subsequent degradation into the vacuole.While it is unmistakable that Atg36 (and Atg30) is essential for pexophagy, it remains unclear whether this protein is an autophagy receptor. This class of molecules has four characteristics (Kraft et al, 2010). First, each autophagy receptor binds a specific cargo. Second, they often interact with adaptor proteins, which function as scaffolds that bring the cargo–receptor complex in contact with the core Atg machinery to allow the specific sequestration of the cargo. Third, they possess at least one LC3-interacting region (LIR) motif that enables them to interact with the LC3/Atg8 pool present in the interior autophagomes and assures the hermetic enwrapping of the cargo into these vesicles. Fourth, autophagy receptors are degraded in the lysosome/vacuole together with the cargo that they bind to. While Atg36 (and Atg30) binds both the cargo and the adaptor protein Atg11, this protein does not appear to be turned over in the vacuole during pexophagy and a LIR motif has not been pinpointed yet. Consequently, it is unclear whether Atg36 is a new type of autophagy receptor or acts together with a not yet identified autophagy receptor involved in pexophagy.A very interesting concept emerging from the work of Motley et al (2012) is that a single protein, that is, Pex3, could be the central regulator of peroxisome homoeostasis (Figure 1). Pex3 is involved in peroxisome biogenesis, segregation and degradation (Bellu et al, 2002; Hoepfner et al, 2005; Farre et al, 2008; Munck et al, 2009; Ma et al, 2011). As a result, the cell could regulate peroxisome abundance by modulating Pex3 function and/or its array of interactions. In this context, it would be particularly interesting to determine whether Pex3 is also the decision maker of a quality control mechanism that eliminates peroxisomes when not correctly assembled and thus dysfunctional, or when not accurately distributed during cell division. Clearly, additional experiments are needed to understand how Pex3 regulates peroxisome homoeostasis, but this protein and this organelle could represent a convenient system to unveil the principles that regulate the steady-state level of other subcellular compartments.     

Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11

The selective autophagy receptors Atg19 and Atg32 interact with two proteins of the core autophagic machinery: the scaffold protein Atg11 and the ubiquitin‐like protein Atg8. We found that the Pichia pastoris pexophagy receptor, Atg30, also interacts with Atg8. Both Atg30 and Atg32 interactions are regulated by phosphorylation close to Atg8‐interaction motifs. Extending this finding to Saccharomyces cerevisiae, we confirmed phosphoregulation for the mitophagy and pexophagy receptors, Atg32 and Atg36. Each Atg30 molecule must interact with both Atg8 and Atg11 for full functionality, and these interactions occur independently and not simultaneously, but rather in random order. We present a common model for the phosphoregulation of selective autophagy receptors.     

Deficiency of the exportomer components Pex1, Pex6, and Pex15 causes enhanced pexophagy in Saccharomyces cerevisiae

Turnover of damaged, dysfunctional, or excess organelles is critical to cellular homeostasis. We screened mutants disturbed in peroxisomal protein import, and found that a deficiency in the exportomer subunits Pex1, Pex6, and Pex15 results in enhanced turnover of peroxisomal membrane structures compared with other mutants. Strikingly, almost all peroxisomal membranes were associated with phagophore assembly sites in pex1Δ atg1Δ cells. Degradation depended on Atg11 and the pexophagy receptor Atg36, which mediates degradation of superfluous peroxisomes. Mutants of PEX1, PEX6, and PEX15 accumulate ubiquitinated receptors at the peroxisomal membrane. This accumulation has been suggested to trigger pexophagy in mammalian cells. We show by genetic analysis that preventing this accumulation does not abolish pexophagy in Saccharomyces cerevisiae. We find Atg36 is modified in pex1Δ cells even when Atg11 binding is prevented, suggesting Atg36 modification is an early event in the degradation of dysfunctional peroxisomal structures in pex1Δ cells via pexophagy.     

The Role of Snx41-Based Pexophagy in Magnaporthe Development

Pexophagy, the degradation of peroxisomes via selective autophagy, depends on Atg20/Snx42 function in Saccharomyces cerevisiae. Besides its role in selective autophagy, Atg20/Snx42 is also involved in an autophagy-independent endosomal retrieval trafficking, in cooperation with two other sorting nexins, Snx41 and Snx4. Recently, we reported that the sorting nexin MoSnx41, which showed high sequence similarity to yeast Snx41 and Snx42/Atg20 proteins, regulates the gamma-glutamyl cycle and GSH production and is essential for conidiation and pathogenicity in Magnaporthe oryzae. Pexophagy was also found to be defective in Mosnx41Δ mutant. These findings indicate that MoSnx41 likely serves combined functions of Snx42/Atg20 and Snx41 in M. oryzae.. In this study, we performed complementation analyses and demonstrate that MoSnx41 alone serves the dual function of protein sorting (ScSnx41) and pexophagy (ScSnx42/Atg20). To study the potential biological function of pexophagy in fungal pathogenic life cycle, we created deletion mutants of potential pexophagy-specific genes, and characterized them in terms of pexophagy, conidiation and pathogenesis. We identified Pex14 as an essential protein for pexophagy in M. oryzae. Overall, our results show that pexophagy per se is not essential for asexual development or virulence in M. oryzae.     

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.     

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

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

Mechanisms of autophagy and pexophagy in yeasts

Autophagy is a process of recycling of the intracellular constituents using vacuoles (lysosomes). General autophagy occurs due to involvement of highly conservative components found in all eukaryotes, from yeasts to higher plants and humans. Autophagy also could be a selective process and be involved in regulation of the cellular number of organelles, including that of peroxisomes. The process of specific autophagic peroxisome degradation is known as pexophagy. Yeasts appear to be convenient model for studying molecular mechanisms of pexophagy, and most known ATG genes (from the term AuTophaGy) were identified in yeast studies. This review examines characteristics of general autophagy, other types of autophagy as well as pexophagy, in particular, functions of Atg proteins in general autophagy and in macro- and micropexophagy. Special attention is given to mechanisms of phagophore assembly, the role of phosphatidylinositol-3-phosphate in pexophagy, the role of peroxines (proteins involved in peroxisome biogenesis) in pexophagy, as well as properties of Atg proteins specifically involved in micropexophagy.     

Pexophagy-linked degradation of the peroxisomal membrane protein Pex3p involves the ubiquitin–proteasome system

Peroxisome autophagy, also known as pexophagy, describes the wholesale degradation of peroxisomes via the vacuole, when organelles become damaged or redundant. In the methylotrophic yeast Hansenula polymorpha, pexophagy is stimulated when cells growing on methanol are exposed to excess glucose. Degradation of the peroxisomal membrane protein Pex3p, a process that does not involve the vacuole, was shown to trigger pexophagy. In this contribution, we have characterised pexophagy-associated Pex3p degradation further. We show that Pex3p breakdown depends on ubiquitin and confirm that Pex3p is a target for ubiquitination. Furthermore, we identify a role for the peroxisomal E3 ligases Pex2p and Pex10p in Pex3p degradation, suggesting the existence of a ubiquitin-dependent pathway involved in removing proteins from the peroxisomal membrane.     

Pexophagy is responsible for 65% of cases of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) is a group of diseases caused by mutations in one of the peroxins, proteins responsible for biogenesis of the peroxisomes. In recent years, it became clear that many peroxins (e.g., PEX3 and PEX14) play additional roles in peroxisome homeostasis (such as promoting autophagic degradation of peroxisomes or pexophagy), which are often opposite to their originally established functions in peroxisome formation and maintenance. Even more interesting, the peroxins that make up the peroxisomal AAA ATPase complex (AAA-complex) in yeast (Pex1, Pex6 and Pex15) or mammals (PEX1, PEX6, PEX26) are responsible for the downregulation of pexophagy. Moreover, this might be even their primary role in human: to prevent pexophagy by removing from the peroxisomal membrane the ubiquitinated peroxisomal matrix protein import receptor, Ub-PEX5, which is also a signal for the Ub-binding pexophagy receptor, NBR1. Remarkably, the peroxisomes rescued from pexophagy by autophagic inhibitors in PEX1^G843D (the most common PBD mutation) cells are able to import matrix proteins and improve their biochemical function suggesting that the AAA-complex per se is not essential for the protein import function in human. This paradigm-shifting discovery published in the current issue of Autophagy has raised hope for up to 65% of all PBD patients with various deficiencies in the AAA-complex. Recognizing PEX1, PEX6 and PEX26 as pexophagy suppressors will allow treating these patients with a new range of tools designed to target mammalian pexophagy.     

Peroxisome Size Provides Insights into the Function of Autophagy-related Proteins

Autophagy is a major pathway of intracellular degradation mediated by formation of autophagosomes. Recently, autophagy was implicated in the degradation of intracellular bacteria, whose size often exceeds the capacity of normal autophagosomes. However, the adaptations of the autophagic machinery for sequestration of large cargos were unknown. Here we developed a yeast model system to study the effect of cargo size on the requirement of autophagy-related (Atg) proteins. We controlled the size of peroxisomes before their turnover by pexophagy, the selective autophagy of peroxisomes, and found that peroxisome size determines the requirement of Atg11 and Atg26. Small peroxisomes can be degraded without these proteins. However, Atg26 becomes essential for degradation of medium peroxisomes. Additionally, the pexophagy-specific phagophore assembly site, organized by the dual interaction of Atg30 with functionally active Atg11 and Atg17, becomes essential for degradation of large peroxisomes. In contrast, Atg28 is partially required for all autophagy-related pathways independent of cargo size, suggesting it is a component of the core autophagic machinery. As a rule, the larger the cargo, the more cargo-specific Atg proteins become essential for its sequestration.     

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.     

Deficiency of the exportomer components Pex1, Pex6, and Pex15 causes enhanced pexophagy in Saccharomyces cerevisiae

Turnover of damaged, dysfunctional, or excess organelles is critical to cellular homeostasis. We screened mutants disturbed in peroxisomal protein import, and found that a deficiency in the exportomer subunits Pex1, Pex6, and Pex15 results in enhanced turnover of peroxisomal membrane structures compared with other mutants. Strikingly, almost all peroxisomal membranes were associated with phagophore assembly sites in pex1Δ atg1Δ cells. Degradation depended on Atg11 and the pexophagy receptor Atg36, which mediates degradation of superfluous peroxisomes. Mutants of PEX1, PEX6, and PEX15 accumulate ubiquitinated receptors at the peroxisomal membrane. This accumulation has been suggested to trigger pexophagy in mammalian cells. We show by genetic analysis that preventing this accumulation does not abolish pexophagy in Saccharomyces cerevisiae. We find Atg36 is modified in pex1Δ cells even when Atg11 binding is prevented, suggesting Atg36 modification is an early event in the degradation of dysfunctional peroxisomal structures in pex1Δ cells via pexophagy.     

The membrane peroxin PEX3 induces peroxisome-ubiquitination-linked pexophagy

Peroxisomes are degraded by a selective type of autophagy known as pexophagy. Several different types of pexophagy have been reported in mammalian cells. However, the mechanisms underlying how peroxisomes are recognized by autophagy-related machinery remain elusive. PEX3 is a peroxisomal membrane protein (PMP) that functions in the import of PMPs into the peroxisomal membrane and has been shown to interact with pexophagic receptor proteins during pexophagy in yeast. Thus, PEX3 is important not only for peroxisome biogenesis, but also for peroxisome degradation. However, whether PEX3 is involved in the degradation of peroxisomes in mammalian cells is unclear. Here, we report that high levels of PEX3 expression induce pexophagy. In PEX3-loaded cells, peroxisomes are ubiquitinated, clustered, and degraded in lysosomes. Peroxisome targeting of PEX3 is essential for the initial step of this degradation pathway. The degradation of peroxisomes is inhibited by treatment with autophagy inhibitors or siRNA against NBR1, which encodes an autophagic receptor protein. These results indicate that ubiquitin- and NBR1-mediated pexophagy is induced by increased expression of PEX3 in mammalian cells. In addition, another autophagic receptor protein, SQSTM1/p62, is required only for the clustering of peroxisomes. Expression of a PEX3 mutant with substitution of all lysine and cysteine residues by arginine and alanine, respectively, also induces peroxisome ubiquitination and degradation, hence suggesting that ubiquitination of PEX3 is dispensable for pexophagy and an endogenous, unidentified peroxisomal protein is ubiquitinated on the peroxisomal membrane.     

Autophagy-Related Pathways and Specific Role of Sterol Glucoside in Yeasts

Recently, we showed that the requirement of sterol glucoside (SG) during pexophagy in yeasts is dependent on the species and the nature of peroxisome inducers. Atg26, the enzyme that converts sterol to SG, is essential for degradation of very large methanol-induced peroxisomes, but only partly required for degradation of smaller-sized oleate- and amine-induced peroxisomes in Pichia pastoris. Moreover, oleate- and amine-induced peroxisomes of another yeast, Yarrowia lipolytica, are degraded by an Atg26-independent mechanism. The same is true for degradation of oleate-induced peroxisomes in Saccharomyces cerevisiae. Here, we review our findings on the specificity of Atg26 function in pexophagy and extend our observations to the role of SG in the cytoplasm to vacuole targeting (Cvt) pathway and bulk autophagy. The results presented here and elsewhere indicate that Atg26 might increase the efficacy of all autophagy-related pathways in P. pastoris, but not in other yeasts. Recently, it was shown that P. pastoris Atg26 (PpAtg26) is required for elongation of the pre-autophagosomal structure (PAS) into the micropexophagic membrane apparatus (MIPA) during micropexophagy. Therefore, we speculate that SG might facilitate elongation of any double membrane from the PAS and this enhancer function of SG becomes essential when extremely large double membranes are formed.

Addendum to:

The Requirement of Sterol Glucoside for Pexophagy in Yeast Is Dependent on the Species and Nature of Peroxisome Inducers

T.Y. Nazarko, A.S. Polupanov, R.R. Manjithaya, S. Subramani and A.A. Sibirny

Mol Biol Cell 2007; 18:106-18