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.     

Highly Oxidized Peroxisomes Are Selectively Degraded via Autophagy in Arabidopsis

The positioning of peroxisomes in a cell is a regulated process that is closely associated with their functions. Using this feature of the peroxisomal positioning as a criterion, we identified three Arabidopsis thaliana mutants (peroxisome unusual positioning1 [peup1], peup2, and peup4) that contain aggregated peroxisomes. We found that the PEUP1, PEUP2, and PEUP4 were identical to Autophagy-related2 (ATG2), ATG18a, and ATG7, respectively, which are involved in the autophagic system. The number of peroxisomes was increased and the peroxisomal proteins were highly accumulated in the peup1 mutant, suggesting that peroxisome degradation by autophagy (pexophagy) is deficient in the peup1 mutant. These aggregated peroxisomes contained high levels of inactive catalase and were more oxidative than those of the wild type, indicating that peroxisome aggregates comprise damaged peroxisomes. In addition, peroxisome aggregation was induced in wild-type plants by exogenous application of hydrogen peroxide. The cat2 mutant also contained peroxisome aggregates. These findings demonstrate that hydrogen peroxide as a result of catalase inactivation is the inducer of peroxisome aggregation. Furthermore, an autophagosome marker, ATG8, frequently colocalized with peroxisome aggregates, indicating that peroxisomes damaged by hydrogen peroxide are selectively degraded by autophagy in the wild type. Our data provide evidence that autophagy is crucial for quality control mechanisms for peroxisomes in Arabidopsis.     

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.     

稻瘟病菌过氧化物酶体产生与降解的关键基因

过氧化物酶体(peroxisomes)是真核细胞中一类单层膜包被的细胞器,参与多种生化代谢.过氧化物酶体起源于内质网,过氧化物酶体形成相关的蛋白称为Peroxin,其编码基因通常写作PEX.细胞中过氧化物酶体的选择性消解称为过氧化物酶体自噬(pexophagy).参与细胞自噬(autophagy)的基因(ATG)大多参与过氧化物酶体自噬.近年来,丝状真菌中过氧化物酶体形成与降解机制的研究进展迅速,相关基因不断被鉴定.本文对相关研究进行了简要评述,并以稻瘟病菌为例,对丝状真菌基因组中可能的PEX和ATG基因进行了检索.发现稻瘟病菌中存在除PEX15,PEX17,PEX18,PEX21,PEX22,ATG19,ATG25,ATG30和ATG31之外的大多数PEX和ATG基因;同时,还存在多个丝状真菌特有的基因.说明过氧化物酶体的产生与消解在酵母、丝状真菌与哺乳动物之间相对保守,同时又各具特性.     

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.     

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.     

Atg26-Mediated Pexophagy Is Required for Host Invasion by the Plant Pathogenic Fungus Colletotrichum orbiculare

The number of peroxisomes in a cell can change rapidly in response to changing environmental and physiological conditions. Pexophagy, a type of selective autophagy, is involved in peroxisome degradation, but its physiological role remains to be clarified. Here, we report that cells of the cucumber anthracnose fungus Colletotrichum orbiculare undergo peroxisome degradation as they infect host plants. We performed a random insertional mutagenesis screen to identify genes involved in cucumber pathogenesis by C. orbiculare. In this screen, we isolated a homolog of Pichia pastoris ATG26, which encodes a sterol glucosyltransferase that enhances pexophagy in this methylotrophic yeast. The C. orbiculare atg26 mutant developed appressoria but exhibited a specific defect in the subsequent host invasion step, implying a relationship between pexophagy and fungal phytopathogenicity. Consistent with this, its peroxisomes are degraded inside vacuoles, accompanied by the formation of autophagosomes during infection-related morphogenesis. The autophagic degradation of peroxisomes was significantly delayed in the appressoria of the atg26 mutant. Functional domain analysis of Atg26 suggested that both the phosphoinositide binding domain and the catalytic domain are required for pexophagy and pathogenicity. In contrast with the atg26 mutant, which is able to form appressoria, the atg8 mutant, which is defective in the entire autophagic pathway, cannot form normal appressoria in the earlier steps of morphogenesis. These results indicate a specific function for Atg26-enhanced pexophagy during host invasion by C. orbiculare.     

植物ATG8结合蛋白

ATG8（自噬相关蛋白8）结合蛋白通过ATG8相互作用基序（ATG8 interaction motif,AIM）或泛素相互作用基序（ubiquitin interaction motif,UIM）与ATG8相互作用,在自噬、选择性自噬和非自噬过程中起关键作用。ATG8结合蛋白在酵母和哺乳动物研究中取得了巨大进展,但在植物领域仍然滞后。本文首先概括了植物ATG8蛋白结构及特征,其次,重点阐述了作为植物选择性自噬受体的ATG8结合蛋白的结构和功能,最后,总结了参与自噬小体闭合、转运和人工合成ATG8结合蛋白研究状况。本文结合最新研究,系统总结了目前发现的植物ATG8结合蛋白结构和功能,以期为植物选择性自噬和自噬的研究提供新思路。     

Disrupting Autophagy Restores Peroxisome Function to an Arabidopsis lon2 Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.     

The autophagy genes atg8 and atg1 affect morphogenesis and pathogenicity in Ustilago maydis

Autophagy is a complex degradative process in which cytosolic material, including organelles, is randomly sequestered within double‐membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, the autophagy genes ATG1 and ATG8 are crucial for autophagy induction and autophagosome assembly, respectively, and their deletion has an impact on the autophagic potential of the corresponding mutant strains. We were interested in the role of autophagy in the development and virulence of U. maydis. Using a reverse genetic approach, we showed that the U. maydis ATG8 orthologue, atg8, is associated with autophagy‐dependent processes. Deletion of atg8 abolished autophagosome accumulation in the vacuoles of carbon‐starved cells and drastically reduced the survival of U. maydisΔatg8 mutant strains during these conditions. In addition, atg8 deletion had an impact on the budding process during saprobic haploid growth. The infection of maize with compatible Δatg8 strains resulted in fewer galled plants, and fungal gall colonization was strongly reduced, as reflected by the very low hyphal density in these tissues. Δatg8 infections resulted in the formation of very few teliospores. To corroborate the role of autophagy in U. maydis development, we also deleted the ATG1 orthologue, atg1. Deletion of atg1 yielded phenotypes similar to the Δatg8 strains during saprobic growth, but of lower magnitude. The Δatg1 strains were only slightly less pathogenic than the wild‐type and teliospore production was not affected. Surprisingly, atg1 deletion in the Δatg8 background exacerbated those phenotypes already observed in the Δatg8 and Δatg1 single‐mutant strains, strongly suggesting an additive phenotype. In particular, the double mutant was completely suppressed for plant gall induction.     

Proteins involved in microbody biogenesis and degradation in Aspergillus nidulans

Fungal microbodies (peroxisomes) are inducible organelles that proliferate in response to nutritional cues. Proteins involved in peroxisome biogenesis/proliferation are designated peroxins and are encoded by PEX genes. An autophagy-related process, termed pexophagy, is responsible for the selective removal of peroxisomes from the cell. Several genes involved in pexophagy are also required for autophagy and are collectively known as ATG genes. We have re-analysed the Aspergillus nidulans genome for the presence of PEX and ATG genes and have identified a number of previously missed genes. Also, we manually determined the correct intron positions in each identified gene. The data show that in A. nidulans and related fungi the basic set of genes involved in peroxisome biogenesis or degradation are conserved. However, both processes have features that more closely resemble organelle formation/degradation in mammals rather than yeast. Thus, filamentous fungi like A. nidulans are ideal model systems for peroxisome homeostasis in man.     

Effect of mutation of C-terminal and heme binding region of Arabidopsis catalase on the import to peroxisomes

We evaluated the import of Arabidopsis catalase to peroxisomes under homogenous transient expression. The amino acids at ?11 to ?4 from the C-terminus are necessary for catalase import. The results are in agreement with the previous work under stable expression. We first demonstrate that heme-binding sites are important for peroxisomal import, suggesting the importance of catalase folding.

Abbreviations: AtCat: Arabidopsis catalase; PTS: peroxisomal targeting signal; PEX: Peroxin     

ATG8 lipidation and ATG8‐mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci

Autophagic recycling of intracellular plant constituents is maintained at a basal level under normal growth conditions but can be induced in response to nutritional demand, biotic stress, and senescence. One route requires the ubiquitin‐fold proteins Autophagy‐related (ATG)‐8 and ATG12, which become attached to the lipid phosphatidylethanolamine (PE) and the ATG5 protein, respectively, during formation of the engulfing vesicle and delivery of its cargo to the vacuole for breakdown. Here, we genetically analyzed the conjugation machinery required for ATG8/12 modification in Arabidopsis thaliana with a focus on the two loci encoding ATG12. Whereas single atg12a and atg12b mutants lack phenotypic consequences, atg12a atg12b double mutants senesce prematurely, are hypersensitive to nitrogen and fixed carbon starvation, and fail to accumulate autophagic bodies in the vacuole. By combining mutants eliminating ATG12a/b, ATG5, or the ATG10 E2 required for their condensation with a method that unequivocally detects the ATG8‐PE adduct, we also show that ATG8 lipidation requires the ATG12–ATG5 conjugate. Unlike ATG8, ATG12 does not associate with autophagic bodies, implying that its role(s) during autophagy is restricted to events before the vacuolar deposition of vesicles. The expression patterns of the ATG12a and ATG12b genes and the effects of single atg12a and atg12b mutants on forming the ATG12–ATG5 conjugate reveal that the ATG12b locus is more important during basal autophagy while the ATG12a locus is more important during induced autophagy. Taken together, we conclude that the formation of the ATG12–ATG5 adduct is essential for ATG8‐mediated autophagy in plants by promoting ATG8 lipidation.     

A new role for ATM in selective autophagy of peroxisomes (pexophagy)

Peroxisomes are autonomously replicating and highly metabolic organelles necessary for β-oxidation of fatty acids, a process that generates large amounts of reactive oxygen species (ROS). Maintaining a balance between biogenesis and degradation of peroxisomes is essential to maintain cellular redox balance, but how cells do this has remained somewhat of a mystery. While it is known that peroxisomes can be degraded via selective autophagy (pexophagy), little is known about how mammalian cells regulate pexophagy to maintain peroxisome homeostasis. We have uncovered a mechanism for regulating pexophagy in mammalian cells that defines a new role for ATM (ATM serine/threonine kinase) kinase as a “first responder” to peroxisomal ROS. ATM is delivered to the peroxisome by the PEX5 import receptor, which recognizes an SRL sequence located at the C terminus of ATM to localize this kinase to peroxisomes. In response to ROS, the ATM kinase is activated and performs 2 functions: i) it signals to AMPK, which activates TSC2 to suppresses MTORC1 and phosphorylates ULK1 to induce autophagy, and ii) targets specific peroxisomes for pexophagy by phosphorylating PEX5 at Ser141, which triggers ubiquitnation of PEX5 at Lys209 and binding of the autophagy receptor protein SQSTM1/p62 to induce pexophagy.     

The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) are metabolic disorders caused by the loss of peroxisomes. The majority of PBDs result from mutation in one of 3 genes that encode for the peroxisomal AAA ATPase complex (AAA-complex) required for cycling PEX5 for peroxisomal matrix protein import. Mutations in these genes are thought to result in a defect in peroxisome assembly by preventing the import of matrix proteins. However, we show here that loss of the AAA-complex does not prevent matrix protein import, but instead causes an upregulation of peroxisome degradation by macroautophagy, or pexophagy. The loss of AAA-complex function in cells results in the accumulation of ubiquitinated PEX5 on the peroxisomal membrane that signals pexophagy. Inhibiting autophagy by genetic or pharmacological approaches rescues peroxisome number, protein import and function. Our findings suggest that the peroxisomal AAA-complex is required for peroxisome quality control, whereas its absence results in the selective degradation of the peroxisome. Thus the loss of peroxisomes in PBD patients with mutations in their peroxisomal AAA-complex is a result of increased pexophagy. Our study also provides a framework for the development of novel therapeutic treatments for PBDs.     

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.     

The ides of MARCH5: The E3 ligase essential for peroxisome degradation by pexophagy

A recent study by Zheng et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202103156) identifies the ubiquitin-protein ligase (E3) MARCH5 as a dual-organelle localized protein that not only targets to mitochondria but also to peroxisomes in a PEX19-mediated manner. Moreover, the authors demonstrate that the Torin1-dependent induction of pexophagy is executed by the MARCH5-catalyzed ubiquitination of the peroxisomal membrane protein PMP70.

Recent research has begun to slowly elucidate the complex processes that underlie selective autophagic degradation of mammalian peroxisomes. The study by Zheng et al. (1) sheds a light on the mechanism underlying pexophagy, which is induced by mTOR (mechanistic target of rapamycin) inactivation (2). The ubiquitously conserved serine/threonine kinase mTOR has a central function in integrating diverse growth signals and orchestrating their physiological effect on a cellular level, while blocking cell growth–restricting mechanisms like the different autophagy pathways (3). Previous work has demonstrated that amino acid starvation could induce mTOR inhibition-dependent peroxisome degradation by up-regulating the activity of the peroxisomal protein ubiquitin (E3) ligase PEX2 (4), which was especially of interest as PEX2 is also required for peroxisomal matrix protein import during the formation of the organelle (5). However, while these data suggested that the dual function of PEX2 might mark it as a point of convergence for the balance of peroxisome formation and degradation, the Zheng et al. study has identified a role for the E3 ligase MARCH5 (membrane-associated RING-CH 5; 1) that aims at a different aspect of peroxisome biology.Zheng et al. identified the peroxisomal proteins PEX3, PEX19, and PMP70 as close interaction partners of MARCH5 (1). The authors could demonstrate a PEX19-dependent localization of a portion of the MARCH5 population to peroxisomes. Here, MARCH5 can bind and polyubiquitinate the abundant peroxisomal membrane protein PMP70. While it is clear that the increased level of polyubiquitinated PMP70 molecules marks peroxisomes for recognition by ubiquitin-binding autophagy receptors that link the target organelle to the autophagosomal membrane, the identity of the E2 enzyme involved in ubiquitin chain generation as well as the ubiquitin adaptors are unknown (Fig. 1). However, based on published research, NBR1 or p62 are good candidates for the adaptors that engage the autophagy machinery (2). Moreover, the Zheng et al. study demonstrates that MARCH5-mediated polyubiquitination of PMP70 is induced by the mTOR inhibitor Torin1. In return, the described Torin1-induced pexophagy was shown to rely on the peroxisomal localization and activity of the catalytic RING domain of MARCH5 (1).Open in a separate windowFigure 1.The small molecule Torin1 can inhibit the kinase mTOR, resulting in a relief of the mTOR-dependent block of MARCH5 targeting to peroxisomes. MARCH5 is inserted into the peroxisomal membrane in a PEX19- and PEX3-dependent manner. MARCH5 ubiquitinates the abundant peroxisomal membrane protein PMP70 with the help of an unknown ubiquitin (Ub)-conjugating enzyme (E2). The ubiquitinated PMP70 molecules are recognized by ubiquitin-binding autophagy receptors, like NBR1 or p62, that link the organelle to the autophagosome, resulting in the autophagic degradation of the peroxisome via pexophagy.It is interesting to note that the opponent of pexophagy-linked ubiquitin signals on peroxisomes was already identified as the deubiquitinating enzyme USP30 (6). This combination is even more relevant when considering that MARCH5 and USP30 were described as an antagonizing enzyme pair that regulates the autophagic degradation of mitochondria via mitophagy (6). The function of MARCH5 is also linked to other mitochondrial ubiquitination factors, like the E3 ligase Parkin. While both enzymes can contribute to mitophagy induction by ubiquitinating proteins of the outer mitochondrial membrane, they can also modify each other. MARCH5 ubiquitinates Parkin in order to restrict the number of Parkin molecules during mitophagy and to prevent Parkin-mediated cell death (7).After mitophagy induction, Parkin can ubiquitinate MARCH5, which results in the p97-mediated membrane extraction of MARCH5 and a PEX3/PEX16-dependent redistribution of MARCH5 to peroxisomes (8). This mechanism was assumed to rescue MARCH5 from degradation by mitophagy. It will be important to elucidate if there is mechanistic overlap between the Parkin-mediated (8) and the Torin1-dependent (1) targeting of MARCH5. Moreover, it will be interesting to determine if MARCH5 is also engaged in an interplay with the peroxisomal E3 ligases PEX2, PEX10, PEX12, or TRIM37.Mitochondria and peroxisomes share basic components of their fission machineries. Both organelles use the membrane proteins FIS1 and mitochondrial fission factor for the targeting of the membrane-constricting GTPase DRP1 (DLP1; 9). In the case of the mitochondria, MARCH5 can ubiquitinate DRP1 and FIS1 for proteasomal degradation in order to limit mitochondrial However, other data indicate the existence of a feedback mechanism, as DRP1 can also negatively influence MARCH5 activity. In addition, MARCH5 not only limits mitochondrial fission, but also represents a basic requirement for this process. This complex relationship of MARCH5 with mitochondrial fission proteins suggests that it performs a central role in the fine-tuning of the basic regulatory aspects of mitochondrial division (10). Therefore, future studies might not only establish a potential role of MARCH5 in peroxisomal fission but might also uncover aspects that could enable further insights into the related process in mitochondria.The different roles of MARCH5 in organelle fission and autophagic degradation could possibly be interconnected in one bipartite reaction sequence. Mitochondrial fission is crucial for mitophagy and enables the removal of damaged sections of mitochondria or the limitation of organelle size for a more efficient engulfment by autophagosomal membranes (11). Therefore, both processes can be functionally interconnected. Interestingly, it has been shown that fission also precedes pexophagy in yeast cells (12), which are thought to use organelle-specific adaptors instead of ubiquitin as a degradation tag. However, these observations suggest that MARCH5 might coordinate peroxisomal fission with pexophagy even in mammalian peroxisomes.In summary, the Zheng et al. study not only identifies a central mechanistic module required for the turnover of mammalian peroxisomes (1) but also raises many interesting questions that will result in further studies dealing with the interplay of the peroxisomal ubiquitination factors, the crosstalk between mitochondria and peroxisomes, and the organization and regulation of the peroxisomal fission machinery as well as the convergence of peroxisomal fission and pexophagy pathways.     

NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog

Macroautophagy/autophagy is a conserved degradation process in eukaryotic cells involving the sequestration of proteins and organelles within double-membrane vesicles termed autophagosomes. In filamentous fungi, its main purposes are the regulation of starvation adaptation and developmental processes. In contrast to nonselective bulk autophagy, selective autophagy is characterized by cargo receptors, which bind specific cargos such as superfluous organelles, damaged or harmful proteins, or microbes, and target them for autophagic degradation. Herein, using the core autophagy protein ATG8 as bait, GFP-Trap analysis followed by liquid chromatography mass spectrometry (LC/MS) identified a putative homolog of the human autophagy cargo receptor NBR1 (NBR1, autophagy cargo receptor) in the filamentous ascomycete Sordaria macrospora (Sm). Fluorescence microscopy revealed that SmNBR1 colocalizes with SmATG8 at autophagosome-like structures and in the lumen of vacuoles. Delivery of SmNBR1 to the vacuoles requires SmATG8. Both proteins interact in an LC3 interacting region (LIR)-dependent manner. Deletion of Smnbr1 leads to impaired vegetative growth under starvation conditions and reduced sexual spore production under non-starvation conditions. The human NBR1 homolog partially rescues the phenotypic defects of the fungal Smnbr1 deletion mutant. The Smnbr1 mutant can neither use fatty acids as a sole carbon source nor form fruiting bodies under oxidative stress conditions. Fluorescence microscopy revealed that degradation of a peroxisomal reporter protein is impaired in the Smnbr1 deletion mutant. Thus, SmNBR1 is a cargo receptor for pexophagy in filamentous ascomycetes.     

Peroxisomal protein PEX13 functions in selective autophagy

PEX13 is an integral membrane protein on the peroxisome that regulates peroxisomal matrix protein import during peroxisome biogenesis. Mutations in PEX13 and other peroxin proteins are associated with Zellweger syndrome spectrum (ZSS) disorders, a subtype of peroxisome biogenesis disorder characterized by prominent neurological, hepatic, and renal abnormalities leading to neonatal death. The lack of functional peroxisomes in ZSS patients is widely accepted as the underlying cause of disease; however, our understanding of disease pathogenesis is still incomplete. Here, we demonstrate that PEX13 is required for selective autophagy of Sindbis virus (virophagy) and of damaged mitochondria (mitophagy) and that disease‐associated PEX13 mutants I326T and W313G are defective in mitophagy. The mitophagy function of PEX13 is shared with another peroxin family member PEX3, but not with two other peroxins, PEX14 and PEX19, which are required for general autophagy. Together, our results demonstrate that PEX13 is required for selective autophagy, and suggest that dysregulation of PEX13‐mediated mitophagy may contribute to ZSS pathogenesis.     

Atg28, a Novel Coiled-Coil Protein Involved in Autophagic Degradation of Peroxisomes in the Methylotrophic Yeast Pichia pastoris

In methylotrophic yeasts, peroxisomes are required for methanol utilization, but are dispensable for growth on most other carbon sources. Upon adaptation of cells grown on methanol to glucose or ethanol, redundant peroxisomes are selectively and quickly shipped to, and degraded in, vacuoles via a process termed pexophagy.

We identified a novel gene named ATG28 (autophagy-related genes) involved in pexophagy in the yeast Pichia pastoris. This yeast exhibits two morphologically distinct pexophagy pathways, micro- and macropexophagy, induced by glucose or ethanol, respectively. Deficiency in ATG28 impairs both pexophagic mechanisms but not general (bulk turnover) autophagy, a degradation pathway in yeast triggered by nitrogen starvation. It is known that the micro-, macropexophagy, and general autophagy machineries are distinct but share some molecular components. The identification of ATG28 suggests that pexophagy may involve species-specific components, since this gene appears to have only weak homologues in other yeasts.