Dataset Information

PpAtg30 tags peroxisomes for turnover by selective autophagy.

ABSTRACT: Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.

SUBMITTER: Farre JC

PROVIDER: S-EPMC3763908 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

PpAtg30 tags peroxisomes for turnover by selective autophagy.

Farré Jean-Claude JC Manjithaya Ravi R Mathewson Richard D RD Subramani Suresh S

Developmental cell 20080301 3

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction ...[more]

PMID: 18331717

Similar Datasets

Project description:The destruction of mitochondria through macroautophagy (autophagy) has been recognised as a major route of mitochondrial protein degradation since its discovery more than 50 years ago, but fundamental questions remain unanswered. First, how much mitochondrial protein turnover occurs through auto-phagy? Mitochondrial proteins are also degraded by nonautophagic mechanisms, and the proportion of mitochondrial protein turnover that occurs through autophagy is still unknown. Second, does auto-phagy degrade mitochondrial proteins uniformly or selectively? Autophagy was originally thought to degrade all mitochondrial proteins at the same rate, but recent work suggests that mitochondrial autophagy may be protein selective. To investigate these questions, we used a proteomics-based approach in the fruit fly Drosophila melanogaster, comparing mitochondrial protein turnover rates in autophagy-deficient Atg7 mutants and controls. We found that ~35% of mitochondrial protein turnover occurred via autophagy. Similar analyses using parkin mutants revealed that parkin-dependent mitophagy accounted for ~25% of mitochondrial protein turnover, suggesting that most mitochondrial autophagy specifically eliminates dysfunctional mitochondria. We also found that our results were incompatible with uniform autophagic turnover of mitochondrial proteins and consistent with protein-selective autophagy. In particular, the autophagic turnover rates of individual mitochondrial proteins varied widely, and only a small amount of the variation could be attributed to tissue differences in mitochondrial composition and autophagy rate. Furthermore, analyses comparing autophagy-deficient and control human fibroblasts revealed diverse autophagy-dependent turnover rates even in homogeneous cells. In summary, our work indicates that autophagy acts selectively on mitochondrial proteins, and that most mitochondrial protein turnover occurs through non-autophagic processes. Abbreviations: Atg5: Autophagy-related 5 (Drosophila); ATG5: autophagy related 5 (human); Atg7: Autophagy-related 7 (Drosophila); ATG7: autophagy related 7 (human); DNA: deoxyribonucleic acid; ER: endoplasmic reticulum; GFP: green fluorescent protein; MS: mass spectrometry; park: parkin (Drosophila); Pink1: PTEN-induced putative kinase 1 (Drosophila); PINK1: PTEN-induced kinase 1 (human); PRKN: parkin RBR E3 ubiquitin protein ligase (human); RNA: ribonucleic acid; SD: standard deviation; Ub: ubiquitin/ubiquitinated; WT: wild-type; YME1L: YME1 like ATPase (Drosophila); YME1L1: YME1 like 1 ATPase (human).

Project description: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.

Dataset Information

PpAtg30 tags peroxisomes for turnover by selective autophagy.

Publications

PpAtg30 tags peroxisomes for turnover by selective autophagy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure