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PMID:10233149

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Citation

Yuan, W, Stromhaug, PE and Dunn, WA Jr (1999) Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein. Mol. Biol. Cell 10:1353-66

Abstract

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.

Links

PubMed PMC25277

Keywords

Adaptation, Physiological; Adenosine Triphosphate/metabolism; Amino Acid Sequence; Autophagy/physiology; Base Sequence; Binding Sites; Catalytic Domain; Fungal Proteins/genetics; Fungal Proteins/metabolism; Glucose/metabolism; Humans; Ligases/genetics; Microbodies/metabolism; Molecular Sequence Data; Mutation; Pichia/genetics; Pichia/metabolism; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Ubiquitin-Activating Enzymes; Ubiquitin-Protein Ligases

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

PICPA:ATG7

involved_in

GO:0000425: pexophagy

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

PICPA:ATG7

involved_in

GO:0000426: micropexophagy

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:ATG7

involved_in

GO:0061025: membrane fusion

ECO:0000304: author statement supported by traceable reference used in manual assertion

P

Seeded From UniProt

complete

HUMAN:ATG7

involved_in

GO:0006464: cellular protein modification process

ECO:0000304: author statement supported by traceable reference used in manual assertion

P

Seeded From UniProt

complete

HUMAN:ATG7

part_of

GO:0005737: cytoplasm

ECO:0000304: author statement supported by traceable reference used in manual assertion

C

Seeded From UniProt

complete

HUMAN:ATG7

enables

GO:0004839: ubiquitin activating enzyme activity

ECO:0000304: author statement supported by traceable reference used in manual assertion

F

Seeded From UniProt

complete


See also

References

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