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

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are s ynthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectivel y sequestered and degraded within the yeast vacuole. Sequestration begins w hen the vacuole changes shape and surrounds the peroxisomes. The opposing m embranes then fuse, engulfing the peroxisome. In this study, we have charac terized a mutant cell line (glucose-induced selective autophagy), gsa7, whi ch is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were una ble to degrade peroxisomal alcohol oxidase. We observed that the peroxisome s were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation o f autophagy but is required for bringing the opposing vacuolar membranes to gether for homotypic fusion, thereby completing peroxisome sequestration. B y sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited s equence homology to a family of ubiquitin-activating enzymes, E1. The knock out mutant gsn7 Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addit ion, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyce s cerevisiae, was capable of rescuing gsa7. We have sequenced the human hom olog 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 doma in and catalytic site of the family of ubiquitin activating enzymes, E1 (UB A1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(Delta ATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester link age with a 25-30 kDa protein. This conjugate was not observed in cells expr essing Gsa7(Delta ATP)-HA or in cells expressing Gsa7(C518S)-HA. Our result s suggest that this unique E1-like enzyme is required for homotypic membran e fusion, a late event in the sequestration of peroxisomes by the vacuole.