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Atg4 plays an important role in efficient expansion of autophagic isolation membranes by cleaving lipidated Atg8 in Saccharomyces cerevisiae.

ABSTRACT: Autophagy, an intracellular degradation system, is highly conserved among eukaryotes from yeast to mammalian cells. In the yeast Saccharomyces cerevisiae, most Atg (autophagy-related) proteins, which are essential for autophagosome formation, are recruited to a restricted region close to the vacuole, termed the vacuole-isolation membrane contact site (VICS), upon induction of autophagy. Subsequently, the isolation membrane (IM) expands and sequesters cytoplasmic materials to become a closed autophagosome. In S. cerevisiae, the ubiquitin-like protein Atg8 is C-terminally conjugated to the phospholipid phosphatidylethanolamine (PE) to generate Atg8-PE. During autophagosome formation, Atg8-PE is cleaved by Atg4 to release delipidated Atg8 (Atg8G116) and PE. Although delipidation of Atg8-PE is important for autophagosome formation, it remains controversial whether the delipidation reaction is required for targeting of Atg8 to the VICS or for subsequent IM expansion. We used an IM visualization technique to clearly demonstrate that delipidation of Atg8-PE is dispensable for targeting of Atg8 to the VICS, but required for IM expansion. Moreover, by overexpressing Atg8G116, we showed that the delipidation reaction of Atg8-PE by Atg4 plays an important role in efficient expansion of the IM other than supplying unlipidated Atg8G116. Finally, we suggested the existence of biological membranes at the Atg8-labeled structures in Atg8-PE delipidation-defective cells, but not at those in atg2Δ cells. Taken together, it is likely that Atg2 is involved in localization of biological membranes to the VICS, where Atg4 is responsible for IM expansion.

SUBMITTER: Hirata E

PROVIDER: S-EPMC5509253 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Project description:The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20(+) is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20(+) gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

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Atg4 plays an important role in efficient expansion of autophagic isolation membranes by cleaving lipidated Atg8 in Saccharomyces cerevisiae.

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