Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation

Abstract

Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1∆ and sjl3∆, and the temperature-sensitive mutant sac1ts/sjl3∆ at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1∆ and sac1ts/sjl3∆ mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1∆ strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1∆ strain, although autophagosomes were present in the cytoplasm. In the sac1ts/sjl3∆ strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3∆ strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis.