Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation |
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| Affiliation: | 1. Department of Molecular and Cell Biology and Biochemistry, Basic Veterinary Science, Faculty of Veterinary Medicine, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan;2. Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan;3. Education and Research Center for Fermentation Studies, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan;1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China;2. Sanya Institute of Nanjing Agricultural University, Sanya, Hainan 572024, China;3. College of Animal Science, Fujian Agriculture & Forestry University, Fuzhou, Fujian 350002, China;1. Departments of Medicine and Physiology and Banting and Best Diabetes Centre, University of Toronto, Toronto, ON, Canada;2. Department of Anatomy, Physiology and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada;3. Maccabi Healthcare Services, Endocrinology Division, Tel Aviv, Israel;1. Rutgers Center for Lipid Research, New Jersey Institute for Food, Nutrition, and Health, Rutgers University, United States of America;2. Department of Nutritional Sciences, Rutgers University, United States of America;3. Departments of Biochemistry and Nutritional Sciences, University of Wisconsin-Madison, United States of America;1. Laboratory of Animal Morphology, Graduate School of Bioagricultural Sciences, Nagoya University, Tokai National Higher Education and Research System, Nagoya, Aichi, Japan;2. Laboratory of Marine Life Science and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan |
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| 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. |
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