Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation
Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation
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磷脂酰肌醇4-磷酸磷酸酶Sac1p和Sjl3p在酵母自噬体形成中的重要作用
DOI：
10.1016/j.bbalip.2022.159184
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Fujita Akikazu
中科院分区：
文献类型：
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作者：
Muramoto Moe;Yamakuchi Yuki;Konishi Rikako;Koudatsu Shiomi;Tomikura Hiromu;Fukuda Kayoko;Kuriyama Sayuri;Kurokawa Yuna;Masatani Tatsunori;Tamaki Hisanori;Fujita Akikazu
自噬受磷酸肌醇调节。我们以前已经证明，磷脂酰肌醇4-磷酸（PtdIns（4）P）是本地化的自噬体膜。此外，在酵母细胞中，磷脂酰肌醇4-激酶Pik 1 p和Stt 4p分别在自噬体的形成及其与空泡的融合中发挥重要作用。在这项研究中，我们分析了PtdIns（4）P磷酸酶在酵母自噬中的主要作用。在限制性温度下，磷酸酶缺失突变体sac 1 ts/sjl 3 ts和温度敏感突变体sac 1 ts/sjl 3 ts的液泡膜、线粒体膜、细胞核膜、内质网膜和质膜上的PtdIns（4）P标记密度显著增加。GFP-Atg 8处理实验表明sac 1 β和sac 1 ts/sjl 3 β突变体中存在缺陷性自噬。与PtdIns（4）P在野生型酵母中自噬体膜的腔小叶中的定位相反，PtdIns（4）P在Sac 1菌株中自噬体膜的腔小叶和胞质小叶中均定位。此外，尽管自噬体存在于细胞质中，但在sac 1菌株中，空泡中的自噬体数量显著减少。在ac 1 ts/sjl 3菌株中，细胞质中的自噬体数量在限制性温度下显著减少。考虑到自噬体和自噬体的数量与野生型酵母相当，我们发现当Sac 1 p和Sj 13 p的PtdIns（4）P磷酸酶活性降低时，自噬体不能形成。总之，这些结果表明磷酸酶对PtdIns（4）P的周转对于自噬体的生物发生是必不可少的。
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 mutantssac1∆ andsjl3∆, and the temperature-sensitive mutantsac1ts/sjl3∆ at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in thesac1∆ andsac1ts/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 thesac1∆ strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in thesac1∆ strain, although autophagosomes were present in the cytoplasm. In thesac1ts/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 thesjl3∆ 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.