Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijyuku, Katsushika-ku, Tokyo 125-8585, Japan.
Institute of Science and Technology Austria, Am Campus 1, A-3400 Klosterneuburg, Austria.
J Cell Sci. 2018 Jan 4;131(1):jcs207696. doi: 10.1242/jcs.207696.
Clathrin-mediated endocytosis requires the coordinated assembly of various endocytic proteins and lipids at the plasma membrane. Accumulating evidence demonstrates a crucial role for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P] in endocytosis but specific roles for phosphatidylinositol-4-phosphate [PtdIns(4)P], other than as the biosynthetic precursor of PtdIns(4,5)P, have not been clarified. In this study we investigated the roles of PtdIns(4)P and PtdIns(4,5)P in receptor-mediated endocytosis through the construction of temperature-sensitive (ts) mutants for the phosphatidylinositol 4-kinases (PI4-kinases) Stt4p and Pik1p and the 1-phosphatidylinositol-4-phosphate 5-kinase [PtdIns(4) 5-kinase] Mss4p. Quantitative analyses of endocytosis revealed that both the and mutants have a severe defect in endocytic internalization. Live-cell imaging of endocytic protein dynamics in and mutants revealed that PtdIns(4)P is required for the recruitment of the α-factor receptor Ste2p to clathrin-coated pits, whereas PtdIns(4,5)P is required for membrane internalization. We also found that the localization to endocytic sites of the ENTH/ANTH domain-bearing clathrin adaptors, Ent1p, Ent2p, Yap1801p and Yap1802p, is significantly impaired in the mutant but not in the mutant. These results suggest distinct roles in successive steps for PtdIns(4)P and PtdIns(4,5)P during receptor-mediated endocytosis.
网格蛋白介导的内吞作用需要各种内吞蛋白和质膜上的脂质在质膜上协调组装。越来越多的证据表明，磷脂酰肌醇-4,5-二磷酸[PtdIns（4,5）P]在胞吞作用中起着关键作用，但除了作为 PtdIns（4,5）P 的生物合成前体外，磷脂酰肌醇-4-磷酸[PtdIns（4）P]的具体作用尚未阐明。在这项研究中，我们通过构建磷脂酰肌醇 4-激酶（PI4-激酶）Stt4p 和 Pik1p 和 1-磷酸磷脂酰肌醇-4-磷酸 5-激酶[PtdIns（4）5-激酶]Mss4p 的温度敏感（ts）突变体，研究了 PtdIns（4）P 和 PtdIns（4,5）P 在受体介导的内吞作用中的作用。内吞作用的定量分析表明，和突变体在内吞内化过程中都存在严重缺陷。和突变体中内吞蛋白动力学的活细胞成像显示，PtdIns（4）P 对于将 α 因子受体 Ste2p 募集到网格蛋白包被的陷窝是必需的，而 PtdIns（4,5）P 对于膜内化是必需的。我们还发现，富含 ENTH/ANTH 结构域的网格蛋白衔接蛋白 Ent1p、Ent2p、 yap1801p 和 yap1802p 的定位到内吞部位在突变体中显著受损，但在突变体中不受影响。这些结果表明，在受体介导的内吞作用中，PtdIns（4）P 和 PtdIns（4,5）P 在连续步骤中具有不同的作用。
