Department of Biochemistry and Molecular Biology, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel; Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
J Biol Chem. 2020 Dec 25;295(52):18076-18090. doi: 10.1074/jbc.RA120.015319. Epub 2020 Oct 21.
α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson's disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P). Our results show that α-Syn colocalizes with PIP and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin-mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI(4,5)P levels on the plasma membrane. In accord with their effects on PI(4,5)P levels, the PD associated A30P, E46K, and A53T mutations in α-Syn further enhance CME in neuronal and nonneuronal cells. However, lysine to glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, which interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI(4,5)P levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn-mediated membrane trafficking.
α-突触核蛋白（α-Syn）是一种与帕金森病（PD）发病机制相关的蛋白质。它是一种内在无序的蛋白质，可与酸性磷脂结合。越来越多的证据支持α-Syn 在膜运输中的作用，包括内吞作用和胞吐作用的机制，尽管α-Syn 在这些机制中的确切作用目前尚不清楚。在这里，我们研究了α-Syn 与酸性磷酸肌醇（PIPs）、磷脂酰肌醇 4,5-二磷酸（PI(4,5)P）和磷脂酰肌醇 3,4-二磷酸（PI(3,4)P）的关联。我们的结果表明，α-Syn 与 PIP 和网格蛋白衔接蛋白 2（AP2）的磷酸化活性形式在网格蛋白包被小窝处共定位。使用转铁蛋白的内吞作用作为网格蛋白介导的内吞作用（CME）的指标，我们发现 α-Syn 参与内吞作用是通过质膜上的 PI(4,5)P 水平特异性介导的。与它们对 PI(4,5)P 水平的影响一致，α-Syn 中的 PD 相关 A30P、E46K 和 A53T 突变进一步增强了神经元和非神经元细胞中的 CME。然而，α-Syn 的 KTKEGV 重复结构域中的赖氨酸到谷氨酸取代，干扰了磷脂结合，在增强 CME 方面无效。我们进一步表明，突触小泡（SV）内吞作用的速率受α-Syn 突变的不同影响，并与它们对 PI(4,5)P 水平的影响相关，除了 A30P 突变。这项研究为 PIPs 在α-Syn 介导的膜运输中的关键作用提供了证据。
