Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut, USA; Marine Biological Laboratory, Woods Hole, Massachusetts, USA; Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut, USA; PREP Program, Yale University, New Haven, Connecticut, USA.
J Biol Chem. 2023 Sep;299(9):105091. doi: 10.1016/j.jbc.2023.105091. Epub 2023 Jul 28.
α-Synuclein and family members β- and γ-synuclein are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. αβγ-synuclein triple knockout neurons exhibit SV endocytosis deficits. Here, we investigated if α-synuclein affects clathrin assembly in vitro. Visualizing clathrin assembly on membranes using a lipid monolayer system revealed that α-synuclein increases clathrin lattices size and curvature. On cell membranes, we observe that α-synuclein is colocalized with clathrin and its adapter AP180 in a concentric ring pattern. Clathrin puncta that contain both α-synuclein and AP180 were significantly larger than clathrin puncta containing either protein alone. We determined that this effect occurs in part through colocalization of α-synuclein with the phospholipid PI(4,5)P2 in the membrane. Immuno-electron microscopy (EM) of synaptosomes uncovered that α-synuclein relocalizes from SVs to the presynaptic membrane upon stimulation, positioning α-synuclein to function on presynaptic membranes during or after stimulation. Additionally, we show that deletion of synucleins impacts brain-derived clathrin-coated vesicle size. Thus, α-synuclein affects the size and curvature of clathrin structures on membranes and functions as an endocytic accessory protein.
α-突触核蛋白和家族成员β-和 γ-突触核蛋白是突触前蛋白，可感知和产生膜曲率，这是突触小泡 (SV) 循环的重要特性。αβγ-突触核蛋白三重敲除神经元表现出 SV 内化缺陷。在这里，我们研究了 α-突触核蛋白是否会影响体外的网格蛋白组装。使用脂质单层系统在膜上可视化网格蛋白组装，结果表明α-突触核蛋白增加了网格蛋白晶格的大小和曲率。在细胞膜上，我们观察到α-突触核蛋白与网格蛋白及其衔接蛋白 AP180 共定位呈同心环模式。含有α-突触核蛋白和 AP180 的网格蛋白斑点明显大于仅含有两种蛋白质之一的网格蛋白斑点。我们确定这种效应部分是通过α-突触核蛋白与膜中磷脂 PI(4,5)P2 的共定位发生的。突触小体的免疫电子显微镜 (EM) 揭示，α-突触核蛋白在刺激后从 SV 重新定位到突触前膜，将α-突触核蛋白定位在刺激期间或之后的突触前膜上发挥作用。此外，我们还表明突触核蛋白的缺失会影响脑源性网格蛋白包被小泡的大小。因此，α-突触核蛋白影响膜上网格蛋白结构的大小和曲率，并作为内吞辅助蛋白发挥作用。
