Holkar Sachin S, Kamerkar Sukrut C, Pucadyil Thomas J
From the Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411 008, India.
From the Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411 008, India
J Biol Chem. 2015 Jun 5;290(23):14267-76. doi: 10.1074/jbc.M115.653394. Epub 2015 Apr 2.
Epsins belong to the family of highly conserved clathrin-associated sorting proteins that are indispensable for clathrin-mediated endocytosis, but their precise functions remain unclear. We have developed an assay system of budded supported membrane tubes displaying planar and highly curved membrane surfaces to analyze intrinsic membrane curvature preference shown by clathrin-associated sorting proteins. Using real-time fluorescence microscopy, we find that epsin preferentially partitions to and assembles clathrin on highly curved membrane surfaces. Sorting of epsin to regions of high curvature strictly depends on binding to phosphatidylinositol 4,5-bisphosphate. Fluorescently labeled clathrins rapidly assemble as foci, which in turn cluster epsin, while maintaining tube integrity. Clathrin foci grow in intensity with a typical time constant of ∼75 s, similar to the time scales for coated pit formation seen in cells. Epsin therefore effectively senses membrane curvature to spatially control clathrin assembly. Our results highlight the potential role of membrane curvature in orchestrating the myriad molecular interactions necessary for the success of clathrin-mediated membrane budding.
Epsin属于高度保守的网格蛋白相关分选蛋白家族，是网格蛋白介导的内吞作用所必需的，但它们的确切功能仍不清楚。我们开发了一种出芽的支持膜管检测系统，该系统展示平面和高度弯曲的膜表面，以分析网格蛋白相关分选蛋白所表现出的内在膜曲率偏好。使用实时荧光显微镜，我们发现epsin优先分配到高度弯曲的膜表面并在其上组装网格蛋白。epsin分选到高曲率区域严格依赖于与磷脂酰肌醇4,5-二磷酸的结合。荧光标记的网格蛋白迅速组装成焦点，进而聚集epsin，同时保持管的完整性。网格蛋白焦点强度的增加具有约75秒的典型时间常数，类似于在细胞中观察到的有被小窝形成的时间尺度。因此，epsin能有效感知膜曲率，以在空间上控制网格蛋白的组装。我们的结果突出了膜曲率在协调网格蛋白介导的膜出芽成功所需的无数分子相互作用中的潜在作用。
