Association of the Endosomal Sorting Complex ESCRT-II with the Vps20 Subunit of ESCRT-III Generates a Curvature-sensitive Complex Capable of Nucleating ESCRT-III Filaments
Association of the Endosomal Sorting Complex ESCRT-II with the Vps20 Subunit of ESCRT-III Generates a Curvature-sensitive Complex Capable of Nucleating ESCRT-III Filaments
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DOI：
10.1074/jbc.m111.266411
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发表时间：
2011-09-30
影响因子：
4.8
通讯作者：
Audhya, Anjon
中科院分区：
文献类型：
--
作者：
Fyfe, Ian;Schuh, Amber L.;Audhya, Anjon
囊泡生物发生和胞质分裂所必需的膜的断裂是由细胞质蛋白介导的，细胞质蛋白包括ESCRT(运输所需的内体分选复合体)机制的成员。在腔内囊泡形成多囊内体的过程中，ESCRT-II复合体启动ESCRT-III亚单位的聚合，而ESCRT-III是膜分裂所必需的。然而，这一过程的空间和时间调节的潜在机制仍然不清楚。在这里，我们证明了纯化的ESCRT-II以曲率依赖的方式与化学定义的膜上的ESCRT-III亚单位Vps20结合。结合脂质体共浮选试验、基于荧光的脂质体相互作用研究和高分辨率原子力显微镜，我们发现ESCRT-II与Vps20之间的相互作用降低了ESCRT-II对平脂双层的亲和力。此外，我们还证明了ESCRT-II和Vps20使Vps32的柔性细丝成核，这些柔韧的细丝作为单一的单体串沿着高度弯曲的膜进行聚合。引人注目的是，Vps32细丝被证明在体外调节膜动力学，这是细胞膜断裂事件的先决条件。我们认为，依赖于曲率的组装途径提供了ESCRT-III的空间调节，以融合并列的曲率升高的双层。
The scission of membranes necessary for vesicle biogenesis and cytokinesis is mediated by cytoplasmic proteins, which include members of the ESCRT (endosomal sorting complex required for transport) machinery. During the formation of intralumenal vesicles that bud into multivesicular endosomes, the ESCRT-II complex initiates polymerization of ESCRT-III subunits essential for membrane fission. However, mechanisms underlying the spatial and temporal regulation of this process remain unclear. Here, we show that purified ESCRT-II binds to the ESCRT-III subunit Vps20 on chemically defined membranes in a curvature-dependent manner. Using a combination of liposome co-flotation assays, fluorescence-based liposome interaction studies, and high-resolution atomic force microscopy, we found that the interaction between ESCRT-II and Vps20 decreases the affinity of ESCRT-II for flat lipid bilayers. We additionally demonstrate that ESCRT-II and Vps20 nucleate flexible filaments of Vps32 that polymerize specifically along highly curved membranes as a single string of monomers. Strikingly, Vps32 filaments are shown to modulate membrane dynamics in vitro, a prerequisite for membrane scission events in cells. We propose that a curvature-dependent assembly pathway provides the spatial regulation of ESCRT-III to fuse juxtaposed bilayers of elevated curvature.