Interaction of the mammalian endosomal sorting complex required for transport (ESCRT) III protein hSnf7-1 with itself, membranes, and the AAA+ ATPase SKD1
Interaction of the mammalian endosomal sorting complex required for transport (ESCRT) III protein hSnf7-1 with itself, membranes, and the AAA+ ATPase SKD1
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DOI：
10.1074/jbc.m413968200
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发表时间：
2005-04-01
影响因子：
4.8
通讯作者：
Hanson, PI
中科院分区：
文献类型：
--
作者：
Lin, Y;Kimpler, LA;Hanson, PI
SKD1/VPS4B 是一种参与多泡体 (MVB) 生物发生的 AAA(+)（与多种细胞活动相关的 ATP 酶）蛋白。在这项研究中，我们发现，ATP 水解缺陷突变体 SKD1(E235Q) 引起的 MVB 生物发生损伤伴随着大型去污剂不溶性蛋白质复合物的组装，该复合物包括运输所需的哺乳动物内体分选复合物的正常可溶性内源成分 (ESCRT) I 和 ESCRT-III 复合物。膜结合 ESCRT-III 复合物被认为是招募 SKD1 到新生 MVB 的底物。为了探索这种关系，我们研究了人类 ESCRT-III 成分 hSnf7-1 和 hVps24、膜和 SKD1 之间的相互作用。我们发现，过表达的 hSnf7-1 的很大一部分与膜相关，在膜上形成一个大的蛋白质复合物，招募 SKD1 并扰乱正常的 MVB 生物发生。过表达的 hVps24 也与膜和扰乱内体结构相关，但仅当与绿色荧光蛋白融合时才发生。结构域分析显示，hSnf7-1 的基本 N 末端一半定位于膜并形成耐洗涤剂的聚合物，其中一些看起来像丝状伪足，延伸到肿胀内体的内腔中或从质膜延伸出来。 hSnf7-1 的 C 端酸性一半不与膜结合，并且是 hSnf7-1 与 SKD1 相互作用所必需的。结合早期的研究，我们的工作表明多种含有 ESCRT-III 的聚合物可以在膜上组装并在 MVB 形成过程中招募 SKD1。
SKD1/VPS4B is an AAA(+) (ATPase associated with a variety of cellular activities) protein involved in multivesicular body (MVB) biogenesis. In this study, we show that the impairment in MVB biogenesis caused by the ATP hydrolysis-deficient mutant SKD1(E235Q) is accompanied by assembly of a large detergent-insoluble protein complex that includes normally soluble endogenous components of mammalian endosomal sorting complex required for transport (ESCRT) I and ESCRT-III complexes. Membrane-bound ESCRT-III complex has been proposed to be the substrate that recruits SKD1 to nascent MVBs. To explore this relationship, we studied interactions among the human ESCRT-III components hSnf7-1 and hVps24, membranes, and SKD1. We found that a significant portion of overexpressed hSnf7-1 associated with membranes where it formed a large protein complex that recruited SKD1 and perturbed normal MVB biogenesis. Overexpressed hVps24 also associated with membranes and perturbed endosome structure but only when fused to green fluorescent protein. Domain analysis revealed that the basic N-terminal half of hSnf7-1 localized to membranes and formed detergent-resistant polymers, some of which looked like filopodia extending into the lumen of swollen endosomes or out from the plasma membrane. The C-terminal acidic half of hSnf7-1 did not associate with membranes and was required for interaction of hSnf7-1 with SKD1. Together with earlier studies, our work suggests that a variety of ESCRT-III-containing polymers can assemble on membranes and recruit SKD1 during formation of the MVB.