Human CHMP6, a myristoylated ESCRT-III protein, interacts directly with an ESCRT-II component EAP20 and regulates endosomal cargo sorting.
Human CHMP6, a myristoylated ESCRT-III protein, interacts directly with an ESCRT-II component EAP20 and regulates endosomal cargo sorting.
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DOI：
10.1042/bj20041227
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发表时间：
2005-04
期刊：
影响因子：
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通讯作者：
Chiharu Yorikawa;H. Shibata;S. Waguri;Kazumi Hatta;Mio Horii;Keiichi Katoh;Toshihide Kobayashi;Y. Uchiyama;M. Maki
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文献类型：
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作者：
Chiharu Yorikawa;H. Shibata;S. Waguri;Kazumi Hatta;Mio Horii;Keiichi Katoh;Toshihide Kobayashi;Y. Uchiyama;M. Maki
CHMP6(带电多囊泡体蛋白6)是酵母VPS(空泡蛋白分类)20的人类同源产物，是ESCRT(运输所需的内体分选复合体)-III的组成部分。在从酵母到人类的各种生物中，CHMP6的各种同源物在每个N-末端都含有N-肉豆蔻酸化的共同序列。人胚胎肾细胞(HEK-293)代谢标记结果显示，[~3H]肉豆蔻酸掺入C端与绿色荧光蛋白(GFP)融合的CHMP6(CHMP6-GFP)。通过免疫共沉淀在HEK-293细胞中表达的表位标记蛋白，观察到CHMP6与另一个ESCRT-III组分CHMP4b/Shax[与Alix相关的Snf7(蔗糖非发酵7)同源物]1，以及与EAP20(ELL相关蛋白20)的相互作用，其中Snf7是人Vps32/Snf7的同源物，EAP20是酵母Vps25的对应物，是ESCRT-II的组成部分。利用从大肠杆菌中纯化的重组蛋白进行的体外下拉实验表明，CHMP6的N端碱性半部分介导了直接的物理相互作用。荧光显微镜分析显示，在HeLa细胞中过表达的CHMP6-GFP呈点状分布于细胞质中，尤其是核周区域。在CHMP6-GFP定位区域观察到LBPA(溶二磷脂酸)，这是MVB(多囊泡体)内部小泡中的一种主要磷脂。标记的EAP20呈弥漫性分布，但与CHMP6-GFP共表达时呈点状分布。CHMP6-GFP过表达导致细胞膜表面转铁蛋白受体减少，但导致转铁蛋白受体在细胞质中聚集。泛素化蛋白和内吞的EGF在CHMP6-GFP表达细胞中持续积累。这些结果表明，CHMP6作为ESCRT-II在子宫内膜上的受体，并调节货物的分拣。
CHMP6 (charged multivesicular body protein 6) is a human orthologue of yeast Vps (vacuolar protein sorting) 20, a component of ESCRT (endosomal sorting complex required for transport)-III. Various CHMP6 orthologues in organisms ranging from yeast to humans contain the N-myristoylation consensus sequence at each N-terminus. Metabolic labelling of HEK-293 (human embryonic kidney) cells showed the incorporation of [3H]myristate into CHMP6 fused C-terminally to GFP (green fluorescent protein) (CHMP6-GFP). Interactions of CHMP6 with another ESCRT-III component CHMP4b/Shax [Snf7 (sucrose non-fermenting 7) homologue associated with Alix] 1, one of three paralogues of human Vps32/Snf7, and with EAP20 (ELL-associated protein 20), a human counterpart of yeast Vps25 and component of ESCRT-II, were observed by co-immunoprecipitation of epitope-tagged proteins expressed in HEK-293 cells. The in vitro pull-down assays using their recombinant proteins purified from Escherichia coli demonstrated direct physical interactions which were mediated by the N-terminal basic half of CHMP6. Overexpressed CHMP6-GFP in HeLa cells exhibited a punctate distribution throughout the cytoplasm especially in the perinuclear area, as revealed by fluorescence microscopic analysis. Accumulation of LBPA (lysobisphosphatidic acid), a major phospholipid in internal vesicles of an MVB (multivesicular body), was observed in the CHMP6-GFP-localizing area. FLAG-tagged EAP20 distributed diffusely, but exhibited a punctate distribution on co-expression with CHMP6-GFP. Overexpression of CHMP6-GFP caused reduction of transferrin receptors on the plasma membrane surface, but caused their accumulation in the cytoplasm. Ubiquitinated proteins and endocytosed EGF continuously accumulated in CHMP6-GFP-expressing cells. These results suggest that CHMP6 acts as an acceptor for ESCRT-II on endosomal membranes and regulates cargo sorting.