CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway
CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway
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DOI：
10.1042/bj20060897
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发表时间：
2006-11-15
影响因子：
4.1
通讯作者：
Maki, Masatoshi
中科院分区：
文献类型：
--
作者：
Horii, Mio;Shibata, Hideki;Maki, Masatoshi
迄今为止报道的所有CHMP（带电多泡体蛋白）都具有共同的特征：它们都含有约100个氨基酸。200个氨基酸残基，具有卷曲螺旋区域，并且具有带电残基的偏置分布（碱性N-末端和酸性C-末端的一半）。酵母直向同源物的CHMP，包括ESCRT-III组件Snf 7，需要货物蛋白的分选多泡体的管腔内囊泡。我们已经表征了一种新的人ESCRT-III相关蛋白，命名为CHMP 7，它由453个氨基酸残基组成。CHMP 7在其C-末端的一半和N-末端的一半分别含有SNF 7结构域和与SNF 7相关的远距离结构域。在先前分类为6个亚家族（CHMP 1-CHMP 6）的10种CHMP蛋白中，CHMP 7的C-末端SNF 7结构域与CHMP 6的SNF 7结构域最相似，其与CHMP 4蛋白和ESCRT-II的组分EAP 20相关。使用过表达Strep标记的CHMP 7和GFP（绿色荧光蛋白）融合的CHMP 4 b（也称为Shax 1）的HEK-293 T（人胚肾）细胞的裂解物的下拉测定揭示了CHMP 7的C-末端一半和CHMP 4 b之间的正相互作用。然而，在CHMP 7和EAP 20之间未观察到相互作用。共聚焦荧光显微镜分析显示，FLAG-CHMP 7在HeLa细胞中弥漫分布在整个细胞质中，但有一些积累，特别是在核周区域。通过CHMP 4 b-GFP或GFP-Vps 4 B（E235 Q）（AAA（与各种细胞活性相关的ATP酶）Vps 4 B的显性负突变体）的过表达，FLAG-CHMP 7的分布被改变为细胞质点状模式，并与它们部分共定位。泛素化蛋白和内吞的EGF在GFP-CHMP 7表达细胞中积累。在从瞬时表达MLV（鼠白血病病毒）Gag蛋白的HEK-293 T细胞释放病毒样颗粒中也观察到过表达的GFP-CHMP 7的显性负效应。这些结果表明，CHMP 7，一种新的CHMP 4相关的ESCRT-III相关蛋白，在内体分选途径中发挥作用。
All CHMPs (charged multivesicular body proteins) reported to date have common features: they all contain approx. 200 amino acid residues, have coiled-coil regions and have a biased distribution of charged residues (basic N-terminal and acidic C-terminal halves). Yeast orthologues of CHMPs, including an ESCRT-III component Snf7, are required for the sorting of cargo proteins to intraluminal vesicles of multivesicular bodies. We have characterized a novel human ESCRT-III-related protein, designated CHMP7, which consists of 453 amino acid residues. CHMP7 contains an SNF7 domain and a distantly SNF7-related domain in its C-terminal half and N-terminal half respectively. Among the ten CHMP proteins classified previously in six subfamilies (CHMP1-CHMP6), the C-terminal SNF7 domain of CHMP7 is most similar to the SNF7 domain of CHMP6, which associates with CHMP4 proteins and EAP20, a component of ESCRT-II. Pull-down assays using lysates of HEK-293T (human embryonic kidney) cells that overexpressed Strep-tagged CHMP7 and GFP (green fluorescent protein)-fused CHMP4b (also named Shax1) revealed a positive interaction between the C-terminal half of CHMP7 and CHMP4b. However, interaction was not observed between CHMP7 and EAP20. Confocal fluorescence microscopic analyses revealed that FLAG-CHMP7 is distributed in HeLa cells diffusely throughout the cytoplasm, but with some accumulation, especially in the perinuclear area. The distribution of FLAG-CHMP7 was altered to a cytoplasmic punctate pattern by overexpression of either CHMP4b-GFP or GFP-Vps4B(E235Q), a dominant-negative mutant of the AAA (ATPase associated with various cellular activities) Vps4B, and partially co-localized with them. Ubiquitinated proteins and endocytosed EGF accumulated in GFP-CHMP7-expressing cells. A dominant-negative effect of overexpressed GFP-CHMP7 was also observed in the release of virus-like particles from HEK-293T cells that transiently expressed the MLV (murine leukaemia virus) Gag protein. These results suggest that CHMP7, a novel CHMP4-associated ESCRT-III-related protein, functions in the endosomal sorting pathway.