Horii Mio, Shibata Hideki, Kobayashi Ryota, Katoh Keiichi, Yorikawa Chiharu, Yasuda Jiro, Maki Masatoshi
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Biochem J. 2006 Nov 15;400(1):23-32. doi: 10.1042/BJ20060897.
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.
迄今报道的所有多囊泡体蛋白（CHMPs）都具有共同特征：它们都含有约200个氨基酸残基，具有卷曲螺旋区域，并且带电残基分布不均（碱性的N端和酸性的C端）。CHMPs的酵母同源物，包括ESCRT-III组分Snf7，是货物蛋白分选至多囊泡体内腔囊泡所必需的。我们鉴定了一种新的人类ESCRT-III相关蛋白，命名为CHMP7，它由453个氨基酸残基组成。CHMP7在其C端和N端分别含有一个SNF7结构域和一个与之远缘相关的结构域。在先前分为六个亚家族（CHMP1-CHMP6）的十种CHMP蛋白中，CHMP7的C端SNF7结构域与CHMP6的SNF7结构域最相似，CHMP6与CHMP4蛋白和ESCRT-II的组分EAP20相关联。使用过表达链霉亲和素标签的CHMP7和绿色荧光蛋白（GFP）融合的CHMP4b（也称为Shax1）的人胚肾（HEK-293T）细胞裂解物进行的下拉试验显示CHMP7的C端与CHMP4b之间存在阳性相互作用。然而，未观察到CHMP7与EAP20之间的相互作用。共聚焦荧光显微镜分析显示，FLAG-CHMP7在HeLa细胞中弥漫性分布于整个细胞质中，但有一些聚集，特别是在核周区域。通过过表达CHMP4b-GFP或GFP-Vps4B（E235Q）（一种与各种细胞活动相关的ATP酶（AAA）Vps4B的显性负突变体），FLAG-CHMP7的分布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表皮生长因子（EGF）在表达GFP-CHMP7的细胞中积累。在瞬时表达鼠白血病病毒（MLV）Gag蛋白的HEK-293T细胞释放病毒样颗粒中也观察到过表达的GFP-CHMP7的显性负效应。这些结果表明，CHMP7是一种新的与CHMP4相关的ESCRT-III相关蛋白，在内体分选途径中起作用。
