The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting
The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting
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DOI：
10.1074/jbc.m301604200
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发表时间：
2003-10-03
影响因子：
4.8
通讯作者：
Maki, M
中科院分区：
文献类型：
--
作者：
Katoh, K;Shibata, H;Maki, M
阿利克斯（ALG-2相互作用蛋白X）是一种95 kDa的蛋白质，通过其C-末端富含脯氨酸的区域与EF-手型Ca 2+结合蛋白ALG-2（骨化相关基因2）相互作用。在这项研究中，我们通过使用酵母双杂交筛选来寻找与人AlixDeltaC（不含C末端区域的截短形式）相互作用的蛋白质，并且我们鉴定了两种类似的人蛋白质CHMP 4a和CHMP 4 b（染色质修饰蛋白;带电多泡体蛋白）作为阿利克斯的新型结合伴侣。通过谷胱甘肽S-转移酶下拉试验和免疫共沉淀实验证实了阿利克斯与CHMP 4 b的相互作用。荧光显微镜分析显示，CHMP 4 b在HeLa细胞中的瞬时表达主要呈点状分布于核周区，并与共表达的阿利克斯共定位。CHMP 4 b的分布与早期和晚期内体标记蛋白EEA 1（早期内体抗原1）和Lamp-1（溶酶体膜蛋白-1）的分布分别部分重叠。CHMP 4 b的瞬时过表达诱导泛素化蛋白的积累，作为点状图案，部分重叠的CHMP 4 b的分布，并抑制内吞的表皮生长因子的消失。相反，在HEK 293细胞中稳定表达的CHMP 4 b在细胞质中弥漫地观察到。然而，在稳定表达CHMP 4 b的细胞中，AlixDeltaC的瞬时过表达诱导了其中CHMP 4 b和AlixDeltaC共定位的囊泡样结构的形成。SKD 1（E235 Q）是AAA型ATP酶SKD 1的显性负性形式，在内吞途径中起关键作用，与CHMP 4 b共免疫沉淀。此外，CHMP 4 b与SKD 1 E235 Q共定位在核周区的点状图案，并且阿利克斯被诱导表现出与SKD 1 E235 Q在HeLa细胞中重叠的点状分布。这些结果表明CHMP 4 b和阿利克斯通过与SKD 1协同作用参与多泡体的形成。
Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca2+-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixDeltaC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC were colocalized. SKD1(E235Q), a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1E235Q as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1E235Q in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1.