The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix/AIP1
The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix/AIP1
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DOI：
10.1093/jb/mvh014
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发表时间：
2004-01-01
影响因子：
2.7
通讯作者：
Maki, M
中科院分区：
文献类型：
--
作者：
Shibata, H;Yamada, K;Maki, M
ag -2是一种Ca2+结合蛋白，属于penta-EF-hand蛋白家族，并以Ca2+依赖的方式与几种蛋白（包括膜联蛋白VII、膜联蛋白X1和Alix/AIP1）结合。利用酵母双杂交系统和生物素标记的ALG-2覆盖实验来表征ALG-2与Alix的相互作用。在Alix的羧基末端富含脯氨酸的区域，794 - 827氨基酸残基对应的区域足以赋予与ALG-2直接作用的能力。该区域包括四串联PxY重复序列。丙氨酸替换表明，该区域的7个脯氨酸残基、PxY重复序列中的4个和PxY重复序列中的4个酪氨酸残基对于与ALG-2的结合亲和力至关重要。内源性ALG-2在Ca2+存在下与flag标记的Alix或flag标记的AlixDeltaEBS（一种缺乏亲内蛋白结合一致序列的缺失突变体）共免疫沉淀，但与flag标记的AlixDeltaABS（另一种缺乏包含氨基酸798-841区域的突变体）共免疫沉淀，这些突变体来自转染了每种flag标记的蛋白表达构建体的HEK293细胞的裂解物。在HEK293细胞中过度表达的flag标记的ALG-2也以Ca2+依赖的方式与Alix共同免疫沉淀，而flag标记的ALG-2（E47A/E114A）是ALG-2的Ca2+结合缺陷突变体，即使在Ca2+存在的情况下，在Alix的免疫沉淀中也未检测到。利用与绿色荧光蛋白（GFP-AlixCT）融合的Alix的羧基端半部分进行荧光显微镜分析，发现HeLa细胞内源性ALG-2与外源性表达的GFP-AlixCT呈点状重叠，GFP-AlixCTDeltaABS在细胞质中弥漫性分布。这些结果表明，ABS在Alix中对AlixCT的有效积累是必需的，并提出了ALG-2通过与Alix的Ca2+依赖性相互作用参与膜运输的可能性。
ALG-2 is a Ca2+-binding protein that belongs to the penta-EF-hand protein family and associates with several proteins, including annexin VII, annexin X1, and Alix/AIP1, in a Ca2+-dependent manner. The yeast two-hybrid system and a biotin-tagged ALG-2 overlay assay were carried out to characterize the interaction between ALG-2 and Alix. The region corresponding to amino acid residues 794 to 827 in the carboxy-terminal proline-rich region of Alix was sufficient to confer the ability to interact directly with ALG-2. This region includes four-tandem PxY repeats. Alanine substitutions indicated that seven proline residues in this region, four in the PxY repeats, and four tyrosine residues in the PxY repeats are crucial for the binding affinity with ALG-2. Endogenous ALG-2 was co-immunoprecipitated in the presence of Ca2+ with FLAG-tagged Alix or FLAG-tagged AlixDeltaEBS, a deletion mutant lacking the endophilin binding consensus sequence, but not with FLAG-tagged AlixDeltaABS, another mutant lacking the region comprising amino acids 798-841, from the lysates of HEK293 cells transfected with each FLAG-tagged protein expression construct. FLAG-tagged ALG-2 overexpressed in HEK293 cells was also co-immunoprecipitated with Alix in a Ca2+-dependent fashion, whereas FLAG-tagged ALG-2(E47A/E114A), a Ca2+-binding deficient mutant of ALG-2, was not detected in the immunoprecipitates of Alix even in the presence of Ca2+. Fluorescent microscopic analyses using the carboxy-terminal half of Alix fused with green fluorescent protein (GFP-AlixCT) revealed that endogenous ALG-2 in HeLa cells exhibits a dot-like pattern overlapping with exogenously expressed GFP-AlixCT, and the distribution of GFP-AlixCTDeltaABS is observed diffusely in the cytoplasm. These results indicate the requirement of ABS in Alix for the efficient accumulation of AlixCT and raise the possibility that ALG-2 participates in membrane trafficking through a Ca2+-dependent interaction with Alix.