Identification of Alix-type and non-Alix-type ALG-2-binding sites in human phospholipid scramblase 3 - Differential binding to an alternatively spliced isoform and amino acid-substituted mutants
Identification of Alix-type and non-Alix-type ALG-2-binding sites in human phospholipid scramblase 3 - Differential binding to an alternatively spliced isoform and amino acid-substituted mutants
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DOI：
10.1074/jbc.m800717200
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发表时间：
2008-04-11
影响因子：
4.8
通讯作者：
Maki, Masatoshi
中科院分区：
文献类型：
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作者：
Shibata, Hideki;Suzuki, Hironori;Maki, Masatoshi
ALG-2是penta-EF-hand蛋白家族的一个原型成员，与Alix在其c端富含pro的区域相互作用，该区域包含四个串联PXY重复序列。人磷脂重组酶3 （PLSCR3）在其n端区域具有类似的序列（ABS-1）。在本研究中，我们发现ag -2与HEK293细胞中表达的PLSCR3以Ca2+依赖的方式相互作用，通过共免疫沉淀、谷胱甘肽s -转移酶（GST）融合ag -2的下拉和生物素标记ag -2的覆盖试验。GST-ALG-2异构体的GST融合蛋白GST-ALG-2（Delta GF122）可以拉低与绿色荧光蛋白（GFP）融合的PLSCR3，但不能拉低GFP Alix。删除含有ABS-1的区域并不足以废除这种结合。第二个ALG-2结合位点（ABS-2）是与ALG-2相互作用所必需的（Delta GF122）。利用合成寡肽和重组蛋白，利用表面等离子体共振生物传感器进行实时相互作用分析，证实了ABS-1与ALG-2、ABS-2与ALG-2以及ALG-2的直接依赖Ca2+结合（Delta GF122）。ABS-2的序列包含多个脯氨酸和两个苯丙氨酸，其中Phe（49）被发现是关键的，因为它被Ala或Tyr取代，导致使用寡肽固定化小珠的下拉实验失去了结合能力。根据与ALG-2（Delta GF122）的结合能力，将ALG-2相互作用蛋白分为两组：(i)异构体非相互作用（ABS-1）型，包括Alix、膜联蛋白A7、膜联蛋白A11和TSG101； （ii）异构体相互作用（ABS-2）型，包括PLSCR3、PLSCR4和Sec31A。使用单氨基酸取代的ALG-2突变体进行的gst -pull - down分析显示，两组之间的结合特异性存在差异，这表明ALG-2配体复合物形成的结构具有灵活性。
ALG-2, a prototypic member of the penta-EF-hand protein family, interacts with Alix at its C-terminal Pro-rich region containing four tandem PXY repeats. Human phospholipid scramblase 3 (PLSCR3) has a similar sequence (ABS-1) in its N-terminal region. In the present study, we found that ALG-2 interacts with PLSCR3 expressed in HEK293 cells in a Ca2+-dependent manner by co-immunoprecipitation, pulldown with glutathione S-transferase (GST) fused ALG-2 and an overlay assay using biotin-labeled ALG-2. The GST fusion protein of an alternatively spliced isoform of ALG-2, GST-ALG-2(Delta GF122), pulled down green fluorescent protein (GFP)-fused PLSCR3 but not GFP Alix. Deletion of a region containing ABS-1 was not sufficient to abrogate the binding. A second ALG-2-binding site (ABS-2) was essential for interaction with ALG-2(Delta GF122). Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca2+-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2(Delta GF122). The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe(49) was found to be critical, because its substitution with Ala or Tyr caused a loss of binding ability by pulldown assays using oligopeptide-immobilized beads. ALG-2-interacting proteins were classified into two groups based on binding ability to ALG-2(Delta GF122): (i) isoform-non-interactive (ABS-1) types, including Alix, annexin A7, annexin A11, and TSG101 and (ii) isoform-interactive (ABS-2) types including PLSCR3, PLSCR4 and Sec31A. GST-pulldown assays using single amino acid-substituted ALG-2 mutants revealed differences in binding specificities between the two groups, suggesting structural flexibility in ALG-2-ligand complex formation.