The mechanism of the Action of the calcium-binding protein ALG-2on the regulation of cell membrane receptor sorting

ALG-2是一种5-EF手性钙结合蛋白。为了探索ALG-2的生理功能，我们寻找了新的ALG-2相互作用蛋白。通过免疫荧光显微镜分析，我们发现参与内质网高尔基体转运的COPII组分Sec31a以钙依赖的方式与ALG-2结合，并与ALG-2共定位于内质网出口部位。令人惊讶的是，ALG-2的选择性剪接的deltaGF-122亚型不与Alix结合，但保留了与Sec31a结合的能力。我们还发现，磷脂扰乱酶3(PLSCR3)有两个ALG-2结合位点：一个(ABS1)类似于Alix结合部位，另一个(ABS2)类似于Sec31a-结合，因为deltaGF-122亚型保留了结合能力。PLSCR3中的第二个ALG-2结合位点不具有作为必需氨基酸残基的Tyr。ALG-2的突变分析表明，ABS I和ABS2可能与ALG-2的不同表面相互作用。我们将ALG-2结合蛋白分为Alix型和非Alix型两类。我们阐明了ALG-2无钙形式和Alix多肽复合体的三维结构。我们分析了HD-PTP和Brox作为含有Brol结构域的Alix的同源基因。HD-PTP和Brox都能结合CHMP4蛋白。HD-PTP具有与ALG-2和内嗜性蛋白相关的富含Pro的区域，而Brox没有这样的区域，但在其C末端具有甲基化位点。Brox被发现以外切体的形式分泌。在ESCRT研究过程中，我们发现了一个新的ESCRT-III相关蛋白，并将其命名为CHMP7。绿色荧光蛋白(GFP)融合CHMP7的过表达抑制了小鼠白血病病毒(MuLV)Gag蛋白的病毒样颗粒(VLP)的释放，提示在货物蛋白的内体分选中起作用

ALG-2 is a penta-EF-hand calcium-binding protein. To explore physiological functions of ALG-2, we searched for new interacting proteins of ALG-2. We identified Sec31A, a component of COPII working in ER-Golgi transport, associates with ALG-2 in a calcium-dependent manner and it colocalized with ALG-2 in the ER exit site by immunofluorescence microscopic analysis. Surprisingly, the alternatively spliced deltaGF-122 isoform of ALG-2 does not bind Alix but retains the capacity of binding to Sec31A. We also found that phospholipid scramblase 3 (PLSCR3) possesses two ALG-2-binding sites : one (ABS1) is similar to Alix-binding site and the other (ABS2) is similar to the Sec31A-binding in that the deltaGF-122 isoform retains the binding capacity. The second ALG-2-binding site in PLSCR3 does not possess Tyr as an essential amino acid residue. Mutational analyses of ALG-2 reveal that ABS I and ABS2 may interact with different surfaces of ALG-2. We classified ALG-2-bidning proteins as two classes : Alix-type and non-Alix type. We elucidated the 3D-structures of ALG-2 in calcium-free form and Alix peptide complex form. We analyzed HD-PTP and Brox as paralogues of Alix that contain Brol domain. Both HD-PTP and Brox bind CHMP4 proteins. While HD-PTP possesses Pro-rich region that associate with ALG-2 and endophilin, Brox lacks such a domain but possesses a famesylation site at its C-terminus. Brox was found to be secreted as an exosome. During the course of ESCRT studies, we identified a novel ESCRT-III related protein and named it CHMP7. Overexpression of green fluorescent protein(GFP) fused CHMP7 suppressed release of virus-like particle (VLP) of murine leukemia virus (MuLV) gag protein, suggesting roles in endosomal sorting of cargo proteins