Shibata Hideki, Inuzuka Tatsutoshi, Yoshida Haruna, Sugiura Hirofumi, Wada Ikuo, Maki Masatoshi
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Biosci Biotechnol Biochem. 2010;74(9):1819-26. doi: 10.1271/bbb.100215. Epub 2010 Sep 7.
ALG-2, a member of the penta-EF-hand protein family, interacts Ca²+-dependently with a COPII component, Sec31A. In this study, we first established HeLa cells stably expressing green fluorescent protein-fused ALG-2 (GFP-ALG-2) and red fluorescent protein-fused Sec31A (Sec31A-RFP). After inducing Ca²+-mobilization, the cytoplasmic distribution of GFP-ALG-2 changed from a diffuse to a punctate pattern, which extensively overlapped with the Sec31A-RFP-positive structures, indicating that ALG-2 is recruited to the endoplasmic reticulum exit sites (ERES) in living cells. Next, overlay experiments with biotin-labeled ALG-2 were done to dissect the ALG-2 binding site (ABS). They revealed that a sequence comprising amino acid residues 839-851 in the Pro-rich region was necessary and sufficient for direct binding to ALG-2. Finally, fluorescence recovery after photobleaching analysis indicated that the ABS deletion reduced the high-affinity population of Sec31A to the ERES, suggesting that the ABS is one of the key determinants of the retention kinetics of Sec31A at ERES.
ALG-2是五聚EF手蛋白家族的成员，它与II型COP（Sec31A）的一个组分以Ca²⁺依赖的方式相互作用。在本研究中，我们首先构建了稳定表达绿色荧光蛋白融合的ALG-2（GFP-ALG-2）和红色荧光蛋白融合的Sec31A（Sec31A-RFP）的HeLa细胞。诱导Ca²⁺动员后，GFP-ALG-2的细胞质分布从弥散型变为点状，与Sec31A-RFP阳性结构广泛重叠，表明ALG-2在活细胞中被募集到内质网出口位点（ERES）。接下来，进行了生物素标记的ALG-2的覆盖实验，以剖析ALG-2结合位点（ABS）。结果显示，富含脯氨酸区域中包含氨基酸残基839 - 851的序列对于直接结合ALG-2是必要且充分的。最后，光漂白后荧光恢复分析表明，ABS缺失降低了Sec31A在内质网出口位点的高亲和力群体，提示ABS是Sec31A在内质网出口位点保留动力学的关键决定因素之一。
