The ALG-2 Binding Site in Sec31A Influences the Retention Kinetics of Sec31A at the Endoplasmic Reticulum Exit Sites as Revealed by Live-Cell Time-Lapse Imaging
The ALG-2 Binding Site in Sec31A Influences the Retention Kinetics of Sec31A at the Endoplasmic Reticulum Exit Sites as Revealed by Live-Cell Time-Lapse Imaging
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DOI：
10.1271/bbb.100215
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发表时间：
2010-09-01
影响因子：
1.6
通讯作者：
Maki, Masatoshi
中科院分区：
文献类型：
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作者：
Shibata, Hideki;Inuzuka, Tatsutoshi;Maki, Masatoshi
ALG-2是penta-EF-hand蛋白家族的成员，与COPII组分Sec31A依赖性地相互作用。在本研究中，我们首先建立了稳定表达绿色荧光蛋白融合ALG-2（GFP-ALG-2）和红色荧光蛋白融合Sec31 A（Sec31 A-RFP）的HeLa细胞。在诱导Ca2+动员后，GFP-ALG-2的胞质分布从弥漫性变为点状，其与Sec31A-RFP阳性结构广泛重叠，表明ALG-2被募集到活细胞中的内质网出口位点（ERES）。接下来，用生物素标记的ALG-2进行覆盖实验以剖析ALG-2结合位点（ABS）。他们揭示了在富含Pro的区域中包含氨基酸残基839 - 851的序列对于直接结合ALG-2是必需的并且是足够的。最后，光漂白分析后的荧光恢复表明，ABS缺失减少了Sec31A的ERES的高亲和力人口，这表明ABS是Sec31A在ERES的保留动力学的关键决定因素之一。
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.