Yamasaki Akinori, Tani Katsuko, Yamamoto Akitsugu, Kitamura Naomi, Komada Masayuki
Department of Biological Sciences, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Mol Biol Cell. 2006 Nov;17(11):4876-87. doi: 10.1091/mbc.e06-05-0444. Epub 2006 Sep 6.
The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca(2+)-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca(2+)-dependent manner and colocalized with Sec31A at ER exit sites. A Ca(2+) binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca(2+) chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca(2+)-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.
从内质网（ER）出口位点出芽形成的运输小泡的形成依赖于由三种成分组成的COPII衣被：小GTP酶Sar1、Sec23/24复合体和Sec13/31复合体。在此，我们提供证据表明凋亡相关基因2（ALG-2），一种功能未知的Ca(2+)结合蛋白，在哺乳动物细胞的ER出口位点调节COPII功能。ALG-2以Ca(2+)依赖的方式与Sec31A的富含脯氨酸区域结合，Sec31A是酵母Sec31普遍表达的哺乳动物同源物，并与Sec31A在ER出口位点共定位。一个不与Sec31A结合的Ca(2+)结合缺陷型ALG-2突变体失去了定位于ER出口位点的能力。Sec31A富含脯氨酸区域的过表达或RNA干扰介导的Sec31A缺失也消除了ALG-2在这些位点的定位。相反，ALG-2的缺失显著降低了与ER出口位点膜相关的Sec31A水平。最后，用细胞可渗透的Ca(2+)螯合剂处理导致ALG-2定位错误，同时ER出口位点的Sec31A水平降低。我们得出结论，ALG-2通过与Sec31A的Ca(2+)依赖相互作用被招募到ER出口位点，进而稳定Sec31A在这些位点的定位。
