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Vol. 17, Issue 11, 4876-4887, November 2006

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The Ca²⁺-binding Protein ALG-2 Is Recruited to Endoplasmic Reticulum Exit Sites by Sec31A and Stabilizes the Localization of Sec31A

Akinori Yamasaki^*, Katsuko Tani, Akitsugu Yamamoto, Naomi Kitamura^*, and Masayuki Komada^*

*Department of Biological Sciences, Tokyo Institute of Technology, Yokohama 226-8501, Japan; School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji 192-0392, Japan; and Department of Bio-science, Nagahama Institute of Bio-science and Technology, Nagahama 526-0829, Japan

Submitted May 22, 2006; Revised August 8, 2006; Accepted August 29, 2006
Monitoring Editor: Randy Schekman

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 Ca²⁺-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 Ca²⁺-dependent manner and colocalized with Sec31A at ER exit sites. A Ca²⁺ 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 Ca²⁺ 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 Ca²⁺-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-05-0444) on September 6, 2006.

Address correspondence to: Masayuki Komada (makomada{at}bio.titech.ac.jp)

Abbreviations used: BAPTA-AM, 1,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid-tetrakis acetoxymethyl ester; BFA, brefeldin A; ER, endoplasmic reticulum; GFP, green fluorescent protein; GST, glutathione S-transferase; LBPA, lysobisphosphatidic acid; MVB, multivesicular body; PCR, polymerase chain reaction; PEF, penta-EF-hand; RNAi, RNA interference; siRNA, small interfering RNA; Vps, vacuolar protein sorting; VSVG, vesicular stomatitis virus G protein.

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