ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in a Ca2+-dependent manner

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Abstract

Intracellular localization of the penta-EF-hand Ca2+-binding protein ALG-2 in HeLa cells was investigated by immunofluorescent confocal microscopy using a polyclonal antibody. In addition to its presence in the nucleus, ALG-2 was found to be distributed in a punctate pattern in the cytoplasm, where it was partly co-stained with an endoplasmic reticulum (ER) exit site marker p125. In vitro GST pull down analysis demonstrated that ALG-2 and its alternatively spliced isoform interact with the COPII component Sec31A in a Ca2+-dependent manner, and a biotin-labeled ALG-2 overlay assay revealed direct binding of ALG-2 to Sec31A. Biochemical and immunofluorescent microscopic analyses showed that ALG-2 was enriched at the Sec31A-localizing membrane compartments upon stimulation with the Ca2+ ionophore A23187. In contrast, treatment of cells with the membrane-permeant Ca2+ chelator BAPTA-AM led to a dispersion of ALG-2 throughout the cells and to a significant loss of Sec31A in the perinuclear region. These findings establish Sec31A as a novel target for ALG-2 and provide a framework for studies on the roles of ALG-2 in ER-Golgi transport.

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Materials and methods

Antibodies. Polyclonal antibodies against the purified recombinant human ALG-2 were raised in rabbits (Keari, Osaka, Japan). The antiserum (5 ml) was mixed with an equal volume of TBS-Ca (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1 mM CaCl2) and loaded onto an affinity column of ALG-2 that had been immobilized to a HiTrap NHS-activated column (1 ml, Amersham–Pharmacia). After washing the column successively with TBS-Ca, antibodies were eluted with 100 mM glycine–HCl, pH 3.0, and then the eluate was

Specificity of the anti-ALG-2 polyclonal antibody and staining pattern of ALG-2 in HeLa cells

A polyclonal anti-ALG-2 antiserum was generated in rabbits by using recombinant human ALG-2 as an antigen, and a specific anti-ALG-2 polyclonal antibody (pAb) was purified by affinity chromatography using an ALG-2-immobilized column in the presence of Ca2+. By Western blotting, a single band at the expected size of ALG-2 was recognized by the purified pAb in HeLa cell lysates, whereas no corresponding band was detected for the lysates from a clonal cell-line of HeLa cells that were stably

Discussion

ALG-2 has been shown to be localized both in the cytoplasm and in the nucleus [12], [18]. In this study, we demonstrated that a subset of ALG-2 localized to p125- and Sec31A-positive structures, which are principally ER exit sites, in HeLa cells under normal cell culture conditions (Fig. 2, Fig. 4A). In BAPTA-AM-treated cells, the staining pattern of p125 was essentially the same as that under normal culture conditions, whereas ALG-2 signals were excluded from the punctate dots of p125 and

Acknowledgments

We thank Dr. K. Tani (Tokyo University of Pharmacy and Life Science) for providing valuable materials. We are grateful to Y. Sano (Nagoya University) and T. Kakiuchi (Nagoya University) for their earlier works related to this study and to Dr. K. Hitomi (Nagoya University) for his encouraging suggestions. This work was supported by a Grant-in-Aid for Scientific Research B (to M.M.) and a Grant-in-Aid for Young Scientist B (to H.S.).

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