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J. Biol. Chem., Vol. 279, Issue 5, 3627-3634, January 30, 2004
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¶
From the
Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan and
Department of Anatomy II, Asahikawa Medical College, Asahikawa, Hokkaido 078-8510, Japan
Granin-family proteins, including chromogranin A (CgA) and secretogranin III (SgIII), are transported to secretory granules (SGs) in neuroendocrine cells. We previously showed that SgIII binds strongly to CgA in an intragranular milieu and targets CgA to SGs in pituitary and pancreatic endocrine cells. In this study, we demonstrated that with a sucrose density gradient of rat insulinoma-derived INS-1 cell homogenates, SgIII was localized to the SG fraction and was fractionated to the SG membrane (SGM) despite lacking the transmembrane region. With depletion of cholesterol from the SGM using methyl-
-cyclodextrin, SgIII was impaired to bind to the SGM. Both SgIII and CgA were solubilized from the SGM by Triton X-100 in contrast to the Triton X-100 insolubility of carboxypeptidase E. SgIII and carboxypeptidase E strongly bound to the SGM-type liposome in intragranular conditions, but CgA did not. Instead, CgA bound to the SGM-type liposome only in the presence of SgIII. Immunocytochemical and pulse-chase experiments revealed that SgIII deleting the N-terminal lipid-binding region missorted to the constitutive pathway in mouse corticotroph-derived AtT-20 cells. Thus, we suggest that SgIII directly binds to cholesterol components of the SGM and targets CgA to SGs in pituitary and pancreatic endocrine cells.
Received for publication, September 11, 2003 , and in revised form, October 31, 2003.
* This work was supported by grants-in-aid and the 21st Century Center of Excellence Program from the Japanese Ministry of Education, Culture, Sports, Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan. Tel.: 81-27-220-8855; Fax: 81-27-220-8896; E-mail: tstake{at}showa.gunma-u.ac.jp.
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