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Originally published In Press as doi:10.1074/jbc.M201191200 on May 21, 2002

J. Biol. Chem., Vol. 277, Issue 33, 29908-29918, August 16, 2002
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Vesicle-associated Membrane Protein-associated Protein-A (VAP-A) Interacts with the Oxysterol-binding Protein to Modify Export from the Endoplasmic Reticulum*

Jessica P. WylesDagger , Christopher R. McMaster, and Neale D. Ridgway§

From the Atlantic Research Center, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada

Oxysterol-binding protein (OSBP) is 1 of 12 related proteins implicated in the regulation of vesicle transport and sterol homeostasis. A yeast two-hybrid screen using full-length OSBP as bait was undertaken to identify partner proteins that would provide clues to the function of OSBP. This resulted in the cloning of vesicle-associated membrane protein-associated protein-A (VAP-A), a syntaxin-like protein implicated in endoplasmic reticulum (ER)/Golgi vesicle transport, and phospholipid regulation in mammalian cells and yeast, respectively. By using a combination of yeast two-hybrid, glutathione S-transferase pull-down and immunoprecipitation experiments, the VAP-A-binding region in OSBP was localized to amino acids 351-442. This region did not include the pleckstrin homology (PH) domain but overlapped with the N terminus of the oxysterol binding and OSBP homology domains. C- and N-terminal truncations or deletions of VAP prevented interaction with OSBP but did not affect VAP multimerization. Although the OSBP PH domain was not necessary for VAP-A binding in vitro, interaction with VAP-A was enhanced in cells by mutation of the conserved PH domain tryptophan (OSBP W174A) or deletion of the C-terminal half of the PH domain (OSBP Delta 132-182). OSBP W174A retained oxysterol binding activity, association with phospholipid vesicles via the PH domain, and localized with VAP in unusual ER-associated structures. At 40 °C, misfolded ts045-vesicular stomatitis virus G protein fused to green fluorescent protein was co-localized with VAP-A/OSBP W174A structures on the ER but was exported to the Golgi when folded normally at 32 °C. A fluorescent ceramide analogue also accumulated in these ER inclusions, and export to the Golgi was partially inhibited as indicated by decreased Golgi staining and a 30% reduction in sphingomyelin synthesis. These studies show that OSBP binding to the ER and Golgi apparatus is regulated by its PH domain and VAP interactions, and the complex is involved at a stage of protein and ceramide transport from the ER.

* This work was supported in part by Program Grant (to N. D. R. and C. R. M.), Scientist (to N. D. R.), and Scholar (to C. R. M.) awards from the Canadian Institutes of Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of Isaac Walton Killiam and Sumner graduate studentships.

§ To whom correspondence should be addressed: Atlantic Research Center, Dept. of Biochemistry and Molecular Biology, Dalhousie University, 5849 University Ave., Halifax, Nova Scotia B3H 4H7, Canada. Tel.: 902-494-7133; Fax: 902-494-1394; E-mail: nridgway@is.dal.ca.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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