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Originally published In Press as doi:10.1074/jbc.M009424200 on January 25, 2001

J. Biol. Chem., Vol. 276, Issue 16, 13127-13135, April 20, 2001
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Gaf-1, a gamma -SNAP-binding Protein Associated with the Mitochondria*

Dong ChenDagger §, Weidong Xu§, Ping HeDagger , Estela E. Medrano, and Sidney W. WhiteheartDagger ||

From the Dagger  Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536 and the  Roy M. and Phyllis Gough Huffington Center on Aging, Departments of Molecular and Cellular Biology and Dermatology, Baylor College of Medicine, Veterans Affairs Medical Center, Houston, Texas 77030

The role of alpha /beta -SNAP (Soluble NSF Attachment Protein) in vesicular trafficking is well established; however, the function of the ubiquitously expressed gamma -SNAP remains unclear. To further characterize the cellular role of this enigmatic protein, a two-hybrid screen was used to identify new, gamma -SNAP-binding proteins and to uncover potentially novel functions for gamma -SNAP. One such SNAP-binding protein, termed Gaf-1 (gamma -SNAP associate factor-1) specifically binds gamma - but not alpha -SNAP. The full-length Gaf-1 (75 kDa) is ubiquitously expressed and is found stoichiometrically associated with gamma -SNAP in cellular extracts. This binding is distinct from other SNAP interactions since no alpha -SNAP or NSF coprecipitated with Gaf-1. Subcellular fractionation and immunofluorescence analysis show that Gaf-1 is peripherally associated with the outer mitochondrial membrane. Only a fraction of gamma -SNAP was mitochondrial with the balance being either cytosolic or associated with other membrane fractions. GFP-gamma -SNAP and the C-terminal domain of Gaf-1 both show a reticular distribution in HEK-293 cells. This reticular structure colocalizes with Gaf-1 and mitochondria as well as with microtubules but not with other cytoskeletal elements. These data identify a class of gamma -SNAP interactions that is distinct from other members of the SNAP family and point to a potential role for gamma -SNAP in mitochondrial dynamics.

* This work was supported by Grant HL56652 from the NHLBI, National Institutes of Health (to S. W. W.), Grant 0020471B from the American Heart Association, Ohio Valley Affiliate (to D. C.), and Grant AG-3663 from the NIA, National Institutes of Health (to E. E. M.).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.

§ The first two authors contributed equally to this work.

|| To whom correspondence should be addressed: Dept. of Biochemistry, University of Kentucky College of Medicine, 800 Rose St., Lexington, KY 40536. Tel.: 859-257-4882; Fax: 859-323-1037; E-mail: whitehe@pop.uky.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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