Journal of Biological Chemistry
Volume 278, Issue 15, 11 April 2003, Pages 13531-13538
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MEMBRANE TRANSPORT STRUCTURE FUNCTION AND BIOGENESIS
Mapping of Functional Domains of γ-SNAP*

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γ-SolubleN-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP) is capable of stabilizing a 20 S complex consisting of NSF, α-SNAP, and SNAP receptors (SNAREs), but its function in vesicular transport is not fully understood. Our two-hybrid analysis revealed that γ-SNAP, unlike α-SNAP, interacts directly with NSF, as well as Gaf-1/Rip11, but not with SNAREs. Gaf-1/Rip11 is a γ-SNAP-associated factor that belongs to the Rab11-interacting protein family. To gain insight into the molecular basis for the interactions of γ-SNAP with NSF and Gaf-1/Rip11, we determined the regions of the three proteins involved in protein-protein interactions. γ-SNAP bound to NSF via its extreme C-terminal region, and the full-length NSF was needed to interact with γ-SNAP. Both the N-terminal and C-terminal regions of γ-SNAP were required for the binding to Gaf-1/Rip11. Gaf-1/Rip11 bound to γ-SNAP via its C-terminal domain comprising a putative coiled-coil region. Although the C-terminal domain of Gaf-1/Rip11 also interacts with Rab11, the binding of γ-SNAP and Rab11 to Gaf-1/Rip11 was not mutually exclusive. Rather, Gaf-1/Rip11 was capable of serving a link between γ-SNAP and Rab11. A complex comprising γ-SNAP and Gaf-1/Rip11 was disassembled in a process coupled to NSF-mediated ATP hydrolysis, suggesting that the interaction between γ-SNAP and Gaf-1/Rip11 is of functional significance.

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Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M213205200

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This work was supported in part by Grants-in-aid for Scientific Research 13680792, 10215205, and 14380339 from the Ministry of Education, Science, Sports and Culture of Japan, and by ONO Medical Research Foundation.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.