The Transmembrane Domain of Vam3 Affects the Composition ofcis- and trans-SNARE Complexes to Promote Homotypic Vacuole Fusion

doi:10.1074/jbc.M209522200

The Transmembrane Domain of Vam3 Affects the Composition ofcis- and trans-SNARE Complexes to Promote Homotypic Vacuole Fusion*

From the ^‡Interdisziplinäres Zentrum für Neurowissenschaften (IZN), University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany, the ^§Biochemie-Zentrum Heidelberg (BZH), University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany, and the ^¶Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85345 Freising, Germany

Abstract

It is presently not clear how the function of SNARE proteins is affected by their transmembrane domains. Here, we analyzed the role of the transmembrane domain of the vacuolar SNARE Vam3 by replacing it by a lipid anchor. Vacuoles with mutant Vam3 fuse poorly and have increased amounts of cis-SNARE complexes, indicating that they are more stable. As a consequence efficientcis-SNARE complex disassembly that occurs at priming as a prerequisite of fusion requires addition of exogenous Sec18.trans-SNARE complexes in this mutant accumulate up to 4-fold over wild type, suggesting that the transmembrane domain of Vam3 is required to transit through this step. Finally, palmitoylation of Vac8, a reaction that also occurs early during priming is reduced by almost one-half. Since palmitoylated Vac8 is required beyondtrans-SNARE complex formation, this may partially explain the fusion deficiency.

Footnotes

↵* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (LA 699/8-1 (to D. L. and C. U.) and by Boehringer Ingelheim Fonds (to L. D.).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.
↵‖ To whom correspondence may be addressed: Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85345 Freising, Germany. Tel.: 49-8161-713500; Fax: 49-8161-4404; E-mail: biopolymere@bl.tum.de (for D. L.) or Biochemie Zentrum Heidelberg, University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Tel.: 49-6221-544180; Fax: 49-6221-544366; E-mail: cu2@ix.urz.uni-heidelberg.de (for C. U.).
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M209522200
↵2 J. Rohde, L. Dietrich, D. Langosch, and C. Ungermann, unpublished results.
Abbreviations:

SNARE

soluble NSF attachment protein receptor (where NSF isN-ethylmaleimide-sensitive factor)

SNAP

soluble NSF attachment protein

TMD

transmembrane domain

PIPES

1,4-piperazinediethanesulfonic acid

BAPTA

1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

GTPγS

guanosine 5′-O-(thiotriphosphate)
- Received September 17, 2002.
The American Society for Biochemistry and Molecular Biology, Inc.