A dual mechanism controlling the localization and function of exocytic v-SNAREs

doi:10.1073/pnas.1431910100

A dual mechanism controlling the localization and function of exocytic v-SNAREs

^*Membrane Traffic and Neuronal Plasticity, Institut National de la Santé et de la Recherche Médicale U536, Institut du Fer-à-Moulin, 75005 Paris, France; ^†Institute of Genetics and Biophysics “A. Buzzati Traverso,” Consiglio Nazionale delle Ricerche, 80125 Naples, Italy; ^‡Unité Mixte de Recherche, 144-Centre National de la Recherche Scientifique, Institut Curie, 75005 Paris, France; ^§Institut National de la Santé et de la Recherche Médicale U528, Institut Curie, 75005 Paris, France; Departments of ^¶Biology and ^∥Bioinformatics, Hybrigenics, 75014 Paris, France; and ^**Molecular Biology and Bioinformatics Unit (MOLBINFO), Department of Biology, University of Padua, 35131 Padua, Italy

Edited by Pietro V. De Camilli, Yale University School of Medicine, New Haven, CT (received for review April 2, 2003)

Abstract

SNARE [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor] proteins are essential for membrane fusion but their regulation is not yet fully understood. We have previously shown that the amino-terminal Longin domain of the v-SNARE TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein)/VAMP7 plays an inhibitory role in neurite outgrowth. The goal of this study was to investigate the regulation of TI-VAMP as a model of v-SNARE regulation. We show here that the Longin domain (LD) plays a dual role. First, it negatively regulates the ability of TI-VAMP and of a Longin/Synaptobrevin chimera to participate in SNARE complexes. Second, it interacts with the adaptor complex AP-3 and this interaction targets TI-VAMP to late endosomes. Accordingly, in mocha cells lacking AP-3δ, TI-VAMP is retained in an early endosomal compartment. Furthermore, TI-VAMPc, an isoform of TI-VAMP lacking part of the LD, does not interact with AP-3, and therefore is not targeted to late endosomes; however, this shorter LD still inhibits SNARE-complex formation. These findings support a mechanism controlling both localization and function of TI-VAMP through the LD and clathrin adaptors. Moreover, they point to the amino-terminal domains of SNARE proteins as multifunctional modules responsible for the fine tuning of SNARE function.

Footnotes

↵ †† To whom correspondence should be addressed. E-mail: galli{at}ifm.inserm.fr.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: NEM, N-ethylmaleimide; SNARE, soluble NSF (NEM-sensitive factor) attachment protein receptor; LD, Longin domain; VAMP, vesicle-associated membrane protein; TI-VAMP, tetanus neurotoxin-insensitive VAMP; TfR, transferrin receptor.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AJ549301).