A dual mechanism controlling the localization and function of exocytic v-SNAREs - PubMed (original) (raw)

. 2003 Jul 22;100(15):9011-6.

doi: 10.1073/pnas.1431910100. Epub 2003 Jul 9.

Rachel Rudge, Marcella Vacca, Graça Raposo, Jacques Camonis, Véronique Proux-Gillardeaux, Laurent Daviet, Etienne Formstecher, Alexandre Hamburger, Francesco Filippini, Maurizio D'Esposito, Thierry Galli

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

Sonia Martinez-Arca et al. Proc Natl Acad Sci U S A. 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-3 delta, 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.

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Figures

Fig. 1.

Fig. 1.

The LD inhibits SNARE-complex formation in vitro and in vivo. (a) Scheme of the structure of TI-VAMP. TMD, transmembrane domain. (b and c) Quantitative in vitro assay to measure the interaction between recombinant syntaxin 1 and SNAP25 with TI-VAMP or ΔLongin-TIVAMP. The graphic shows one representative experiment of three independent experiment. (d and e) HeLa cells transfected with GFP-TIVAMP or GFP-ΔLongin-TIVAMP (d) or with GFP-Syb2 or GFP-Longin/Syb2 (e), and treated as described in Fig. 6_c_, were lysed and immunoprecipitated with an anti-GFP antibody. Coimmunoprecipitated SNAREs were detected by Western blot and quantified by densitometry. The histograms show the statistical analyses of at least three independent experiments. *, P < 0.05, Student's t test.

Fig. 4.

Fig. 4.

Direct interaction between AP-3δ and the LD. (a) Results of a yeast two-hybrid screening using either the Longin or the cytoplasmic domain of Drosophila or human TI-VAMP as baits. SNAP-29, a yet unknown partner of TI-VAMP, was also detected both in the human and in the_Drosophila_ screening, and biochemical experiments validated this interaction (unpublished observations). The red and green lines highlight the intersection and total coverage of all prey clones identified in the yeast two-hybrid screen, respectively. (b) HeLa cells transfected with the GFP-fusion constructs indicated were crosslinked, lysed, and immunoprecipitated with an anti-GFP antibody. Coimmunoprecipitated proteins were detected by Western blot. (c) HeLa cells fixed and double stained for endogenous TI-VAMP and AP-1, AP-2, and AP-3 are shown. (Bar, 15 μm.)

Fig. 2.

Fig. 2.

The LD controls the intracellular localization of TI-VAMP. (a) HeLa cells transfected with GFP-TIVAMP or GFP-ΔLongin/TIVAMP were either directly fixed (control) or fixed after sucrose treatment (sucrose). Note the swelling of GFP-TIVAMP-positive vesicles, but not that of GFP-ΔLongin-TIVAMP-positive vesicles (Insets). (Bar, 10 μm; bar in Insets,7 μm.) (b) Ultrathin cryosections of MDCK cells expressing GFP-TIVAMP and GFP-Δ Longin-TIVAMP were ImmunoGold-labeled with anti-GFP antibodies and PAG10. TI-VAMP localizes to the limiting membrane of lysosomal compartments (arrows). Occasionally, TI-VAMP is detected in small closely apposed vesicles (arrowheads). GFP-Δ Longin-TIVAMP localizes to tubular vesicular membrane structures (arrows). (Bars, 200 nm.)

Fig. 3.

Fig. 3.

The LD controls the intracellular localization of v-SNAREs. HeLa cells transfected with the constructs indicated were fixed and stained for the endosomal markers CD63 and TfR. Arrows point to structures labeled by both the GFP-fusion protein and either CD63 or TfR. Arrowheads point to structures positive for only one of the proteins. (Bar, 13 μm.)

Fig. 5.

Fig. 5.

Localization and function of TI-VAMP depends on its interaction with functional AP-3 complex. (a) Mocha cells were transfected with either GFP-TIVAMP or GFP-ΔLongin-TIVAMP alone, or were cotransfected with AP-3δ. GFP-TIVAMP and GFP-ΔLongin-TIVAMP were detected by direct GFP fluorescence (green), whereas AP-3δ (blue) and TfR (red) were detected by coimmunolabeling. Merge, the level of colocalization between the TfR and GFP-TIVAMP or GFP-ΔLongin-TIVAMP. (b) Scheme of the structure of TI-VAMP compared with TI-VAMPc. Isoform c lacks residues 28–68 (red box). (c) HeLa cells transfected with GFP-TIVAMPc were fixed and stained for endogenous CD63. (d) HeLa cells transfected with the GFP-fusion constructs indicated were crosslinked, lysed, and immunoprecipitated with an anti-GFP antibody. Coimmunoprecipitated proteins were detected by Western blot. (e) HeLa cells transfected with the GFP-fusion constructs indicated were incubated with NEM as in Fig. 6_c_, lysed, and immunoprecipitated with a mouse monoclonal anti-GFP antibody. Coimmunoprecipitated SNAREs were detected by Western blot. (Bar in a, 10 μm; bar in c, 13 μm.)

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