Syntaxin 7, syntaxin 8, Vti1 and VAMP7 (vesicle-associated membrane protein 7) form an active SNARE complex for early macropinocytic compartment fusion in Dictyostelium discoideum.
The macropinocytic pathway in Dictyostelium discoideum is organized linearly. After actin-driven internalization, fluid material passes sequentially from endosomes to lysosomes, where molecules are degraded and absorbed. Residual material is exocytosed via post-lysosomal compartments. Syntaxin 7 is a SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) protein that is present and active in D. discoideum endosomes [Bogdanovic, Bruckert, Morio and Satre (2000) J. Biol. Chem. 275, 36691-36697]. Here we report the identification of its main SNARE partners by co-immunoprecipitation and MS peptide sequencing. The syntaxin 7 complex contains two co-t-SNAREs [Vti1 (Vps10p tail interactor 1) and syntaxin 8] and a v-SNARE [VAMP7 (vesicle-associated membrane protein 7)] (where t-SNAREs are SNAREs of the target compartment and v-SNAREs are SNAREs present in donor vesicles). In endosomes and in vitro, syntaxin 7, Vti1 and syntaxin 8 form a complex that is able to bind VAMP7. Antibodies to syntaxin 8 and a soluble recombinant VAMP7 fragment both inhibit in vitro reconstituted D. discoideum endosome fusion. The lysosomal content of syntaxin 7, Vti1, syntaxin 8 and VAMP7 is low compared with that in endosomes, implying a highly active recycling or retention mechanism. A likely model is that VAMP7 is a v-SNARE present on vesicles carrying lysosomal enzymes, and that the syntaxin 7-Vti1-syntaxin 8 t-SNARE complex is associated with incoming endocytic material.
Bogdanovic
Aleksandra
A
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Département de Réponse et Dynamique Cellulaires, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.
Bennett
Nelly
N
Kieffer
Sylvie
S
Louwagie
Mathilde
M
Morio
Takahiro
T
Garin
Jérôme
J
Satre
Michel
M
Bruckert
Franz
F
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D004023:Dictyostelium / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010873:Pinocytosis; D015800:Protozoan Proteins / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C121241:VAMP7 protein, human; C491419:VTI1A protein, human; D033921:Vesicular Transport Proteins