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.
- membrane fusion
Abbreviations used: GST, glutathione S-transferase; GTP[S], guanosine 5′-[γ-thio]triphosphate; HRP, horseradish peroxidase; MALDI—TOF, matrix-assisted laser-desorption ionization—time-of-flight; NSF, N-ethylmaleimide-sensitive fusion protein; PNS, post-nuclear supernatant; SNAP, soluble NSF attachment protein; SNAP-25 (-23), synaptosome-associated protein of 25 (23)kDa; SNARE, SNAP receptor; t-SNARE, SNARE of the target compartment; v-SNARE, SNARE present in donor vesicles; Syn7, syntaxin 7; Syn8, syntaxin 8; VAMP, vesicle-associated membrane protein; Vti1, Vps10p tail interactor 1.
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