sequential analysis of trans-snare formation in intracellular membrane fusion在细胞内的膜融合trans-snare形成的序列分析.pdfVIP

Sequential Analysis of Trans-SNARE Formation in Intracellular Membrane Fusion 1 1 2 2 2 Kannan Alpadi , Aditya Kulkarni , Veronique Comte , Monique Reinhardt , Andrea Schmidt , Sarita 1 2 1 Namjoshi , Andreas Mayer , Christopher Peters * ´ 1Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America, 2 Departement de ´ Biochimie, Universite de Lausanne, Epalinges, Switzerland Abstract SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q , Q , and Q ) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis- a b c SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Qa SNARE, leaving behind a QbcR subcomplex. This subcomplex serves as an acceptor for a Qa SNARE from the opposite membrane, leading to Q -Q R trans-complexes. Activity tests of vacuoles with diagnostic a bc distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the