A possible predocking attachment site for N-ethylmaleimide-sensitive fusion protein. Insights from in vitro endosome fusion

María I. Colombo, Moges Taddese, Sidney W. Whiteheart, Philip D. Stahl

Research output: Contribution to journalArticlepeer-review

49 Scopus citations

Abstract

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ubiquitous protein required for multiple vesicular transport events. We have investigated the role of the two nucleotide-binding regions of NSF in endosomal fusion by analyzing NSF routants in a cell-free system. Our results indicate that mutations on the first ATP-binding domain, that render a protein defective in either ATP binding or ATP hydrolysis, results in almost complete inhibition of endosomal fusion. A mutation in the second ATP-binding site of NSF was only slightly inhibitory. The inhibitory effect was observed only when the mutant proteins were added at early times during the fusion reaction indicating that NSF may be required for an early step during the docking/fusion process. Binding studies using Western blotting reveal that the binding of NSF mutants to endosomal membranes is differentially affected by Ca 2+. Our results indicate that NSF, depending on its nucleotide state, may interact with membranes via an alternate mechanism. Our findings suggest the existence of a predocking binding site either independent of the docking complex or a site that leads to the formation of the SNAP-SNARE complex (e.g. 20 S particle).

Original languageEnglish
Pages (from-to)18810-18816
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number31
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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