rsly1 Binding to Syntaxin 5 Is Required for Endoplasmic Reticulum-to-Golgi Transport but Does Not Promote SNARE Motif Accessibility

Antionette L. Williams, Sebastian Ehm, Noëlle C. Jacobson, Dalu Xu, J. C. Hay

Research output: Contribution to journalArticlepeer-review

Abstract

Although some of the principles of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function are well understood, remarkably little detail is known about sec1/munc18 (SM) protein function and its relationship to SNAREs. Popular models of SM protein function hold that these proteins promote or maintain an open and/or monomeric pool of syntaxin molecules available for SNARE complex formation. To address the functional relationship of the mammalian endoplasmic reticulum/Golgi SM protein rsly1 and its SNARE binding partner syntaxin 5, we produced a conformation-specific monoclonal antibody that binds only the available, but not the cis-SNARE-complexed nor intramolecularly closed form of syntaxin 5. Immunostaining experiments demonstrated that syntaxin 5 SNARE motif availability is nonuniformly distributed and focally regulated. In vitro endoplasmic reticulum-to-Golgi transport assays revealed that rsly1 was acutely required for transport, and that binding to syntaxin 5 was absolutely required for its function. Finally, manipulation of rsly1-syntaxin 5 interactions in vivo revealed that they had remarkably little impact on the pool of available syntaxin 5 SNARE motif. Our results argue that although rsly1 does not seem to regulate the availability of syntaxin 5, its function is intimately associated with syntaxin binding, perhaps promoting a later step in SNARE complex formation or function.

Original languageEnglish
Pages (from-to)162-175
Number of pages14
JournalMolecular Biology of the Cell
Volume15
Issue number1
DOIs
StatePublished - Jan 2004

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