rsly1 binding to syntaxin 5 is required for endoplasmic reticulum-to-Golgi transport but does not promote SNARE motif accessibility.
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.
Williams
Antionette L
AL
University of Michigan, Department of Molecular, Cellular, and Developmental Biology, Ann Arbor, Michigan 48109-1048, USA.
Ehm
Sebastian
S
Jacobson
Noëlle C
NC
Xu
Dalu
D
Hay
Jesse C
JC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D017874:Immediate-Early Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D051938:Munc18 Proteins; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D050765:Qa-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; C106568:Scfd1 protein, rat; D033921:Vesicular Transport Proteins