title: The N-terminal peptide of the syntaxin Tlg2p modulates binding of its closed conformation to Vps45p.
abstract: The Sec1/Munc18 (SM) protein family regulates intracellular trafficking through interactions with individual SNARE proteins and assembled SNARE complexes. Revealing a common mechanism of this regulation has been challenging, largely because of the multiple modes of interaction observed between SM proteins and their cognate syntaxin-type SNAREs. These modes include binding of the SM to a closed conformation of syntaxin, binding to the N-terminal peptide of syntaxin, binding to assembled SNARE complexes, and/or binding to nonsyntaxin SNAREs. The SM protein Vps45p, which regulates endosomal trafficking in yeast, binds the conserved N-terminal peptide of the syntaxin Tlg2p. We used size exclusion chromatography and a quantitative fluorescent gel mobility shift assay to reveal an additional binding site that does not require the Tlg2p N-peptide. Characterization of Tlg2p mutants and truncations indicate that this binding site corresponds to a closed conformation of Tlg2p. Furthermore, the Tlg2p N-peptide competes with the closed conformation for binding, suggesting a fundamental regulatory mechanism for SM-syntaxin interactions in SNARE assembly and membrane fusion.
authors: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors: Melonnie L M
authors: ML
authors: Furgason
authors: Chris
authors: C
authors: MacDonald
authors: Scott G
authors: SG
authors: Shanks
authors: Sean P
authors: SP
authors: Ryder
authors: Nia J
authors: NJ
authors: Bryant
authors: Mary
authors: M
authors: Munson
mesh_terms: D001667:Binding, Competitive; D002942:Circular Dichroism; D024202:Electrophoretic Mobility Shift Assay; D015151:Immunoblotting; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C112252:TLG2 protein, S cerevisiae; C092934:VPS45 protein, S cerevisiae; D033921:Vesicular Transport Proteins