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.
| Original language | English |
|---|---|
| Pages (from-to) | 14303-8 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 106 |
| Issue number | 34 |
| DOIs | |
| Publication status | Published - 25 Aug 2009 |
Keywords
- Binding, Competitive
- Circular Dichroism
- Electrophoretic Mobility Shift Assay
- Immunoblotting
- Kinetics
- Models, Molecular
- Mutation
- Protein Binding
- Protein Conformation
- Protein Structure, Tertiary
- Qa-SNARE Proteins
- Recombinant Proteins
- Saccharomyces cerevisiae
- Saccharomyces cerevisiae Proteins
- Vesicular Transport Proteins