Conformational regulation of SNARE assembly and disassembly in vivo.
SNAP receptor (SNARE) proteins function in intracellular trafficking by forming complexes that bridge vesicle and target membranes prior to fusion. Biochemical studies indicate that the entry of certain SNARE proteins into complexes is inhibited by intramolecular interactions that generate a closed conformation. For example, an essential N-terminal regulatory domain of the yeast plasma membrane SNARE Sso1p sequesters the C-terminal SNARE motif and prevents it from binding to its assembly partners Sec9p and Sncp. Here, we introduce mutations into Sso1p that cause it to remain constitutively open. These open mutants can functionally substitute for wild-type Sso1p protein in vivo, demonstrating that inhibition of SNARE assembly is not the essential function of the N-terminal regulatory domain. Furthermore, the open mutants suppress sec9--4, a mutation that causes a severe defect in SNARE assembly. Elevated levels of SNARE complexes are observed in cells expressing the open mutants. In the presence of sufficient Sec9p, these complexes accumulate to levels that cause severe growth defects. Similarly, overexpression of the open mutants in yeast carrying mutations in the SNARE disassembly machinery impairs growth. Our findings indicate that elevated levels of SNARE complexes can be toxic and that these levels are normally controlled by the SNARE disassembly machinery, by the limited availability of Sec9p, and by the closed conformation of Sso1p.
Munson
Mary
M
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Hughson
Frederick M
FM
D005656:Fungal Proteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D009154:Mutation; D011487:Protein Conformation; D050765:Qa-SNARE Proteins; D050767:Qc-SNARE Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050767:Qc-SNARE Proteins; C090117:SEC9 protein, S cerevisiae; D050600:SNARE Proteins; C084006:SSO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins