title: A random mutagenesis approach to isolate dominant-negative yeast sec1 mutants reveals a functional role for domain 3a in yeast and mammalian Sec1/Munc18 proteins.
abstract: SNAP receptor (SNARE) and Sec1/Munc18 (SM) proteins are required for all intracellular membrane fusion events. SNAREs are widely believed to drive the fusion process, but the function of SM proteins remains unclear. To shed light on this, we screened for dominant-negative mutants of yeast Sec1 by random mutagenesis of a GAL1-regulated SEC1 plasmid. Mutants were identified on the basis of galactose-inducible growth arrest and inhibition of invertase secretion. This effect of dominant-negative sec1 was suppressed by overexpression of the vesicle (v)-SNAREs, Snc1 and Snc2, but not the target (t)-SNAREs, Sec9 and Sso2. The mutations isolated in Sec1 clustered in a hotspot within domain 3a, with F361 mutated in four different mutants. To test if this region was generally involved in SM protein function, the F361-equivalent residue in mammalian Munc18-1 (Y337) was mutated. Overexpression of the Munc18-1 Y337L mutant in bovine chromaffin cells inhibited the release kinetics of individual exocytosis events. The Y337L mutation impaired binding of Munc18-1 to the neuronal SNARE complex, but did not affect its binary interaction with syntaxin1a. Taken together, these data suggest that domain 3a of SM proteins has a functionally important role in membrane fusion. Furthermore, this approach of screening for dominant-negative mutants in yeast may be useful for other conserved proteins, to identify functionally important domains in their mammalian homologs.
authors.affiliation: Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdom.
authors.forename: Alan
authors.initials: A
authors.lastname: Boyd
authors.forename: Leonora F
authors.initials: LF
authors.lastname: Ciufo
authors.forename: Jeff W
authors.initials: JW
authors.lastname: Barclay
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Graham
authors.forename: Lee P
authors.initials: LP
authors.lastname: Haynes
authors.forename: Mary K
authors.initials: MK
authors.lastname: Doherty
authors.forename: Michèle
authors.initials: M
authors.lastname: Riesen
authors.forename: Robert D
authors.initials: RD
authors.lastname: Burgoyne
authors.forename: Alan
authors.initials: A
authors.lastname: Morgan