Abstract

Membrane trafficking is an essential cellular process where tethering factors offer the first level of specificity for the proper localization of vesicles to target membranes. The localization of the tethers themselves may depend on their associations with a unique subset of lipids or activated GTPases. The TRAPP vesicle tethering complex localizes to the Golgi in an unknown manner but its localization may be mediated by the Bet3 protein. To gain insight into the mechanism of localization of TRAPP a synthetic lethal yeast screen with the bet3-4 mutant was employed. The screen is based on a plasmid-dependent assay which exploits the adenine biosynthesis pathway and red/white colony sectoring. A Saccharomyces cerevisiae bet3-4 ade2 ade3 mutant strain with a counter-selectable plasmid harbouring the BET3 and ADE3 genes was randomly mutated with ethyl methanesulfonate (EMS). Double mutants, identified as uniformly red colonies, were obtained and initially screened for the presence of a mutation in either BET3 or another gene encoding a TRAPP subunit. Mutants that were judged to be in non-TRAPP genes were then screened with a yeast library to try to identify the mutated gene. One synthetic lethal mutant was suppressed by VPS16 , a gene encoding a component of another vesicle tethering complex. Since VPS16 does not suppress bet3-4 and since sequencing failed to reveal any mutations in VPS16 , our results suggest that the defective gene in this particular mutant can be suppressed by VPS16 . The identity of the mutated gene remains unknown at present.