Killer-toxin-resistant kre12 mutants of Saccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

Abstract

The Saccharomyces cerevisiae killer toxin K1 is a secreted alpha/beta-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to beta-1,6-D-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of the KRE genes whose products are involved in synthesis and/or assembly of cell wall beta-D-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strains indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), rendering kre12 mutants incapable of binding significant amounts of toxin to the membrane. Since kre12 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from a kre12 mutant and from an isogenic Kre12(+) strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in the kre12 mutant. A model for K1 killer toxin action is presented in which the gene product of KRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.