KILLER-TOXIN-RESISTANT KRE12 MUTANTS OF SACCHAROMYCES-CEREVISIAE - GENETIC AND BIOCHEMICAL-EVIDENCE FOR A SECONDARY K1 MEMBRANE-RECEPTOR

The Saccharomyces cerevisiae killer toxin K1 is a secreted alpha/beta- heterodimeric protein toxin that kills sensitive yeast cells in a rece ptor-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, th e killer toxin is transferred to the cytoplasmic membrane, subsequentl y 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 ge netics and successive back-crossings to sensitive wild-type strains in dicated that this toxin resistance is due to mutation(s) in a single c hromosomal yeast gene (KRE12), rendering kre12 mutants incapable of bi nding significant amounts of toxin to the membrane. Since kre12 mutant s showed normal toxin binding to the cell wall, but markedly reduced m embrane binding, we isolated and purified cytoplasmic membranes from a kre12 mutant and from an isogenic Kre12(+) strain and analyzed the me mbrane protein patterns by 2D-electrophoresis using a combination of i soelectric focusing and SDS-PAGE. Using this technique, three differen t proteins (or subunits of a single multimeric protein) were identifie d 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 bi nding to the membrane and subsequent ion channel formation.