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Saccharomyces cerevisiae K1 killer strains are infected by the M1 double-stranded RNA virus encoding a secreted protein toxin that kills sensitive cells by disrupting cytoplasmic membrane function. Toxin binding to spheroplasts is mediated by Kre1p, a cell wall protein initially attached to the plasma membrane by its C-terminal GPI anchor. Kre1p binds toxin directly. Both cells and spheroplasts of Δkre1 mutants are completely toxin resistant; binding to cell walls and spheroplasts is reduced to 10% and

The preprotoxin gene of the 1.9 kb M1 dsRNA genome from type I killer yeast has been sequenced employing a partial-length cDNA derived from an in vivo transcript. A single open reading frame, commencing with AUG at M1 dsRNA bases 14-16, terminates with UAG at 963-965 and codes for a 316 amino acid protein, believed to be identical to the 34 kd preprotoxin species, M1-P1, synthesized by in vitro translation of denatured M1 dsRNA. N-terminal sequencing of M1-P1 confirms this prediction. Secreted toxin is shown to consist of two dissimilar, disulfide-bonded subunits, alpha and beta, of apparent size 9.5 and 9.0 kd, respectively, whose N-terminal sequences are also found in the predicted preprotoxin sequence. Its proposed domains consist of delta, a 44 amino acid N-terminal segment, followed by alpha and beta, which are separated by gamma, a large central glycosylated segment. Processing sites, domain functions, and the potential role of gamma in immunity are discussed.

The M species (medium sized) dsRNA (1.1–1.4 × 10 6 daltons) isolated from a toxin-producing yeast killer strain (K + R + ) and three related, defective interfering (suppressive) S species dsRNAs of the yeast killer-associated cytoplasmic multicomponent viral-like particle system were analyzed by in vitro translation in a wheat germ cell-free protein synthesis system. Heat-denatured M species dsRNA programmed the synthesis of two major polypeptides, M-P1 (32,000 daltons) and M-P2 (30,000 daltons). M-P1 has been shown by the criteria of proteolytic peptide mapping and cross-antigenicity to contain the 12,000 dalton polypeptide corresponding to the in vivo produced killer toxin, thus establishing that it is the M species dsRNA which carries the toxin gene. An M species dsRNA obtained from a neutral strain (K − R + ) also programmed the in vitro synthesis of a polypeptide identical in molecular weight to M-P1, thus indicating that the cytoplasmic determinant of the mutant neutral phenotype is either a simple point mutation in the dsRNA toxin gene or a mutation in a dsRNA gene which is required for functional toxin production. In vitro translation of each of the three different suppressive S dsRNAs resulted in the production of a polypeptide (S-P1) of approximately 8000 daltons instead of the 32,000 dalton M-P1 polypeptide programmed by M dsRNA. This result is consistent with the heteroduplex analysis of these dsRNAs by Fried and Fink (1978), which shows retention of M dsRNA ends, accompanied by large internal deletions in each of the S dsRNAs translated.

The type 1 killer phenotype in S. cerevisiae, mediated by secretion of an 11.5 kilodalton (kd) protein toxin, is cytoplasmically determined by the 1.9 kb M1-dsRNA plasmid. Maintenance of M1-dsRNA is dependent on the 4.5 kb L1-dsRNA because L1 encodes the capsid protein of the virus-like particles that separately encapsidate both dsRNA species. We have shown that in vitro translation of denatured M1-dsRNA produces M1-P1, a 32 kd protein containing the toxin peptides. We now demonstrate the presence of an unstable, 42 kd, membrane-associated, glycosylated protoxin in killer cells, probably derived from M1-P1 by cotranslational processing, and glycosylation. In vitro cotranslational processing of M1-P1, derived both from in vivo mRNAs and from denatured M1-dsRNA, produces a product resembling protoxin. Processing involves loss of 1.6 kd of protein, presumably an N-terminal leader peptide, and glycosylation. This information, together with data on in vitro expression of suppressive deletion mutants of M1-dsRNA, allows construction of testable models for the functional sequence of M1-P1 and for its maturation to toxin.