Translation of the L-species dsRNA genome of the killer-associated virus-like particles of Saccharomyces cerevisiae

Virus-like particles containing the L (P1)-species of double-stranded RNA (dsRNA) were isolated from Saccharomyces cerevisiae, and the translational activity of the virus-like particle-derived dsRNA was analyzed in the wheat germ cell-free system. Denaturation of the dsRNA immediately prior to in vitro translation resulted in the synthesis of one major and at least three minor polypeptides, whereas undenatured dsRNA, as expected, did not stimulate [35S]methionine incorporation into polypeptides, but actually slightly inhibited endogenous activity. The major in vitro translation product of the denatured L-dsRNA was shown to be identical with the major L-dsRNA containing virus-like particle capsid polypeptide on the basis of three criteria: co-electrophoresis on sodium dodecyl sulfate polyacrylamide gels, immunoprecipitation, and tryptic peptide analysis. We have therefore established that the L-dsRNA genome encodes the major virus-like particle capsid polypeptide. This result adds considerable support to the hypothesis that the L-dsRNA genome acts as a helper genome to the smaller (1.6 x 10(6) dalton) M-dsRNA genome in killer strains of yeast by providing the M-dsRNA containing virus-like particles with their major coat protein

A glycosylated protoxin in killer yeast: models for its structure and maturation

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

Translational analysis of the killer-associated virus-like particle dsRNA genome of S. cerevisiae: M dsRNA encodes toxin

The M species (medium sized) dsRNA (1.1-1.4 x 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 ihe 12,000 dalton polypeptide corresponding to the in vivo produced killer toxin, thus establishing thiat 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

Sequence of the preprotoxin dsRNA gene of type I killer yeast: multiple processing events produce a two-component toxin

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

Yeast FKBP-13 is a Membrane-Associated FK506-Binding Protein Encoded by the Nonessential Gene FKB2

The immunosuppressants FK506 and rapamycin prevent T-cell activation and also inhibit the growth of certain strains of the yeast Saccharomyces cerevisiae. It has previously been shown that yeast contains a 12-kDa cytosolic FK506-binding protein (yFKBP-12), which also possesses peptidylprolyl cis-trans isomerase activity, and that fkb1 strains lacking yFKBP-12 are resistant to rapamycin and sensitive to FK506. The absence of yFKBP-12 permitted the detection and isolation of a second FK506- and rapamycin-binding protein, which is about 13 kDa in size (yFKBP-13) and membrane-associated. Purified yFKBP-13 binds FK506 with 15-fold lower affinity than yFKBP-12 and has peptidylprolyl cis-trans isomerase activity with a similar substrate profile. The sequence of the first 37 N-terminal amino acids was determined, and the yFKBP-13 gene (FKB2) was cloned and sequenced. A hydrophobic putative signal sequence precedes the N terminus of the mature protein. yFKBP-13 most closely resembles the membrane-associated human FKBP-13, which also possesses a signal peptide, whereas yFKBP-12 most closely resembles human FKBP-12. fkb2 and fkb1 fkb2 mutants are viable and unaltered in their sensitivity to FK506, suggesting that yeast possesses an additional target for this drug. Furthermore, fkb2 null mutations confer no change in rapamycin sensitivity. These findings show that yFKBP-13 and yFKBP- 12 have distinct functions within the cell