MOLECULAR BASIS OF YEAST KILLER PROTOXIN GENE EXPRESSION

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We are attempting to elucidate basic molecular mechanisms underlying the production and specific immunity to a mycoviral toxin in the type 1 killer dsRNA system in S. cerevisiae. Our approach is a coordinate one utilizing both biochemical and genetic techniques. We anticipate that results from these studies will provide new insights into a variety of interrelated aspects of protein secretion and endoproteolysis in yeast relevant to protein processing in higher eucaryotes, and will further develop our understanding of the molecular biology of this mycoviral system, particularly with respect to mechanisms of toxin action and immunity function. The K1 system consists of a segmented dsRNA genome packaged cytoplasmically in virus-like particles (VLPs). The two predominant constituents are a 4.7 kilobase pair (kb) L-dsRNA, which encodes the major VLP capsid protein, and a 1.9 kb M-dsRNA. In previous studies we identified a 34.8 kilodalton (kd) preprotoxin as the primary translation product of M-dsRNA. Through gene fusion experiments employing a cDNA clone of the preprotoxin gene, we determined that both toxin and immunity determinants reside in this single molecule. The toxin consists of two 9.0-9.5 kd components, Alpha and Beta derived from a 43 kd glycosylated protoxin which has the sequence Delta-Alpha-Gamma-Beta. Delta is a 44 amino acid N-terminal leader, and Gamma, about 103 amino acids, is the site of glycosylation. Maturation follows the standard yeast secretory pathway defined by the SEC genes, and involves the additional function of chromosomal KEX (killer expression) and REX (resistance expression) loci. We plan to continue our studies by addressing several specific issues. We will evaluate the structure and function of the protein domains of protoxin by site-specific mutagenesis. This will include identification of the cellular component responsible for immunity. We will define components that interact and participate in toxin and immunity production and function by isolation and characterization of second-site suppressors of various protoxin mutants. Finally, we will analyze the role of the N-terminal Delta sequence and the central Gamma sequence in intracellular transit and processing of the preprotoxin.