SEQUENCE OF THE M28 DSRNA - PREPROTOXIN IS PROCESSED TO AN ALPHA BETAHETERODIMERIC PROTEIN TOXIN/

The killer and immunity phenotypes of K28 killer strains of Saccharomy ces cerevisiae are determined by the 1.75-kb M28 dsRNA virus. In the p lus strand, M28p, the K28 preprotoxin gene, comprises bases 13-1047 an d is followed, after an additional 85 bases, by a 63-bp poly(A) sequen ce and a 553-base 3'-sequence. This 3'-sequence contains two potential stem-loop structures predicted to bind the L-A encoded cap-pol protei n, initiating encapsidation; high-level expression results in curing o f M1 dsRNA. Expression of M28p confers the complete K28 killer and imm unity phenotype on a cell lacking M28 dsRNA. K28 toxin is a disulfide- bonded heterodimer of alpha (10.5 kDa) and beta (11 kDa) components wh ose N-termini correspond to M28p residues 50-61 and 246-257, respectiv ely. alpha is preceded by a potentially redundant pair of secretion si gnal peptides; deletion of the first reduces toxin secretion by 75%. W hile M28p bears no sequence similarity to M1p, the K1 preprotoxin, the predicted patterns of processing by glycosylation and cleavage are re markably similar. The beta N- and C-termini are probably processed by Kex2p and Kex1p, respectively; the mechanism of cleavage at the less t ypical sites bounding the alpha component is under investigation. Whil e a kex2 Delta mutation prevents toxin secretion, secreted toxin retai ns 20% activity in a kex1 Delta mutant. Neither mutation affects immun ity. (C) 1995 academic Press, Inc.