Mutational analysis of K28 preprotoxin processing in the yeast Saccharomyces cerevisiae

摘要

K28 killer strains of Saccharomyces cerevisiae are permanently infected with a cytoplasmic persisting dsRNA virus encoding a secreted α/β heterodimeric protein toxin that kills sensitive cells by cell-cycle arrest and inhibition of DNA synthesis. In vivo processing of the 345 aa toxin precursor (preprotoxin; pptox) involves multiple internal and carboxy-terminal cleavage events by the prohormone convertases Kex2p and Kex1p. By site-directed mutagenesis of the preprotoxin gene and phenotypic analysis of its in vivo effects it is now demonstrated that secretion of a biological active virus toxin requires signal peptidase cleavage after Gly36 and Kex2p-mediated processing at the α subunit N terminus (after Glu-Arg49), the α subunit C terminus (after Ser-Arg149) and at the β subunit N terminus (after Lys-Arg245). The mature C terminus of the β subunit is trimmed by Kex1p, which removes the terminal Arg345 residue, thus uncovering the toxin’s endoplasmic reticulum targeting signal (HDEL) which – in a sensitive target cell – is essential for retrograde toxin transport. Interestingly, both toxin subunits are covalently linked by a single disulfide bond between α-Cys56 and β-Cys340, and expression of a mutant toxin in which β-Cys340 had been replaced by Ser340 resulted in the secretion of a non-toxic α/β heterodimer that is blocked in retrograde transport and incapable of entering the yeast cell cytosol, indicating that one important in vivo function of β-Cys340 might be to ensure accessibility of the toxin’s β subunit C terminus to the HDEL receptor of the target cell.