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Mutational analysis of K28 preprotoxin processing in the yeast Saccharomyces cerevisiae

Angewandte Molekularbiologie, Universität des Saarlandes, FR 8.3, Gebäude 2, Postfach 151150, D-66041 Saarbrücken, Germany¹

Author for correspondence: Manfred J. Schmitt. Tel: +49 681 302 4730. Fax: +49 681 302 4710. e-mail: mjs{at}microbiol.uni-sb.de

K28 killer strains of Saccharomyces cerevisiae are permanently infected with a cytoplasmic persisting dsRNA virus encoding a secreted /ß 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 subunit N terminus (after Glu-Arg⁴⁹), the subunit C terminus (after Ser-Arg¹⁴⁹) and at the ß subunit N terminus (after Lys-Arg²⁴⁵). The mature C terminus of the ß 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linked by a single disulfide bond between -Cys⁵⁶ and ß-Cys³⁴⁰, and expression of a mutant toxin in which ß-Cys³⁴⁰ had been replaced by Ser³⁴⁰ resulted in the secretion of a non-toxic /ß heterodimer that is blocked in retrograde transport and incapable of entering the yeast cell cytosol, indicating that one important in vivo function of ß-Cys³⁴⁰ might be to ensure accessibility of the toxin’s ß subunit C terminus to the HDEL receptor of the target cell.

Keywords: Kex2p endopeptidase, preprotoxin processing, C-terminal HDEL motif

Abbreviations: ER, endoplasmic reticulum; MBA, methylene blue agar; pptox, preprotoxin; SP, signal peptidase

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