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Volume 148, Issue 5

Mutational analysis of K28 preprotoxin processing in the yeast Free

Abstract

K28 killer strains of 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. 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 effects it is now demonstrated that secretion of a biological active virus toxin requires signal peptidase cleavage after Gly and Kex2p-mediated processing at the α subunit N terminus (after Glu-Arg), the α subunit C terminus (after Ser-Arg) and at the β subunit N terminus (after Lys-Arg). The mature C terminus of the β subunit is trimmed by Kex1p, which removes the terminal Arg 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linked by a single disulfide bond between α-Cys and β-Cys, and expression of a mutant toxin in which β-Cys had been replaced by Ser resulted in the secretion of a non-toxic α/β heterodimer that is blocked in retrograde transport and incapable of entering the yeast cell cytosol,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 function of β-Cys might be to ensure accessibility of the toxin’s β subunit C terminus to the HDEL receptor of the target cell.

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Keyword(s): C-terminal HDEL motif , ER, endoplasmic reticulum , Kex2p endopeptidase , MBA, methylene blue agar , pptox, preprotoxin , preprotoxin processing and SP, signal peptidase
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2002-05-01
2023-10-01
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Mutational analysis of K28 preprotoxin processing in the yeast Saccharomyces cerevisiae
Microbiology 148, 1317 (2002); https://doi.org/10.1099/00221287-148-5-1317
/content/journal/micro/10.1099/00221287-148-5-1317
/content/journal/micro/10.1099/00221287-148-5-1317
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