Mutational analysis of K28 preprotoxin processing in the yeast Saccharomyces cerevisiae.
K28 killer strains of Saccharomyces cerevisiae are permanently infected with a cytoplasmic persisting dsRNA virus encoding a secreted alpha/beta 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 Gly(36) and Kex2p-mediated processing at the alpha subunit N terminus (after Glu-Arg(49)), the alpha subunit C terminus (after Ser-Arg(149)) and at the beta subunit N terminus (after Lys-Arg(245)). The mature C terminus of the beta subunit is trimmed by Kex1p, which removes the terminal Arg(345) 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disulfide bond between alpha-Cys(56) and beta-Cys(340), and expression of a mutant toxin in which beta-Cys(340) had been replaced by Ser(340) resulted in the secretion of a non-toxic alpha/beta heterodimer that is blocked in retrograde transport and incapable of entering the yeast cell cytosol, indicating that one important in vivo function of beta-Cys(340) might be to ensure accessibility of the toxin's beta subunit C terminus to the HDEL receptor of the target cell.
Riffer
Frank
F
Angewandte Molekularbiologie, Universität des Saarlandes, FR 8.3, Gebäude 2, Postfach 151150, D-66041 Saarbrücken, Germany.
Eisfeld
Katrin
K
Breinig
Frank
F
Schmitt
Manfred J
MJ
D004220:Disulfides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017353:Gene Deletion; D055646:Killer Factors, Yeast; D008565:Membrane Proteins; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D009842:Oligopeptides / Q000378:metabolism; D010641:Phenotype; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism
D004220:Disulfides; D055646:Killer Factors, Yeast; D008565:Membrane Proteins; D009183:Mycotoxins; D009842:Oligopeptides; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; C077480:histidyl-aspartyl-glutamyl-leucine; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; C026515:type I signal peptidase