Substitution of cysteines in the yeast viral killer toxin K1 precursor reveals novel insights in heterodimer formation and immunity.

The killer toxin K1 is a virally encoded fungal A/B toxin acting by disrupting plasma membrane integrity. The connection of α and β constitutes a critical feature for toxin biology and for decades the formation of three disulphide bonds linking the major toxin subunits was accepted as status quo. Due to the absence of experimental evidence, the involvement of each cysteine in heterodimer formation, K1 lethality and immunity was systematically analysed. Substitution of any cysteine in α led to a complete loss of toxin dimer secretion and toxicity, whereas K1 toxin derivatives carrying mutations of C248, C312 or the double mutation C248-312 were active against spheroplasted cells. Importantly, substitution of the C95 and C107 in the toxin precursor completely abolished the mediation of functional immunity. In contrast, K1 toxicity, i.e. its ionophoric effect, does not depend on the cysteine residues at all. In contrast to the literature, our data imply the formation of a single disulphide bond involving C92 in α and C239 in β. This finding not only refines the current model stated for decades but also provides new opportunities to elucidate the mechanisms underlying K1 toxicity and immunity at the molecular level.