Abstract
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.
Highlights
The secretion of proteinaceous compounds is a conserved evolutionary phenomenon and a wide-spread biological feature
We propose the formation of only one disulphide bond linking the major toxin subunits involving C92 and C239, thereby refining the model of K1 dimer formation and function/s of its subunits stated in the literature for decades
As classical A/B toxins, the mature heterodimers consist of one α and β subunit, respectively, covalently linked via at least one disulphide bond[21]
Summary
The secretion of proteinaceous compounds is a conserved evolutionary phenomenon and a wide-spread biological feature. Killer yeast necessitate a particular self-immunity system against their own toxin as they possess the same receptors on their cell surface and are in principle as susceptible to their toxin as sensitive cells. Expression of the toxin precursor in a sensitive cell is sufficient to confer immunity to externally applied K1, clearly pointing to a mechanism involving proteins of the secretory pathway and the precursor itself[12,13]. In the more neutral pH of the ER, thiol residues in the β subunit are deprotonated and thereby able to attack one of the remaining sulfuric residues in a nucleophilic fashion This process results in a rearrangement of the disulphide bonds leading to the separation of the toxin subunits and the release of α into the cytoplasm. We propose the formation of only one disulphide bond linking the major toxin subunits involving C92 and C239, thereby refining the model of K1 dimer formation and function/s of its subunits stated in the literature for decades
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