Defining the domain organization of K1-like killer toxins

Antifungal killer toxins produced by yeasts undergo post-translational processing to enable the formation of mature and active toxins. The preprocessed, immature K1 killer toxin from Saccharomyces cerevisiae has four different domains (delta, alpha, gamma, and beta) that are cleaved to produce a mature toxin consisting of a disulfide-linked alpha and beta heterodimer. K1 homologs from various yeast species of the Saccharomycotina have been previously identified by primary sequence homology. Primary sequence analysis has identified putative cleavage sites across all homologs, with some conservation with cleavage sites identified in K1 and the likely importance of the conserved carboxypeptidases Kex1 and Kex2. The identification of sites of proteolytic processing has enabled an accurate definition of the domain boundaries of delta, alpha, gamma, and beta domains in all K1 homologs. Functional assays revealed that many K1 homologs contain cytotoxic alpha domains. Interestingly, several killer toxins that lacked measurable antifungal activity still exhibited toxic alpha domains when expressed independently. These findings suggest that alpha domain toxicity is conserved across K1 homologs, and that variations in post-translational processing and domain interactions may influence full-length toxin functionality. This work provides insights into the evolutionary conservation and diversity of yeast killer toxins.