Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin.

Abstract

Strains of Saccharomyces cerevisiae harboring M1‐dsRNA, the determinant of type 1 killer and immunity phenotypes, secrete a dimeric 19‐kd toxin that kills sensitive yeast cells by the production of cation‐permeable pores in the cytoplasmic membrane. The preprotoxin, an intracellular precursor to toxin, has the domain sequence delta‐alpha‐gamma‐beta where alpha and beta are the 9.5‐and 9.0‐kd subunits of secreted toxin. Plasmids containing a partial cDNA copy of M1, in which alpha, gamma, and beta are fused to the PH05 promoter and signal peptide, have previously been shown to express phosphate‐repressible toxin production and immunity. Here the construction of a complete DNA copy of the preprotoxin gene and its mutagenesis are described. Analysis of the expression of these mutants from the PH05 promoter elucidates the functions of the preprotoxin domains. delta acts as a leader peptide and efficiently mediates the secretion, glycosylation and maturation of killer toxin. Mutations within the beta subunit indicate it to be essential for binding of toxin to and killing of whole cells but unnecessary for the killing of spheroplasts. Mutations within the putative active site of alpha prevent killing of both cells and spheroplasts. The probable role of beta is therefore recognition and binding to the cell wall receptor whereas alpha is the active ionophore. Mutations within alpha causing loss of toxicity also cause loss of immunity, while the mutants described within gamma and beta retain partial or complete immunity. Expression of gamma without alpha or beta confers no phenotype. The immunity determinant may minimally consist of the alpha domain and the N‐terminal portion of gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

“…The role of the alpha polypeptide in S. cerevisiae kl toxin (4,19,45) is the interaction with the cell membrane that kills the cell by disrupting ion transport (8,12,23). This is similar to the mode of action of the cytotoxins (25,38 The primary sequence similarity is most evident in the region of residues 26 to 45 of KP6 beta (Fig.…”

“…This suggests a relatedness of the killer toxin beta polypeptides. The kl beta polypeptide is known to be responsible for interaction with the S. cerevisiae cell wall, facilitating interaction of the alpha polypeptide with the cell membrane (45). Therefore, this may also be the role of the KP6 and KP1 beta polypeptides.…”

Mutational analysis of the functional domains of yeast K1 killer toxin.

“…In addition, strains defective in the processing proteases encoded by the KEX2 or KEXI genes, which are required for the maturation of active toxin, retain immunity (7,38). Models for conferring immunity hypothesize that immunity to the toxin occurs by the precursor or some derivative competing with the mature toxin for binding to a membrane receptor (2,32). However, no convincing evidence for a membrane receptor exists, and the precursor component could directly interfere with the ability of the toxin to form ion channels (for a review, see reference 6).…”

“…Results of such studies and from prior work indicate that the toxin and the component responsible for immunity are both encoded by the preprotoxin gene and act at the level of the plasma membrane. Although the precise protein product that confers immunity has not been determined, site-directed mutagenesis of the preprotoxin gene maps the immunity domain within the region encoding the a subunit (2,32,43). Since some of the mutations which fail to allow precursor processing retain immunity, it was suggested that the precursor can function as the immunity component (2,32).…”

“…Although the precise protein product that confers immunity has not been determined, site-directed mutagenesis of the preprotoxin gene maps the immunity domain within the region encoding the a subunit (2,32,43). Since some of the mutations which fail to allow precursor processing retain immunity, it was suggested that the precursor can function as the immunity component (2,32). In addition, strains defective in the processing proteases encoded by the KEX2 or KEXI genes, which are required for the maturation of active toxin, retain immunity (7,38).…”