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

S L Sturley, Q Elliot, J LeVitre

    The EMBO Journal
    |December 1, 1986
    PubMed

    Insights

    This study reveals the Saccharomyces cerevisiae killer toxin

    Area of Science:

    • * Molecular biology
    • * Yeast genetics
    • * Protein biochemistry

    Background:

    • * Saccharomyces cerevisiae strains carrying M1-dsRNA exhibit killer and immunity phenotypes.
    • * These strains secrete a 19-kd dimeric toxin that forms pores in yeast cell membranes.
    • * The toxin precursor, preprotoxin, has a delta-alpha-gamma-beta domain structure.

    Purpose of the Study:

    • * To elucidate the functions of preprotoxin domains through mutagenesis.
    • * To understand the roles of alpha, beta, and gamma domains in toxin activity and immunity.
    • * To identify the minimal sequence required for immunity.

    Main Methods:

    • * Construction and mutagenesis of a complete DNA copy of the preprotoxin gene.
    • * Expression of mutants using the PH05 promoter and signal peptide.
    • * Analysis of toxin production, secretion, cell killing, and immunity phenotypes.

    Main Results:

    • * The delta domain acts as a leader peptide for secretion, glycosylation, and maturation.
    • * The beta subunit is crucial for whole-cell binding and killing but not spheroplast killing.
    • * The alpha subunit's active site is essential for killing both cells and spheroplasts; mutations abolish toxicity and immunity.
    • * The gamma and beta subunits contribute to immunity; alpha and N-terminal gamma may form the immunity determinant.

    Conclusions:

    • * The beta subunit mediates cell wall receptor binding, while alpha functions as the ionophore.
    • * The alpha subunit is critical for both toxicity and immunity.
    • * The alpha domain and N-terminal gamma may constitute the minimal immunity determinant.

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