Intracellular expression of Kluyveromyces lactis toxin γ subunit mimics treatment with exogenous toxin and distinguishes two classes of toxin‐resistant mutant

Abstract

The Kluyveromyces lactis toxin is a heterotrimeric protein which irreversibly arrests proliferation of sensitive Saccharomyces cerevisiae cells in the G1 phase of the cell cycle. By expressing the gamma subunit of the toxin in sensitive yeast cells from a conditional promoter, it was previously demonstrated that it alone is required for inhibition (Tokunaga et al. (1989). Nucleic Acids Res. 17, 3435-3446). Here we show that, like native exogenous toxin, intracellular gamma subunit expression promotes a striking arrest of sensitive cells in G1. However, unlike the G1 arrest caused by native toxin, that induced by the gamma subunit alone does not result in reduced cellular viability and is fully and rapidly reversible, suggesting that the G1 arrest and the irreversibility of action may reflect different aspects of the toxin's interaction with sensitive cells. We have selected a large number of S. cerevisiae mutants which are highly resistant to the toxin in order to study its mode of action in more detail. Complementation analysis demonstrated that all but one of the mutants were recessive and these defined four separate genes. Members of two complementation groups concurrently acquired resistance to intracellular gamma subunit expression, suggesting that they contain a modified toxin target site. The other two genes appear to be required for entry of the gamma subunit into the sensitive cells since these mutants, while refractory to exogenous toxin, were fully sensitive to intracellular gamma subunit expression.