Transduction of killing action of killer protein to sensitive yeast strains

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Antagonistic interactions between yeasts by secreted proteinaceous toxins appear to be quite high in natural habitat. To know the molecular mechanisms of yeast killer proteins, we screened a set of Saccharomyces cerevisiae mutants, individually deleted for 4901 yeast genes, for altered sensitivity against purified killer proteins of Kluyveromyces lactis (zymocin).Zymocin, atrimeric(α,β,γ)protein toxin complex, inhibits proliferation of S. cerevisiae cells. We present an analysis of kti6 mutants, which resist exogenous zymocin but are sensitive to intracellular expression of itsinhibitory γ- toxinsubunit, suggesting that KTI6 encodes a factor needed for toxin entry into the cell. Consistent with altered cell surface properties, kti6 cells resist hygromycinB, syringomycinE, and nystatin, antibiotics that require intact membrane potentials or provok emembrane disruption. KTI6 is allelic toI PT1, coding formannosyl-diinositolphospho-ceramide [M(IP)2C] synthase, which produces M(IP)2C, the … More major plasma membrane sphingolipid. kti6 membranes lack M(IP)2C and sphingolipid mutants that have reduced levels of M(IP)2C precursors, including the sphingolipid building block ceramide survive zymocin. Inaddition, kti6/ipt1 cells allow zymocin docking but prevent import of its toxic γ-subunit. Genetic analysis indicates that Kti6 is likely to act upstream of lipid raft proton pump Kti10/Pma1, apreviously identified zymocin sensitivity factor. Insum, M(IP)2C operates in a plasma membrane step that follows recognition of cell wall chitin by zymocin but precedes the involvement of elongator, the potential toxin target.To see the localization of zymocin subunits on the zyocin treated cell, we fractionated the cell lysate. We analyzed the presence of each a, b and g subunits on each fractions by Western-blotting with anti-a, b, g subunit antibody, respectively. a and g subunits were detected in the insoluble fraction of both in cell wall fraction and membrane fraction, respectively. However, β subunit was not detected. These data shows that both a and y subunits invade in the sensitive cell, through membrane. Less