Kluyveromyces lactis zymocin, a trimeric (␣␤␥) protein toxin complex, inhibits proliferation of Saccharomyces cerevisiae cells. Here we present an analysis of kti6 mutants, which resist exogenous zymocin but are sensitive to intracellular expression of its inhibitory ␥-toxin subunit, suggesting that KTI6 encodes a factor needed for toxin entry into the cell. Consistent with altered cell surface properties, kti6 cells resist hygromycin B, syringomycin E, and nystatin, antibiotics that require intact membrane potentials or provoke membrane disruption. KTI6 is allelic to IPT1, coding for mannosyl-diinositolphospho-ceramide [M(IP) 2 C] synthase, which produces M(IP) 2 C, the major plasma membrane sphingolipid. kti6 membranes lack M(IP) 2 C and sphingolipid mutants that have reduced levels of M(IP) 2 C precursors, including the sphingolipid building block ceramide survive zymocin. In addition, kti6/ipt1 cells allow zymocin docking but prevent import of its toxic ␥-subunit. Genetic analysis indicates that Kti6 is likely to act upstream of lipid raft proton pump Kti10/Pma1, a previously identified zymocin sensitivity factor. In sum, M(IP) 2 C 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.

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The G1 cell cycle arrest imposed by Kluyveromyces lactis zymocin on Saccharomyces cerevisiae requires a functional RNA polymerase II (pol II) TOT/Elongator complex. In a study of zymocin's mode of action, genetic scenarios known to impair transcription or affect the pol II machinery itself were found to elicit hypersensitivity to zymocin. Thus, mutations in components of SAGA, SWI/SNF, Mediator and Ccr4-Not, complexes involved in transcriptionally relevant functions such as nucleosome modification, chromatin remodelling and formation of the preinitiation complex, make yeast cells hypersensitive to the lethal effects of zymocin. The defects at the level of transcriptional elongation displayed by rtf1Delta, ctk1, fcp1 and rpb2 mutants also result in zymocin hypersensitivity. Intriguingly, inactivation of histone deacetylase (HDAC) activity, which is expected to reduce the demand for the histone acetyltransferase (HAT) function of TOT/Elongator, also reduces sensitivity to zymocin. Thus, zymocin interferes with pol II-dependent transcription, and this effect requires the HAT function of TOT, presumably while the Elongator complex is associated with pol II.