Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin Target (TOT), I used mTn3::tagging to isolate zymocin resistant tot mutants from budding yeast. Of these it could identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)-associated Elongator complex of RNA-polymerase-II holoenzyme. In addition TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with Elongator. Two more Elongator subunit genes, ELP6 (TOT6) and ELP4 (TOT7) have been identified. Other than the typical elp ts-phenotype, tot deletions results in a complex phenotype including resistance to zymocin hypersensitivity towards caffeine and calcofluor white as well as slow groth and a G1 cell cycle delay. Intriguingly, strains lacking Non-Elongator HATs (gcn5Δ, hat1Δ, hpa3Δ and sas3Δ) or Non-Elongator transcription elongator factors TFIIS (dst1Δ) and Spt4p (spt4Δ) cannot confer resistance towards the K. lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signaling toxitity of the K. lactis zymocin. Elevated TOT4 copy number also results in resistance to zymocin and shows an intermediate tot phenotype, wich include mild sensitivities toward caffein and calcofluor white, suggesting that TOT4 influences Elongator function. As shown by co-immunprecipitation, Tot4p interacts with Elongator and Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4. Besides its role as a HAT of Tot3p, Tot3p assists subunit communication within Elongator by mediating Tot2p-Tot4p, Tot2p-Tot5p and Tot2p-Tot1p protein-protein interactions. TOT1 is essential for Tot4p-Tot2p and Tot4p-Tot3p interactions and TOT2 is essential for Tot4p-Tot5p and Tot3p-Tot4p interaction. The latter was lost with a C-terminal Tot2p truncation, the former was affected by progressively truncating TOT1. Despite being dispensable for Tot4p-Tot2p interaction, the extreme C-terminus of Tot1p may play a role in Elongator function, as its truncation confers zymocin resitance. The ability of Tot2p to interact with the HAT subunit Tot3p of Elongator and with subunit Tot5p is dependent on Tot1. Also, the association of core-Elongator Tot1-3p with HAP (Tot5-7p), the second three subunit subcomplex of Elongator, was found to be sensitive to loss of TOT1/ELP1 gene funktion. Structural integrity of the HAP complex itself requires the TOT7/ELP4, TOT5/ELP5 and TOT6/ELP6 genes, and tot6Δ as well as tot7Δ cells can no longer promote interaction between Tot5p/Elp5p and Tot2p/Elp2p. The Association between Elongator and Tot4p requires Tot1-3p/Elp1-3p and Tot5p/Elp5p, indicating that this contact requires a preassembled holo-Elongator complex. |