SummaryZymocin-induced cell death in Saccharomyces cerevisiae requires the toxin-target (TOT) effector Elongator, a protein complex with functions in transcription, exocytosis and tRNA modification. In line with the latter, trm9 ∆ ∆ ∆ ∆ cells lacking a tRNA methylase specific for wobble uridine (U 34 ) residues survive zymocin and in excess, the Trm9 substrate tRNA Glu copies zymocin protection of Elongator mutants. Phenotypes typical of a tot3/elp3 ∆ ∆ ∆ ∆ Elongator mutant are absent from trm9 ∆ ∆ ∆ ∆ cells but copied in a tot3 ∆ ∆ ∆ ∆ trm9 ∆ ∆ ∆ ∆ double mutant suggesting that Elongator acts upstream of Trm9. Consistent with Elongator-dependent tRNA modification being more important to mRNA decoding than Trm9, SUP4 and SOE1 tRNA suppressors are highly sensitive to loss of Elongator and tRNA U 34 hypomodification. As Trm9 overexpression counteracts the effect of high-copy tRNA Glu

Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator-dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.

SummaryIn yeast, the role for the Elongator complex in tRNA anticodon modification is affected by phosphorylation of Elongator subunit Elp1. Thus, hyperphosphorylation of Elp1 due to inactivation of protein phosphatase Sit4 correlates with Elongator-minus phenotypes including resistance towards zymocin, a tRNase cleaving anticodons of Elongator-dependent tRNAs. Here we show that zymocin resistance of casein kinase hrr25 mutants associates with hypophosphorylation of Elp1 and that nonsense suppression by the Elongator-dependent SUP4 tRNA is abolished in hrr25 or sit4 mutants. Thus changes that perturb the evenly balanced ratio between hyper-and hypophosphorylated Elp1 forms present in wild-type cells lead to Elongator inactivation. Antagonistic roles for Hrr25 and Sit4 in Elongator function are further supported by our data that Sit4 inactivation is capable of restoring both zymocin sensitivity and normal ratios between the two Elp1 forms in hrr25 mutants. Hrr25 binds to Elongator in a fashion dependent on Elongator partner Kti12. Like sit4 mutants, overexpression of Kti12 triggers Elp1 hyperphosphorylation. Intriguingly, this effect of Kti12 is blocked by hrr25 mutations, which also show enhanced binding of Kti12 to Elongator. Collectively, our data suggest that rather than directly targeting Elp1, the Hrr25 kinase indirectly affects Elp1 phosphorylation states through control of Sit4-dependent dephosphorylation of Elp1.

SummaryThe alpha-tubulin suppressor 1 (ATS1) gene and the killer toxin-insensitive 13 (KTI13) locus from Saccharomyces cerevisiae are allelic. The Ats1/Kti13 gene product interacts with the cell polarity factor Nap1 and promotes growth inhibition of S. cerevisiae by zymocin, a tRNAse toxin complex from Kluyveromyces lactis. Kti13 removal causes zymocin resistance, a trait that is typical of defects in the Elongator complex. Here, we show that Kti13 co-purifies with the Elongator partner protein Kti11 and that the Kti11 interaction, not the Nap1 partnership, requires the C-terminus of Kti13. Moreover, Kti13 functionally relates to roles of the Elongator complex in tRNA wobble uridine modification, tRNA suppression of nonsense (SUP4) and missense (SOE1) mutations and tRNA restriction by zymocin. Also, inactivation of Kti13 or Elongator rescues the thermosensitive growth defect of secretory mutants (sec2-59 ts , sec12-4 ts ), suggesting that Kti13 and Elongator affect secretion processes that depend on the GTP exchange factors Sec2 and Sec12 respectively. Distinct from tandem deletions in KTI13 and Elongator genes, a kti13Dkti11D double deletion induces synthetic sickness or lethality. In sum, our data suggest that Kti13 and Kti11 support Elongator functions and that they both share Elongator-independent role(s) that are important for cell viability.

Zymocin, a toxic protein complex produced by Kluyveromyces lactis, inhibits cell cycle progression in Saccharomyces cerevisiae. In studying its action, a resistant mutant ( kti14-1) was found to express the totphenotype typical of totD cells, toxin target (TOT) mutants that are impaired in RNA polymerase II Elongator function. Phenotypic analysis of a kti14-1 tot3D double mutant revealed a functional link between KTI14 and TOT/Elongator. Unlike totD cells, the kti14-1 mutant is sensitive to the drug methylmethane sulfonate (MMS), indicating that, besides being affected in TOT function, kti14-1 cells are also compromised in DNA repair. Single-copy complementation identified HRR25, which codes for casein kinase I (CKI), as KTI14. Kinase-minus hrr25 mutations (K38A and T176I) conferred zymocin resistance, while deletion of the other yeast CKI genes (YCK1)3) had no effect. A mutation in KTI14 that truncates the P/Q-rich C-terminus of Hrr25p also dissociates MMS sensitivity from zymocin resistance; this mutant is resistant to the toxin, but shows normal sensitivity to MMS. Thus, although kinase-minus mutations are sufficient to protect yeast cells from zymocin, toxicity is also dependent on the integrity of the C-terminal region of Hrr25p, which has been implicated in determining the substrate specificity or localization of Hrr25p.

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.

SummaryZymocin, a three-subunit ( a a a ab b b bg g g g ) toxin complex from Kluyveromyces lactis , imposes a cell cycle block on Saccharomyces cerevisiae . Phenotypic analysis of the resistant kti10 mutant implies a membrane defect, suggesting that KTI10 represents a gene involved early in the zymocin response. Consistently, KTI10 is shown here to be allelic to PMA1 encoding H +

Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator-dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast-plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.