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

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct6A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct6A37 or Ψ38/39 and mcm5U34 or s2U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNALysUUU or tRNAGlnUUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm5U34 or s2U34, and this defect can be rescued by overexpression of tRNAGlnUUG. Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research.

The phytohormone abscisic acid (ABA) plays an important role in modulating plant growth, development, and stress responses. In a genetic screen for mutants with altered drought stress responses, we identified an ABA-overly sensitive mutant, the abo1 mutant, which showed a drought-resistant phenotype. The abo1 mutation enhances ABA-induced stomatal closing and increases ABA sensitivity in inhibiting seedling growth. abo1 mutants are more resistant to oxidative stress than the wild type and show reduced levels of transcripts of several stress-or ABA-responsive genes. Interestingly, the mutation also differentially modulates the development and growth of adjacent guard cells. Map-based cloning identified ABO1 as a new allele of ELO2, which encodes a homolog of Saccharomyces cerevisiae Iki3/Elp1/Tot1 and human IB kinase-associated protein. Water stress caused by drought and soil salinity is an important environmental factor that limits the productivity and distribution of plants. The cellular and molecular mechanisms of plant responses to water stress have been analyzed extensively (48,59,63). Water stress can induce the accumulation of the phytohormone abscisic acid (ABA) (59). ABA plays a vital role in triggering stomatal closure, which reduces transpirational water loss and constitutes an essential part of plant drought tolerance (48,58,63). Analysis of Arabidopsis thaliana mutants has defined several ABA response loci that encode proteins such as protein phosphatases and kinases, which greatly affect guard cell movement (10,45,58).Recent studies indicate that transcripts of protein-coding genes are regulated at all steps of RNA metabolism, from transcription initiation to RNA processing (50). A great deal of information about plant transcriptional regulators that bind the promoters to initiate gene transcription in response to water stress has been collected (61). In contrast, much less is known about proteins involved in RNA processing (30). Nevertheless, recent studies point to a central role of RNA processing in regulating ABA sensitivity and osmotic stress responses. The RNA-binding protein FCA was reported to be an ABA receptor, although it appears to function in ABA regulation of flowering rather than in seed dormancy or drought tolerance (44). ABH1, a cap-binding protein, functions in early ABA signaling (20). A recessive mutation in the SAD1 gene encoding an Sm-like snRNP required for mRNA splicing, export, and degradation rendered plants hypersensitive to ABA and drought (56). The Arabidopsis HYL1 gene encodes a nuclear double-stranded RNA-binding protein. A knockout mutation of the HYL1 gene caused abnormal development, increased sensitivity to abscisic acid, and reduced sensitivity to auxin and cytokinin (33). HYL1 controls gene expression likely through microRNA-mediated gene regulation, although the targeted genes related to ABA sensitivity are still unknown (18). AKIP1 isolated from Vicia faba is a single-stranded RNAbinding protein which can bind to a dehydrin mRNA after phosphorylation by an ABA-...

Diphthamide is a highly modified histidine residue in eukaryal translation elongation factor 2 (eEF2) that is the target for irreversible ADP ribosylation by diphtheria toxin (DT). In Saccharomyces cerevisiae, the initial steps of diphthamide biosynthesis are well characterized and require the DPH1-DPH5 genes. However, the last pathway step—amidation of the intermediate diphthine to diphthamide—is ill-defined. Here we mine the genetic interaction landscapes of DPH1-DPH5 to identify a candidate gene for the elusive amidase (YLR143w/DPH6) and confirm involvement of a second gene (YBR246w/DPH7) in the amidation step. Like dph1-dph5, dph6 and dph7 mutants maintain eEF2 forms that evade inhibition by DT and sordarin, a diphthamide-dependent antifungal. Moreover, mass spectrometry shows that dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2, demonstrating failure to complete the final amidation step. Consistent with an expected requirement for ATP in diphthine amidation, Dph6 contains an essential adenine nucleotide hydrolase domain and binds to eEF2. Dph6 is therefore a candidate for the elusive amidase, while Dph7 apparently couples diphthine synthase (Dph5) to diphthine amidation. The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7. Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal −1 frameshifting and altered responses to translation inhibitors. Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, our work paves the way for a detailed mechanistic understanding of diphthamide formation.