Cell proliferation control using yeast as model eukaryotes

Over the last 10 years, we have focussed on the study of yeast cell proliferation, particularly in response to zymocin which is a trimeric (abg) toxin complex from Kluyveromyces lactis that kills Saccharomyces cerevisiae. The overall goal of our research into this yeast pathosystem has been to use the power of budding yeasts as eukaryotic models to provide insights into organismic interaction and microbial competition. Using molecular techniques, we have genetically dissected the lethal response towards zymocin, which results in a G1 cell cycle arrest, into a multi-step pathway that involves ~30 genes. It initiates with docking of zymocin onto cell wall chitin and chitinolysis by zymocin's a-subunit. Subsequently, early events that mediate import and activation of zymocin's lethal g-toxin subunit require plasma membrane sphingolipid synthesis and the proton pump Pma1. Intracellularly, g-toxicity requires the Elongator complex (Elp1-6), Elongator-related proteins (Kti11-14: k iller t oxin i nsensitive) and tRNA methylase Trm9. These zymocin players were isolated in our group using genome-wide transposon mutagenesis to identify toxin resistant S. cerevisiae mutants or single-copy complementation of existing kti mutants generated in the group of Stark at Dundee. As shown by the Svejstrup group, Elongator is a histone acetylase (HAT) that partners with RNA polymerase II. By virtue of its HAT activity, Elongator is thought to assist transcription by RNA polymerase II through chromatin. In further support of a transcriptional role, Elongator binds unspliced nascent mRNAs that emanate from the transcription machinery in the nucleus. 

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Elongator function in zymocin-dependent growth inhibition

Apart from this, Elongator is related to processes as diverse as tubulin and tRNA modification. Whether these depend on Elongator's transcriptional functions is not known. Strikingly, however, the group of Byström has shown that Elongator's role in tRNA modification is required for the generation of modified uridines (U34) at the wobble position of tRNA anticodons including 5-methoxycarbonyl-methyl-2-thiouridine (mcm5s2U34). Although it is unclear how Elongator takes part in U34 modification , it is required for suppression of non-/missense mutations by U34-carrying tRNA suppressors. So Elongator may influence anticodon/codon interaction, tRNA decoding and thereby mRNA translation. In further support of a link between Elongator and tRNA functioning, zymocin's g-toxin is a tRNase that cleaves tRNA anticodons that carry mcm5s2U34 . Since the full modification depends on Elongator and tRNA methylase Trm9, this provides an explanation why Elongator and trm9 mutants survive zymocin: they efficiently protect against the tRNase attack of g -toxin. Our recent data that expression of ELO2, the Arabidopsis homolog of the yeast Elongator subunit Elp1, reconfers zymocin sensitivity to a yeast elp1 mutant, imply that Elongator roles in tRNA modification may have been conserved among eukaryotes. Moreover, we have provided preliminary evidence that Elongator function is subject to phosphomodulation of Elp1.

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Kti11 – a versatile partner of protein complexes with roles in cell proliferation

Intriguingly, Elongator partner protein Kti11 not only mediates cell death by zymocin but is also known as Dph3. Dph3 is one of five components that are required for diphthamide synthesis on translational elongation factor EF2. Although the physiological role of diphthamide is unknown, this exotic protein modification serves as the target for lethal ADP-ribosylation of EF2 by bacterial toxins including diphtheria toxin and exotoxin A. So, Kti11/Dph3 appears to be a versatile partner of proteins involved in cell proliferation and growth. Not surprisingly, the mammalian homolog of DPH1, encoding a partner of Kti11/Dph3, is known as a tumour suppressor gene in ovarian cancer (OVCA1) formation.

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