The role of protein methylation in growth of Saccharomyces cerevisiae and a disruption mutant of SAH1
DeShield, Harven Victor
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Growth of a disruption mutant of S-adenosylhomocysteine hydrolase (Δ SAH1) in S.cerevisiae is severely defective. A prolonged lag precedes log growth, the rate of log growth is 6.7-fold less than WT, and the number of cells at stationary phase for ΔSAH1 is 5-fold less than WT. These effects are due to inhibiting methyltransferases (MTases) by the accumulation of SAH. Cells were labeled with 3 H-CH 3 -SAM and 3 H-Leu to determine the effects of the ΔSAH1 mutation on protein methylation and synthesis. Total protein methylation is inhibited 3.5-fold and protein synthesis is 50% of WT. Single and double mutants of the MTases; RMT1, HSL7, and SET1 showed defective growth and cold-sensitivity, but none of these mutants were as severely affected as Δ SAH1. A BLAST search identified DED1, an RNA helicase that is essential for translation initiation as a possible MTase substrate. Ded1p is methylated in WT and barely detectable in Δ SAH1. Cln3p plays a key role in the initiation of the cell cycle, and is known to be hypersensitive to decreased protein synthesis. Cln3p levels in Δ SAH1 were barely detectable by Western blots or Cln3-Cdc28 kinase assays. Transfection of Δ SAH1 cells with CLN3-1 leads to increased Cln3p levels and complete reversal of the growth, methylation, and protein synthesis defects of Δ SAH1. Cln3p is known to initiate the cell cycle by stimulating the phosphorylation of Whi5p. Whi5p inhibits transcription, and its phosphorylation leads to its dissociation from the essential transcription factor, SBF. The effects of CLN3-1 on Whi5 phosphorylation paralleled its effects on growth indicating that the effects of CLN3-1 were on Cln3p levels and activation of the cell cycle. The results of this thesis indicate a role for protein and RNA methylation in regulating transcription, translation, cell growth, and the cell cycle.