Abstract

The enzyme ATP (CTP): tRNA-specific tRNA nucleotidyltransferase (tRNA-NT) is an essential enzyme in eukaryotes and some prokaryotes and plays a critical role in tRNA maturation. This enzyme is responsible for the addition of AMP and CMP residues to the 3’-ends of tRNA molecules to allow for their aminoacylation and subsequent use in protein synthesis. The tRNA-NT (Cca1) in Saccharomyces cerevisiae (yeast), although encoded by a single nuclear gene (CCA1), functions in multiple cellular compartments (cytosol, nucleus, and mitochondrion). Here we used mass spectrometry to explore both cis- and trans-acting factors that could provide clues to the importance of the function and localization of this enzyme in Saccharomyces cerevisiae. We characterized post-translational modifications on the native enzyme and analyzed the proteomes of native and temperature-sensitive (ts) yeast strains at permissive (20oC and 30oC) and quasi-restrictive (33oC) temperatures.
Our experiments identified amino acids cysteine 307, lysine 312 and tyrosine 317 in one α-helix as sites of post-translational modifications in early log phase showing acetylation, acetylation and phosphorylation, respectively. As these modifications were present, this may suggest that these post-translational modifications play a role in protein function or localization.
As we have identified a mutation in the gene coding for tRNA-NT that leads to a temperature-sensitive phenotype, we set out to do a comparison of the proteome profiles of this strain and the native yeast strain from which it was derived. This study revealed specific differences between the native and ts strains and between the different temperatures tested. Levels of stress-response proteins (Ctt1, Ddp1, Gad1, Prx1, Sod2, Tdh1, Uga1, and Uga2) were increased in the variant but not the native strain at 33°C but not in either the native or the variant at any other temperature tested (20°C or 30°C). This suggests that in the ts strain, the stress response is induced to combat the loss of activity associated with the variant tRNA-NT. In addition, a number of mitochondrial or ribosomal proteins such as Aim24, Atp20, Cox2, Cox5A, Mhr1, Nuc1, Rip1, Drs1, Edc3, Hca4, Nog2, Nsa2, Rlp7, Sod1, Sof1, Tsr4, Utp23 whose levels differ in the native and variant proteomes were identified, suggesting pathways to be explored to explain the ts phenotype.