The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization.

We report that the temperature-sensitive (ts) phenotype in Saccharomyces cerevisiae associated with a variant tRNA nucleotidyltransferase containing an amino acid substitution at position 189 results from a reduced ability to incorporate AMP and CMP into tRNAs. We show that this defect can be compensated for by a second-site suppressor converting residue arginine 64 to tryptophan. The R64W substitution does not alter the structure or thermal stability of the enzyme dramatically but restores catalytic activity in vitro and suppresses the ts phenotype in vivo. R64 is found in motif A known to be involved in catalysis and nucleotide triphosphate binding while E189 lies within motif C previously thought only to connect the head and neck domains of the protein. Although mutagenesis experiments indicate that residues R64 and E189 do not interact directly, our data suggest a critical role for residue E189 in enzyme structure and function. Both R64 and E189 may contribute to the organization of the catalytic domain of the enzyme. These results, along with overexpression and deletion analyses, show that the ts phenotype of cca1-E189F does not arise from thermal instability of the variant tRNA nucleotidyltransferase but instead from the inability of a partially active enzyme to support growth only at higher temperatures.

The folding capacity of the mature domain of the dual-targeted plant tRNA nucleotidyltransferase influences organelle selection.

tRNA-NTs (tRNA nucleotidyltransferases) are required for the maturation or repair of tRNAs by ensuring that they have an intact cytidine-cytidine-adenosine sequence at their 3'-termini. Therefore this enzymatic activity is found in all cellular compartments, namely the nucleus, cytoplasm, plastids and mitochondria, in which tRNA synthesis or translation occurs. A single gene codes for tRNA-NT in plants, suggesting a complex targeting mechanism. Consistent with this, distinct signals have been proposed for plastidic, mitochondrial and nuclear targeting. Our previous research has shown that in addition to N-terminal targeting information, the mature domain of the protein itself modifies targeting to mitochondria and plastids. This suggests the existence of an as yet unknown determinate for the distribution of dual-targeted proteins between these two organelles. In the present study, we explore the enzymatic and physicochemical properties of tRNA-NT variants to correlate the properties of the enzyme with the intracellular distribution of the protein. We show that alteration of tRNA-NT stability influences its intracellular distribution due to variations in organelle import capacities. Hence the fate of the protein is determined not only by the transit peptide sequence, but also by the physicochemical properties of the mature protein.

Dual targeting of the tRNA nucleotidyltransferase in plants: not just the signal.

Enzymes involved in tRNA maturation are essential for cytosolic, mitochondrial, and plastid protein synthesis and are therefore localized to these different compartments of the cell. Interestingly, only one isoform of tRNA nucleotidyltransferase (responsible for adding the 3'-terminal cytidine-cytidine-adenosine to tRNAs) has been identified in plants. The present study therefore explored how signals contained on this enzyme allow it to be distributed among the different cell compartments. It is demonstrated that the N-terminal portion of the protein acts as an organellar targeting signal and that differential use of multiple in-frame start codons alters the localization of the protein. Moreover, it is shown that the mature domain has a major impact on the distribution of the protein within the cell. These data indicate that regulation of dual localization involves not only specific N-terminal signals, but also additional factors within the protein or the cell.

Characterization of a gene encoding tRNA nucleotidyltransferase from Candida glabrata.

A gene encoding ATP (CTP):tRNA nucleotidyltransferase (EC2.7.7.25) was isolated from Candida (Torulopsis) glabrata by complementation in Saccharomyces cerevisiae. The predicted amino acid sequence of the protein revealed a large region with high sequence similarity to members of the Class II group of the nucleotidyltransferase superfamily and an N-terminal region characteristic of a mitochondrial targeting sequence. The essential role of the carboxylates within the conserved DXD and RRD motifs was confirmed by mutagenesis. C. glabrata strains bearing truncated CCA1 genes that lacked sequences encoding the putative mitochondrial targeting peptide were unable to grow on non-fermentable carbon sources but were able to grow on a fermentable carbon source. These results suggest that, as in S. cerevisiae, the C. glabrata CCA-adding enzyme is a sorting isozyme that functions in multiple cellular compartments. Mapping of the 5'-ends of primary transcripts of CCA1 revealed multiple transcription start sites located both upstream of and between two in-frame start codons. When the cells were cultured on a non-fermentable carbon source the longer transcripts appeared more abundant, suggesting that the choice of transcription start sites was influenced by carbon source. The shorter transcripts, which lacked sequences encoding the mitochondrial targeting information, were more predominant in cells grown on glucose. These observations suggest that expression of CCA-adding isozymes in C. glabrata may be regulated. The DNA sequence has been assigned GenBank Accession No. AF098803.