Localization of the Arabidopsis tRNA nucleotidyltransferase in plant cells and characterization of a novel Arabidopsis protein (Gim1p) interacting with tRNA nucleotidyltransferase

The enzyme tRNA nucleotidyltransferase catalyzes the addition of CMP and AMP residues to the 3 ' end of immature tRNAs. In eukaryotic cells, the nuclear, mitochondrial and chloroplast genomes all encode tRNAs and tRNA nucleotidyltransferase is therefore required in these compartments as well as in the cytosol. In yeast, one gene codes for the tRNA nucleotidyltransferase that functions in the nucleus, mitochondrion and cytosol. As a single gene coding for tRNA nucleotidyltransferase has been identified in Arabidopsis , we were interested in determining whether its gene product(s) is(are) targeted to multiple locations. Protoplast transformation experiments using a green fluorescent protein:tARNA nucleotidyltransferase fusion protein suggests that, as in yeast, both mitochondrial and nuclear targeting information are encoded by the Arabidopsis tRNA nucleotidyltransferase. In addition, this protein also appears to be targeted to the chloroplast. The Arabidopsis homologue of the yeast Gim1p which has been shown in a yeast two-hybrid assay to interact with the Arabidopsis tRNA nucleotidyltransferase (Gu, 2000), complemented a cold and benomyl-sensitive defect in the yeast GIM1 gene. The two-hybrid interaction between the Arabidopsis Gim1p homologue and the Arabidopsis tRNA nucleotidyltransferase was shown to be dependent on the presence of the yeast Gim5p. This observation may suggest that the interaction between Gim1p and tRNA nucleotidyltransferase requires the G[barbelow]enes I[barbelow]nvolved in M[barbelow]icrotubule assembly C[barbelow]omplex (GIMC) of which both Gim1p and Gim5p are components

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