Abstract

The enzyme ATP(CTP): tRNA-specific tRNA nucleotidyltransferase (also known as Cca1, the CCA-adding enzyme) adds to all tRNAs the 3’-cytidine, cytidine, and adenosine (CCA) tail required for aminoacylation. A single essential nuclear gene, CCA1, in Saccharomyces cerevisiae codes for versions of this protein found in the cytosol, nucleus and mitochondrion (Chen et al., 1992). Here, we use mass spectrometry to explore factors involved in the distribution and activity of this enzyme. These factors may be cis-acting, e.g., post-translational modifications (PTMs) of Cca1, or trans-acting, e.g., accessory proteins involved in Cca1 activity or which serve to direct Cca1 to specific locations in the cell.
Specific PTMs, i.e., phosphorylation and/or acetylation, were identified on amino acids S4, T5, T7, S12, T17, T19, and S21 at the N-terminus of Cca1 and also on K57, K118, K150, K328, T407, and K507. Most of these PTMs are reported for the first time in this study. These PTMs might impact enzyme localization (as the N-terminal cluster of PTMs is near the known mitochondrial targeting signal) or activity as some post-translationally modified amino acids (e.g., K118, K150) are in important functional domains of the protein. Differences between the PTMs detected at specific positions in different cellular fractions, suggest the role of these modifications in Cca1 localization. Moreover, some conserved PTMs (e.g., K150, T407, and K507) across different conditions and fraction might implicate these specific modifications in enzyme activity.
As Cca1 is an essential enzyme present in different parts of the cell (mitochondria, nucleus and cytosol), we identified proteins interacting with it that might mediate its function or localization. Among the proteins identified were the karyopherins Kap95, Kap104 and Kap123, which allowed us to predict a nuclear localization signal in a eukaryotic CCA-adding enzyme for the first time. Additionally, Cca1 was shown to interact with other enzymes required for tRNA maturation and repair.