engleski

C-terminal extensions of eukaryotic seryl-tRNA synthetases: impact on protein function and stability

Eukaryotic seryl-tRNA synthetases (SerRS) contain a positively charged C-terminal extension, which is not found in the prokaryotic counterparts. It was previously shown that the removal of the whole C-terminal appendix from yeast SerRS affected both the stability of the enzyme and its affinity for the substrates, suggesting that the C-terminal extension is important in maintaining the overall structure of the synthetase. Yeast two-hybrid screen of Saccharomyces cerevisiae cDNA library identified Pex21p, a protein related to peroxisome biogenesis, as an interacting partner of yeast SerRS. In present work, functional aspects of eukaryotic SerRS extensions and their impact on protein stability were studied on yeast and maize SerRSs as representative eukaryotic enzymes. A series of maize and yeast SerRS variants was generated, comprising partial deletions and mutations in the C-terminal region. Mutational screening, protein expression and thermal denaturation studies revealed that the proximal region of extension appended to the enzyme core is indispensable for the stability of maize and yeast SerRS. Catalytic properties of shortened SerRS variants were not affected by truncations if the short peptide proximal to the catalytic core was left intact. Shortened SerRS variants readily formed complexes with cognate tRNA, as shown by electrophoretic mobility shift assay. Results of kinetic and gel-shift experiments led to conclusion that C-terminal extensions of eukaryotic SerRSs do not function as auxiliary tRNA-binding domains, in spite of their basic character. Complementation experiments of the Saccharomyces cerevisiae strain with disrupted SerRS gene demonstrated that truncated variants of yeast and maize SerRS are functional in vivo as well. Yeast SerRS binds peroxin Pex21p, while moderately homologous maize SerRS does not. In order to map Pex21p interaction domain, yeast SerRS was progressively shortened starting from C-terminus and tested for ability to interact with Pex21p in yeast two-hybrid and pull-down assay. The interaction with Pex21p was lost upon removal of the C-terminal extension. Therefore, the presence of yeast SerRS extension was requisite for the interaction with Pex21p. To explore whether yeast SerRS extension is a standalone Pex21p-binding domain, extension of maize SerRS was replaced with extension of yeast counterpart. No interaction between such chimeric proteins and Pex21p could be observed, implying that additional regions of yeast SerRS are involved in Pex21p binding. In conclusion, the proximal region of eukaryotic SerRS C-terminal extensions is indispensable for protein stability, while the remaining part of the extension resides available for other functions, such as species-specific protein:protein interactions.

seryl-tRNA synthetase; aaRS extensions; protein stability; peroxin; protein-protein interaction

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