Tomato EF-Tsmt, a functional mitochondrial translation elongation factor from higher plants

Ethylene-induced ripening in tomato (Lycopersicon esculentum) resulted in the accumulation of a transcript designated LeEF-Tsmt that encodes a protein with significant homology to bacterial Ts translational elongation factor (EF-Ts). Transient expression in tobacco and sunflower protoplasts of full-length and truncated LeEF-Tsmt- GFP fusion constructs and confocal microscopy observations clearly demonstrated the targeting of LeEF-Tsmt to mitochondria and not to chloroplasts and the requirement for a signal peptide for the proper sorting of the protein. Escherichia coli recombinant LeEF-Tsmt co-eluted from Ni-NTA resins with a protein corresponding to the molecular weight of the elongation factor EF-Tu of E. coli, indicating an interaction with bacterial EF-Tu. Increasing the GDP concentration in the extraction buffer reduced the amount of EF-Tu in the purified LeEF-Tsmt fraction. The purified LeEF-Tsmt stimulated the poly(U)-directed polymerization of phenylalanine 10-fold in the presence of EF-Tu. Furthermore, LeEF-Tsmt was capable of catalysing the nucleotide exchange reaction with E. coli EF-Tu. Altogether, these data demonstrate that LeEF-Tsmt encodes a functional mitochondrial EF-Ts. LeEFTsmt represents the first mitochondrial elongation factor to be isolated and functionally characterized in higher plants