Purification of the yeast mitochondrial methionyl‐tRNA synthetase

Abstract

Yeast-mitochondria1 methionyl-tRNA synthetase was purified 1060-fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four-step procedure based on affinity chromatography (heparin-Ultrogel, tRNAMe'-Sepharose, Agarose-hexyl-AMP) to yield a single polypeptide of high specific activity (1800 U/mg). Like the cytoplasmic methionyl-tRNA synthetase ( M , 85 000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size ( M , 65 000). In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152000) made up of identical subunits. The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNA'" are similar to those of the cytoplasmic isoenzyme. However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E. coli tRNAMe'. Furthermore, polyclonal antibodies raised against the two proteins did not show any cross-reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants. Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases are different proteins coded for by two distinct nuclear genes.Like the yeast cytoplasmic aminoacyl-tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin. This distinguishes them from the corresponding enzymes of E. coli. Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.The components of the yeast mitochondrial translation apparatus are of dual origin. While the mitochondrial (mt) DNA codes for a complete set of transfer RNAs and the 15s and 21s ribosomal RNAs, the proteins required for mitochondrial protein synthesis such as the ribosomal proteins, the aminoacyl-tRNA synthetases, and the translation factors are coded for by nuclear genes. The sole exception is the ribosomal protein varl which is encoded by the mt DNA (for reviews, see [l, 21).Aminoacyl-tRNA synthetases constitute a divergent family of enzymes differing in size and subunit structure but catalyzing the same reaction, the selective attachment of amino acids to their cognate tRNAs. The molecular and catalytic properties of these enzymes have been extensively studied over the past 20 years [3, 41. However, information about mitochondrial aminoacyl-t RNA synthetases remained scarce until recently. Previous studies on phenylalanyl-, leucyl-, and methionyl-tRNA synthetases from yeast mitochondria and cytoplasm indicated that the two isoenzymes exhibit distinctive properties [5 -71. By contrast, no significant differences could be observed between the mitochondrial and cytoplasmic valyl-tRNA synthetases IS]. Recently, cloning and molecular analysis of genes coding for mitochondrial aminoacyl-tRNA synthetases revealed the existence of two classes of nuclea...

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian andE.colimethionyl-tRNA synthetases

“…Finally, the mitochondrial MTS from yeast has been reported to aminoacylate both the E. coli initiator and elongator tRNAMet, as efficiently as the two mitochondrial tRNAsMet (20,22). Interestingly, the mitochondrial elongator tRNAsMet sequenced up to now display the 7-nucleotide anticodon loop (33).…”

“…In particular, elongator tRNAMet from rabbit liver has been reported not to bind MTSEC better than a non-Met tRNA (17). In addition, yeast mitochondrial and wheat germ chloroplastic MTS aminoacylate as efficiently E. coli tRNAsMet as their cognate tRNAs (20)(21)(22). Finally, eukaryotic cytoplasmic MTS aminoacylate the mitochondrial, choroplastic and E.coli tRNAsMet with a low efficiency (20-22) whereas mammalian cytoplasmic MTS efficiently aminoacylates cytoplasmic tRNAMet from yeast (23).…”

Characterization of the Human Mitochondrial Methionyl-tRNA Synthetase

“…The domain organization of human mtMetRS most closely resembles that of the S. cereVisiae and C. albicans mitochondrial MetRSs. Human mtMetRS lacks the approximately 100 amino acid C-terminal extensions found in many bacterial MetRSs and thus functions as a monomer, similar to the characterized mitochondrial MetRSs (11,12). Despite the lack of a high degree of sequence similarity to E. coli MetRS (Table 1), Swiss-Model was able to model a significant region of the catalytic core of human mtMetRS based on the crystal structure of both E. coli and T. thermophilus MetRS (Figure 4A).…”

“…The human mitochondrial PheRS, LeuRS, and IleRS and the bovine mitochondrial SerRS have been studied biochemically ( − ). The mitochondrial MetRSs from Saccharomyces cerevisiae and the pathogenic fungus Candida albicans have been identified and characterized as well ( , ). Mitochondrial synthetases are encoded in the nucleus and contain mitochondrial import sequences for transport into the organelle ().…”

Structural characterization of free-state and product-stateMycobacterium tuberculosismethionyl-tRNA synthetase reveals an induced-fit ligand-recognition mechanism

“…The following references are cited in the Supporting Information for this article: Kalogerakos et al (1980), Kohda et al (1987), Nureki et al (1993), Schmitt et al (1997) and Schwob et al (1988).…”