Characterization of MSM1, the structural gene for yeast mitochondrial methionyl‐tRNA synthetase

Abstract

Respiratory-deficient mutants of Saccharomyces cerevisiae assigned to p e t complementation group G72 are impaired in mitochondrial protein synthesis. The loss of this activity has been correlated with the inability of the mutants to acylate the two methionyl-tRNAs of yeast mitochondria. A nuclear gene (MSM1) capable of complementing the respiratory deficiency has been cloned by transformation of the G72 mutant C122/U3 with a yeast genomic library. In situ disruption of the M S M l gene in a wild-type haploid strain of yeast induces a respiratory-deficient phenotype but does not affect the ability of the mutant to grow on fermentable substrates indicating that the product of M S M l functions only in mitochondrial protein synthesis. Mitochondria1 extracts prepared from the mutant with the disrupted copy of MSMl were found to be defective in acylation of the two mitochondrial methionyl-tRNAs thereby confirming the identity of M S M l as the structural gene for the mitochondrial methionyl-tRNA synthetase. The sequence of the protein encoded by MSMI is similar to the Escherichia coli and yeast cytoplasmic methionyl-tRNA synthetases. Based on the primary-sequence similarities of the three proteins, the mitochondrial enzyme appears to be more related to the bacterial than to the yeast cytoplasmic methionyl-tRNA synthetase.Yeast mitochondria contain a complete set of aminoacyltRNA synthetases that catalyze the acylation of the 23 endogenous cognate tRNAs [l]. In contrast to the tRNA substrates whose genes are located in mitochondrial DNA [l, 21, the synthetases are all encoded by nuclear genes. Only the mitochondrial and cytoplasmic histidyl-and valyl-tRNA synthetases of yeast are presently known to be products of common genes [3,4]. All the other mitochondrial synthetases studied to date have been found to be encoded by genes distinct from those coding for the similar cytoplasmic enzymes As part of a study aimed at characterizing the aminoacyltRNA synthetases of mitochondria, we have screened a collection of nuclear respiratory-deficient mutants of yeast for strains defective in acylation of mitochondrial tRNAs [S]. In the present communication, we report the properties of a pet complementation group whose members are impaired in the acylation of the two mitochondrial methionyl-tRNAs. A gene capable of restoring the respiratory deficiency of a representative mutant from this complementation group has been cloned by transformation with a yeast genomic library. The cloned gene designated as M S M l codes for a protein with M , 66 558. The amino acid sequence of the MSMI product derived from the gene sequence is similar to the Escherichia coli [9] and yeast methionyl-tRNA synthetases [lo]. These results indicate that both mitochondrial methionyl-tRNA isoacceptors are acylated by a single enzyme whose gene is distinct from the gene coding for the cytoplasmic methionyl-tRNA synthetase.[S -81.Correspondence fo A. Tzagoloff, Department of Biological Sciences. Columbia University, New York, NY 10027, USA Ahhrevintions. pet, ...

Structural similarities in glutaminyl- and methionyl-tRNA synthetases suggest a common overall orientation of tRNA binding.

“…The region of Met-tRNA synthetase located at this position in the model (residues 356-360) forms the sequence Arg-Tyr-Tyr-Tyr-Thr and is thus similarly capable of polar interaction with the tRNA. This sequence is well-conserved in Met-tRNA synthetase enzymes from other organisms (25,26).…”

Identification of residues involved in the binding of methionine byEscherichia coli methionyl‐tRNA synthetase

“…The michaelian parameters of the homogeneous H301A, W305F, W305A and V298A enzymes in the [32P]pPi-ATP exchange and tRNAr M~t aminoacylation reactions were determined (Table I) [18], Bacillus stearothermophilus [3], Thermus thermophilus [19], Sacharomyces cerevisiae mitochondrion [20] and Sacharomyces cerevh siae cytoplasm [21] were aligned. Shown at the bottom is a consensus sequence obtained through the selection of those residues conserved in at least three out ofthe five compared sequences.…”

General Structure/Function Properties of Microbial Methionyl‐Trna Synthetases

“…Alignment of MetRS sequences from Bacillus subtilis (Ogasarawa et al, 1994), Thermus thermophilus (Nureki et al, 1991), Haemophilus influenzae (Fleishman et al, 1995), Mycoplasma genitulium (Fraser et al, 1995), yeast cytoplasm (Walter et al, 1983) and the yeast mitochondrion (Tzagoloff et al, 1989) actually shows that the peptide regions studied in the present work are conserved (Fig. 1).…”

“…The sequences are those of the enzymes from B. sfearofhermophil~is (Mechulam et al, 1991 b), E. coli (Dardel et al, 1984), B. subti1i.y (Ogasarawa et al, 1994), 7: thermophilus (Nureki et al, 1991). yeast mitochondrion (Tzagoloff et at., 1989), H. inj2uenzae (Fleishman et al, 1995), M. genitulium (Fraser et al, 1995), yeast cytoplasm (Walter et al, 1983). chemical properties resembling those of its E. coli counterpart (Kalogerakos et al, 1980;Mayaux and Blanquet, 1981;Mechulam et al, 1991b), although the two enzymes share only 27% identical residues (Fig.…”