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Characterization of MSM1, the structural gene for yeast mitochondrial methionyl-tRNA synthetase
A Tzagoloff1, A Vambutas, A Akai
1Department of Biological Sciences, Columbia University, New York, NY 10027.
Abstract:
Respiratory-deficient mutants of Saccharomyces cerevisiae assigned to pet 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 MSM1 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 MSM1 functions only in mitochondrial protein synthesis. Mitochondrial extracts prepared from the mutant with the disrupted copy of MSM1 were found to be defective in acylation of the two mitochondrial methionyl-tRNAs thereby confirming the identity of MSM1 as the structural gene for the mitochondrial methionyl-tRNA synthetase. The sequence of the protein encoded by MSM1 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.
Insights
Researchers identified the MSM1 gene, crucial for mitochondrial protein synthesis in yeast. This gene encodes the mitochondrial methionyl-tRNA synthetase, essential for respiratory function.
Area of Science:
- Molecular biology
- Yeast genetics
- Mitochondrial biogenesis
Background:
- Respiratory-deficient Saccharomyces cerevisiae mutants (G72) exhibit impaired mitochondrial protein synthesis.
- This impairment is linked to defects in methionyl-tRNA acylation within yeast mitochondria.
Purpose of the Study:
- To identify the nuclear gene responsible for complementing the respiratory deficiency in G72 mutants.
- To elucidate the function of the identified gene in mitochondrial protein synthesis.
Main Methods:
- Cloning of the nuclear gene (MSM1) via yeast genomic library transformation.
- In situ gene disruption of MSM1 in a wild-type yeast strain.
- Analysis of respiratory phenotype and growth on fermentable substrates.
- Biochemical assays on mitochondrial extracts to assess methionyl-tRNA acylation.
Main Results:
- The cloned gene, MSM1, complements the respiratory deficiency in G72 mutants.
- Disruption of MSM1 leads to respiratory deficiency but retains fermentative growth.
- Mitochondrial extracts from MSM1-disrupted mutants show defects in methionyl-tRNA acylation.
- MSM1 is confirmed as the structural gene for mitochondrial methionyl-tRNA synthetase.
Conclusions:
- The MSM1 gene product is essential for mitochondrial protein synthesis in yeast.
- The mitochondrial methionyl-tRNA synthetase shares sequence similarities with bacterial and cytoplasmic counterparts.
- The mitochondrial enzyme shows greater evolutionary relatedness to bacterial methionyl-tRNA synthetase than to the yeast cytoplasmic enzyme.