NAM9 Nuclear Suppressor of Mitochondrial Ochre Mutations in Saccharomyces cerevisiae Codes for a Protein Homologous to S4 Ribosomal Proteins from Chloroplasts, Bacteria, and Eucaryotes

Abstract

We report the genetic characterization, molecular cloning, and sequencing of a novel nuclear suppressor, the NAM9 gene from Saccharomyces cerevisiae, which acts on mutations of mitochondrial DNA. The strain NAM9-1 was isolated as a respiration-competent revertant of a mitochondrial mit mutant which carries the V25 ochre mutation in the oxi1 gene. Genetic characterization of the NAM9-1 mutation has shown that it is a nuclear dominant omnipotent suppressor alleviating several mutations in all four mitochondrial genes tested and has suggested its informational, and probably ribosomal, character. The NAM9 gene was cloned by transformation of the recipient oxi1-V25 mutant to respiration competence by using a gene bank from the NAM9-1 rho o strain. Orthogonal-field alternation gel electrophoresis analysis and genetic mapping localized the NAM9 gene on the right arm of chromosome XIV. Sequence analysis of the NAM9 gene showed that it encodes a basic protein of 485 amino acids with a presequence that could target the protein to the mitochondrial matrix. The N-terminal sequence of 200 amino acids of the deduced NAM9 product strongly resembles the S4 ribosomal proteins from chloroplasts and bacteria. Significant although less extensive similarity was found with ribosomal cytoplasmic proteins from lower eucaryotes, including S. cerevisiae. Chromosomal inactivation of the NAM9+ gene is not lethal to the cell but leads to respiration deficiency and loss of mitochondrial DNA integrity. We conclude that the NAM9 gene product is a mitochondrial ribosomal counterpart of S4 ribosomal proteins found in other systems and that the suppressor acts through decreasing the fidelity of translation.

The Single Amino Acid Changes in the Yeast Mitochondrial S4 Ribosomal Protein Cause Temperature-Sensitive Defect in the Accumulation of Mitochondrial 15S rRNA

“…The results are illustrated in Figure . Another single amino acid substitution (Ser 82 → Leu) in the S4 domain of MNA9/Mna6p was also identified by Boguta et al ( , ) using a ts respiratory defect yeast mutant harboring the mutation. This mutation suppresses the ochre mutation in the mt genome by increasing the translational ambiguity of mitoribosome ().…”

“…The cloned MNA6 gene carries a 1458 bp open reading frame that codes for a basic protein of estimated molecular mass 56 kDa. The protein sequence homology searches of the GeneBanks revealed that the MNA6 encodes an S4-like r-protein (see below) and is also identical to the previously isolated NAM9 gene (GeneBank Accession number M60730) (17). To confirm the genetic identity of the MNA6 and NAM9 genes, the yeast strain with the disrupted nam9:URA3 gene (MATR his3 leu2 ura3 nam9:URA3) (provided by Dr. Magdalena Boguta, Warsaw, Poland) was crossed with the ts mna6 mutant (MATa his1 trp1).…”

“…The wild type (wt) and four mutant alleles of the MNA6 gene have been cloned and sequenced. The cloned gene appears to be identical to the previously isolated NAM9 gene (17). For simplicity, the MNA6/NAM9 gene will be referred to as MNA6 throughout this paper.…”