The single amino acid changes in the yeast mitochondrial S4 ribosomal protein cause temperature-sensitive defect in the accumulation of mitochondrial 15S rRNA

Four different mutant alleles of a nuclear gene (MNA6), which lose mt 15S rRNA at nonpermissive temperature (36 degrees C), were previously generated by EMS mutagenesis of Saccharomyces cerevisiae. To understand the biochemical basis for the loss of 15S rRNA in these mutants, the wild-type and mutant alleles of the MNA6 gene were isolated and characterized. The DNA sequencing of the cloned MNA6 gene revealed that it has an open reading frame specifying a 486 amino acid polypeptide, which appears to be a yeast mt homologue of the S4 r-protein family. The large size of this yeast S4 homologue is due to a nonhomologous long C-terminal extension. The MNA6 gene also appeared to be identical to the previously isolated yeast NAM9 gene. The in vitro expression under coupled transcription-translation reaction conditions followed by mt import demonstrated that MNA6 indeed encodes a similar to 56 kDa protein targeted to the mitochondria. We have also demonstrated by Western blot analysis using anti-Mna6p antibody that Mna6p is associated with the small subunit of mitoribosomes. The sequence analysis of the four mutant mna6 alleles revealed that Leu(109) -> Phe, Arg(111) -> Lys, Pro(424) -> Leu, or Pro(438)->Leu amino acid substitution in Mna6p causes temperature-dependent loss of the 15S rRNA. These mutations do not affect the mitochondrial import or accumulation of Mna6p. Rather the evidence paints to an inability of mutant MnaGp to be assembled into the mitoribosomes of cells grown at 36 degrees C.