The single amino acid changes in the yeast mitochondrial S4 ribosomal protein cause temperature-sensitive defect in the accumulation of mitochondrial15S rRNA

Four different mutant alleles of a nuclear gene (MNA6), which lose mt 15S r RNA at nonpermissive temperature (36 degrees C), were previously generated by EMS mutagenesis of Saccharomyces cerevisiae. To understand the biochemic al basis for the loss of 15S rRNA in these mutants, the wild-type and mutan t alleles of the MNA6 gene were isolated and characterized. The DNA sequenc ing of the cloned MNA6 gene revealed that it has an open reading frame spec ifying a 486 amino acid polypeptide, which appears to be a yeast mt homolog ue 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 appea red to be identical to the previously isolated yeast NAM9 gene. The in vitr o expression under coupled transcription-translation reaction conditions fo llowed by mt import demonstrated that MNA6 indeed encodes a similar to 56 k Da protein targeted to the mitochondria. We have also demonstrated by Weste rn blot analysis using anti-Mna6p antibody that Mna6p is associated with th e small subunit of mitoribosomes. The sequence analysis of the four mutant mna6 alleles revealed that Leu(109) -> Phe, Arg(111) -> Lys, Pro(424) -> Le u, or Pro(438)->Leu amino acid substitution in Mna6p causes temperature-dep endent loss of the 15S rRNA. These mutations do not affect the mitochondria l import or accumulation of Mna6p. Rather the evidence paints to an inabili ty of mutant MnaGp to be assembled into the mitoribosomes of cells grown at 36 degrees C.