Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins inSaccharomyces cerevisiae

Abstract

The genes for two large subunit proteins, YmL8 and YmL20, of the mitochondrial ribosome of Saccharomyces cerevisiae were cloned by hybridization with synthetic oligonucleotide mixtures corresponding to their N-terminal amino acid sequences. They were termed MRP-L8 and MRP-L20, respectively, and their nucleotide sequences were determined using a DNA sequencer. The MRP-L8 gene was found to encode a 26.8-kDa protein whose deduced amino acid sequence has a high degree of similarity to ribosomal protein L17 of Escherichia coli. The gene MRP-L20 was found to encode a 22.3-kDa protein with a presequence consisting of 18 amino acid residues. By Southern blot hybridization to the yeast chromosomes separated by field-inversion gel electrophoresis, the MRP-L8 and MRP-L20 genes were located on chromosomes X and XI, respectively. Gene disruption experiments indicate that their products, YmL8 and YmL20 proteins, are essential for the mitochondrial function and the absence of these proteins causes instability of the mitochondrial DNA.

Mitochondrial transport of mitoribosomal proteins, YmL8 and YmL20, in Saccharomyces cerevisiae

“…The location, direction and sequence of each primer are shown in Table 1. The sizes of P7 and P8 were determined by agarose gel electrophoresis (Kitakawa et al, 1990 T4 DNA polymerase was added at a final concentration of 0.03 unit/pl and the mixture was incubated at 37°C for 30 min. After extraction with phenol, phenolkhloroform and chloroform, the amplified DNA was precipitated with ethanol, washed with 70% ethanol and redissolved in 25 p1 water.…”

“…It was reported that YmL8 protein had no N-terminal presequence and that its N-terminal and C-terminal regions were similar to E. coli ribosomal proteins L17 and S13, respectively (Kitakawa et al, 1990). YmL8 protein is larger than the E. coli proteins and has an extended C-terminal region.…”

“…All components except for the RNAs and one protein are encoded in the nucleus. About 20 nuclear genes for mitoribosomal proteins have so far been cloned and characterized (Myers et al, 1987;Partaledis and Mason, 1988;Fearon and Mason, 1988;Grohmann et al, 1989;Matsushita et al, 1989;Dang and ElIis, 1990;McMullin et al, 1990;Kitakawa et al, 1990;Graack et al, 1991;Kang et al, 1991a,b;Boguta et al, 1992;Davis et al, 1992;Fearon and Mason, 1992;Graack et al, 1992). Most of these mitoribosomal protein genes have been found to be essential for mitochondrial function.…”

“…Therefore, we were interested to investigate further on the function of the presequences of mitoribosomal proteins. Among the yeast mitoribosomal proteins which do not possess any presequence, YmL8 and YmL33 were found to have a considerable degree of sequence similarity in their N-terminal regions to Escherichia coli ribosomal proteins L17 and L30, respectively (Kitakawa et al, 1990;Kang et al, 1991b). These yeast mitoribosomal proteins are much larger than their E. coli counterparts by having longer C-terminal regions.…”

“…digested with HindII. It was then ligated with a 2.5-kb Him dIII-PvuII fragment of pL8EP which contained the MRP-L8 gene (Kitakawa et al, 1990). One of the resultant plasmids, termed pYML8link, was digested with BamHI and K m I and the 3' end of MRP-L.8 was deleted with exo-EXPERIMENTAL PROCEDURES…”