Abstract
MRP20 and MRP49 are proteins of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae. Their genes were identified through immunological screening of a genomic library in the expression vector lambda gt11. Nucleotide sequencing revealed that MRP49 is tightly linked to TPK3 and encodes a 16-kDa, basic protein with no significant relatedness to any other known protein. MRP20 specifies a 263-amino-acid polypeptide with sequence similarity to members of the L23 family of ribosomal proteins. The levels of the mRNAs and proteins for both MRP20 and MRP49 were regulated in response to carbon source. In [rho0] strains lacking mitochondrial rRNA, the levels of the two proteins were reduced severalfold, presumably because the unassembled proteins are unstable. Null mutants of MRP20 converted to [rho-] or [rho0], a characteristic phenotype of mutations in essential genes for mitochondrial translation. Inactivation of MRP49 caused a cold-sensitive respiration-deficient phenotype, indicating that MRP49 is not an essential ribosomal protein. The mrp49 mutants were defective in the assembly of stable 54 S ribosomal subunits at the nonpermissive temperature. With the results presented here, there are now published sequences for 14 yeast mitochondrial ribosomal proteins, only five of which bear discernable relationships to eubacterial ribosomal proteins.
Highlights
From the Department of Biochemistry and Program in Molecular and Cellular Biology,University of Massachusetts, Amherst, Massachusetts 01003
Identification of MRP20 and MRP49"Monoclonal antibodies specific for the MRP20 (33 kDa) andMRP49 (16 kDa) proteins of the mitochondrial 54 S ribosomal subunit were used to identify immunoreactive recombinants in a yeast genomic library constructed in the expression vector Xgtll (Young and Davis, 1983)
A 124-kDa protein that cross-reacted with both the MRP49 mAb and a mAb to P-galactosidase was detected in immunoblots of total protein extractedfrom XMRP49-infectedE. coli cells, indicating that a P-galactosidase"RP49 fusion protein was expressed from an in-frame fusion between lac[2] and the coding region of MRP49
Summary
The specificity of the monoclonal antibodies was determined by immunoblotting as described previously (Partaledis and Mason, 1988).The MRP20 and MRP49 proteins were named on the basis of their electrophoretic mobilities in SDS-polyacrylamide gels as the 20th and 49th largest of the total proteins of the mitochondrial ribosome. Identification of MRP20 and MRP49"Monoclonal antibodies specific for the MRP20 (33 kDa) andMRP49 (16 kDa) proteins of the mitochondrial 54 S ribosomal subunit were used to identify immunoreactive recombinants in a yeast genomic library constructed in the expression vector Xgtll (Young and Davis, 1983).
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