A mitochondrial protein from Neurospora crassa detected both on ribosomes and in membrane fractions: Analysis of the gene, the message, and the protein

Abstract

Abstract We have isolated clones representing at least three nuclear genes for mitochondrial ribosomal proteins from Neurospora crassa by screening a lambda gt11 cDNA library with an antiserum against a mixture of these proteins. The cDNA and genomic DNA sequence for one of these genes, mrp-3, was determined. The MRP3 protein was purified by immune-affinity chromatography, using a monoclonal antibody probe, and subjected to amino acid sequence analysis to identify the mature amino terminus and a prospective mitochondrial-targeting presequence. MRP3 was identified as the largest, least basic protein detected from the small subunit of ribosomes which had been salt-washed and fractionated on sucrose gradients. However, the mRNA and protein products of mrp-3 were found to be present in excess over those of other N. crassa mitoribosomal protein genes. Using a solution hybridization/S1 nuclease assay, we found three-fold- more mRNA for mrp-3 than for another mito-ribosomal protein gene. In addition, a 30- to 50-fold excess of non-ribosomal MRP3 protein was discovered. The additional protein was localized in mitochondrial membrane fractions; none was detected in matrix fractions after removal of the ribosomes. An immunologically related protein was detected in ribosome and membrane fractions of mitochondria from Saccharomyces cerevisiae. The functional significance of this dual localization remains an enigma.