Objective
The NAM1 gene in Saccharomyces cerevisiae was isolated as a multicopy suppressor of mitochondrial mutations affecting the splicing of group I or group II mitochondrial introns. Northern blot analysis with 9 different mitochondrial probes, specific for 7 separate transcription units, suggested that the NAM1 protein is not a general mitochondrial transcription factor but that it is selectively and predominantly required for the processing and/or for the stability of tha atp6 transcripts and of 2 intron-containing pre-messenger ribonucleic acids (mRNA).
A fusion plasmid allowing the expression of the NAM1 protein (NAM1p) in Escherichia coli was constructed, after which, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) purification of the resultant protein was used to obtain antibodies which recognised primarily a single protein under Western blot conditions. Submitochondrial localization experiments have shown that NAM1p is a soluble protein of the mitochondrial matrix. It is proposed that NAM1p could be an RNA-convoying protein stabilizing and directing mitochondrial transcripts towards the inner face of the inner membrane where translation and assembly seem to occur.
In order to find other gene products interacting with NAM1, a search was made for genes which, expressed on multicopy plasmids, compensated for the respiratory deficiency of nam1-inactivated strains. A 3.8 kb yeast genomic fragment LM9, carrying such a suppressor gene was isolated from a wild type genomic bank after screening for functional complementation of strains deleted-disrupted in NAM1. Sequencing and subcloning the fragment have shown that an us open reading frame (ORF), encoding a putative protein of 225 amino acids, bears the suppressor activity. The function of that protein is under investigation.
Funding Scheme
CSC - Cost-sharing contractsCoordinator
91198 Gif Sur Yvette
France
