Partial purification and biochemical characterization of CBP1 from Saccharomyces cerevisiae

Abstract

The product of the yeast nuclear gene CBP1 is a mitochondrial protein which is necessary for the stability of the mitochondrial-encoded cytochrome b (cob) messenger RNA. In this dissertation I describe the partial purification, and the biochemical characterization of the CBP1 protein. Using a monoclonal antibody directed against CBP1, I have shown that CBP1 is a mitochondrial protein. I found that CBP1 was undetectable in a crude mitochondrial fraction isolated from a wild-type strain. However, a 66 kDa immunoreactive peptide was detected in a purified mitochondrial fraction, and was not present in a control strain. That CBP1 is a mitochondrial protein and contains an amino terminal leader peptide which is removed during import was confirmed by in vitro import of ³⁵S-labeled CBP1 into isolated mitochondria. I found that the mature protein product was 66 kDa, whereas the precursor protein migrated with the mobility of a 68 kDa polypeptide. This was in contrast to the published DNA sequence from which one can derive a molecular weight of 76 kDa for CBP1. It was determined from electrophoretic analysis of a nested group of carboxyl-terminal truncated peptides which were translated in vitro, that this aberrant electrophoretic mobility is due to a property of the carboxyl terminal region of the primary sequence which contains numerous basic residues. Having identified the CBP1 gene product in wild-type yeast, I wanted to determine the role of CBP1 in stabilizing the cob transcript. Using wild-type mitochondrial extracts containing CBP1, and extracts from a bacterial strain overexpressing CBP1, I tested different substrates derived from the 5'-untranslated leader of cob in three different types of assays; gel retardation, RNA-protein UV-crosslinking, and in scission assays in which I examined the possibility that CBP1 is responsible for generating the mature 5'-end of the cob mRNA. I was unable to show a direct interaction between CBP1 and the 5'-untranslated leader of cob mRNA in vitro. However, I was able to identify an exonuclease activity in a purified mitochondrial fraction from a wild-type yeast strain, which was not present in a control strain which harbored a deletion in the CBP1 gene.