Partial purification and biochemical characterization of CBP1 from Saccharomyces cerevisiae
AuthorWeber, Eric Renato
AdvisorDieckmann, Carol L.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractThe 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.
Degree ProgramMolecular and Cellular Biology