Identification of the CBP1 polypeptide in mitochondrial extracts from Saccharomyces cerevisiae

CBP1 is a nuclearly encoded yeast protein required for stability of mitochondrial cytochrome b pre-mRNA. Previous studies have shown that CBP1 stabilizes the cytochrome b transcripts via interaction with the 5'-end. For the present study, both rabbit polyclonal and mouse monoclonal antibodies against CBP1 were prepared using a trpE-CBP1 fusion polypeptide as a source of antigen. CBP1 was undetectable in a crude mitochondrial fraction from a wild-type strain by Western blot assay, but a 66-kDa immunoreactive protein was detected in a more purified fraction. The 66-kDa protein was absent in the equivalent fraction from a strain with a deletion in CBP1. Assignment of Mr = 66,000 to the mature CBP1 polypeptide was verified by Western analysis of mitochondria from a strain which over-expresses CBP1. Mitochondrial localization was verified by transcribing CBP1 in vitro with T3 polymerase, translating the artificial mRNA in a rabbit reticulocyte system and importing 35S-CBP1 precursor polypeptides into isolated mitochondria. The mature protein product was 66 kDa, whereas the precursor protein migrated as if it were 68 kDa rather than 76 kDa as predicted from the sequence. Analysis of polypeptides truncated at the carboxyl terminus showed that CBP1 polypeptides migrate anomalously fast in the Laemmli system due to a property of the carboxyl two-thirds of the primary sequence, several sections of which are extremely basic.