Abstract
In Saccharomyces cerevisiae, the nuclear-encoded protein Cbp1 promotes stability and translation of mitochondrial cytochrome b transcripts through interaction with the 5' untranslated region. Fusion of a biotin binding peptide tag to the C terminus of Cbp1 has now allowed detection in mitochondrial extracts by using peroxidase-coupled avidin. Cbp1 is associated with the mitochondrial membranes when high ionic strength extraction conditions are used. However, the protein is easily solubilized by omitting salt from the extraction buffer, which suggests Cbp1 is loosely associated with the membrane through weak hydrophobic interactions. Gel filtration analysis and blue native PAGE showed that Cbp1 is part of a single 900,000-Da complex. The complex was purified using the biotin tag and a sequence-specific protease cleavage site. In addition to Cbp1, the complex contains several polypeptides of molecular weights between 113 and 40 kDa. Among these, we identified another message-specific factor, Pet309, which promotes the stability and translation of mitochondrial cytochrome oxidase subunit I mRNA. A hypothesis is presented in which the Cbp1-Pet309 complex contains several message-specific RNA binding proteins and links transcription to translation of the mRNAs at the membrane.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Buffers
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Chromatography, Gel
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Intracellular Membranes / metabolism
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Membrane Proteins / chemistry
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Membrane Proteins / genetics
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Membrane Proteins / isolation & purification
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Membrane Proteins / metabolism*
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Mitochondrial Proteins / chemistry
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Mitochondrial Proteins / genetics
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Mitochondrial Proteins / isolation & purification
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Mitochondrial Proteins / metabolism*
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Molecular Weight
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Multiprotein Complexes
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Peptide Initiation Factors
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Protein Binding / drug effects
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RNA / metabolism*
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RNA, Mitochondrial
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RNA-Binding Proteins / chemistry
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / isolation & purification
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RNA-Binding Proteins / metabolism*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / isolation & purification
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Saccharomyces cerevisiae Proteins / metabolism*
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Salts / chemistry
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Salts / pharmacology
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Solubility
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Sonication
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Substrate Specificity
Substances
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Buffers
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Membrane Proteins
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Mitochondrial Proteins
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Multiprotein Complexes
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PET309 protein, S cerevisiae
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Peptide Initiation Factors
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RNA, Mitochondrial
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RNA-Binding Proteins
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Saccharomyces cerevisiae Proteins
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Salts
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cbp1 protein, S cerevisiae
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RNA