Abstract
The mitochondrial degradosome (mtEXO), the main RNA-degrading complex of yeast mitochondria, is composed of two subunits: an exoribonuclease encoded by the DSS1 gene and an RNA helicase encoded by the SUV3 gene. We expressed both subunits of the yeast mitochondrial degradosome in Escherichia coli, reconstituted the complex in vitro and analyzed the RNase, ATPase and helicase activities of the two subunits separately and in complex. The results reveal a very strong functional interdependence. For every enzymatic activity, we observed significant changes when the relevant protein was present in the complex, compared to the activity measured for the protein alone. The ATPase activity of Suv3p is stimulated by RNA and its background activity in the absence of RNA is reduced greatly when the protein is in the complex with Dss1p. The Suv3 protein alone does not display RNA-unwinding activity and the 3' to 5' directional helicase activity requiring a free 3' single-stranded substrate becomes apparent only when Suv3p is in complex with Dss1p. The Dss1 protein alone does have some basal exoribonuclease activity, which is not ATP-dependent, but in the presence of Suv3p the activity of the entire complex is enhanced greatly and is entirely ATP-dependent, with no residual activity observed in the absence of ATP. Such absolute ATP-dependence is unique among known exoribonuclease complexes. On the basis of these results, we propose a model in which the Suv3p RNA helicase acts as a molecular motor feeding the substrate to the catalytic centre of the RNase subunit.
Publication types
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Research Support, N.I.H., Intramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphate / metabolism
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Catalytic Domain
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DEAD-box RNA Helicases / metabolism
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Endoribonucleases / genetics*
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Endoribonucleases / isolation & purification
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Endoribonucleases / metabolism
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Endoribonucleases / physiology*
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Escherichia coli
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Exoribonucleases / metabolism
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Genes, Fungal / physiology
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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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Mitochondrial Proteins / physiology*
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Multienzyme Complexes / genetics*
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Multienzyme Complexes / isolation & purification
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Multienzyme Complexes / metabolism
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Multienzyme Complexes / physiology*
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Polyribonucleotide Nucleotidyltransferase / genetics*
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Polyribonucleotide Nucleotidyltransferase / isolation & purification
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Polyribonucleotide Nucleotidyltransferase / metabolism
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Polyribonucleotide Nucleotidyltransferase / physiology*
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Protein Subunits / metabolism
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RNA Helicases / genetics*
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RNA Helicases / isolation & purification
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RNA Helicases / metabolism
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RNA Helicases / physiology*
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RNA-Binding Proteins / metabolism
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae Proteins / metabolism
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Transformation, Bacterial
Substances
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Mitochondrial Proteins
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Multienzyme Complexes
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Protein Subunits
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RNA-Binding Proteins
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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degradosome
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Adenosine Triphosphate
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Polyribonucleotide Nucleotidyltransferase
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Endoribonucleases
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Exoribonucleases
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SUV3 protein, S cerevisiae
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DEAD-box RNA Helicases
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RNA Helicases