Abstract
We report the functional characterization of RPN6, an essential gene from Saccharomyces cerevisiae encoding the proteasomal subunit Rpn6p. For this purpose, conditional mutants that are able to grow on galactose but not on glucose were obtained. When these mutants are shifted to glucose, Rpn6p depletion induces several specific phenotypes. First, multiubiquitinated proteins accumulate, indicating a defect in proteasome-mediated proteolysis. Second, mutant yeasts are arrested as large budded cells with a single nucleus and a 2C DNA content; in addition, the spindle pole body is duplicated, indicating a general cell cycle defect related to the turnover of G(2)-cyclins after DNA synthesis. Clb2p and Pds1p, but not Sic1p, accumulate in the arrested cells. Depletion of Rpn6p affects both the structure and the peptidase activity of proteasomes in the cell. These results implicate Rpn6p function in the specific recognition of a subset of substrates and point to a role in maintaining the correct quaternary structure of the 26 S proteasome.
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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CDC28 Protein Kinase, S cerevisiae / metabolism
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Cell Cycle Proteins / metabolism
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Cyclin B / metabolism
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Cysteine Endopeptidases / chemistry
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Cysteine Endopeptidases / metabolism
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DNA, Complementary / metabolism
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Endopeptidases / chemistry*
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Endopeptidases / metabolism
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Escherichia coli / metabolism
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Flow Cytometry
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Fluorescent Antibody Technique, Indirect
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G2 Phase
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Galactose / pharmacology
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Gene Library
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Genotype
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Glucose / pharmacology
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Humans
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Microscopy, Electron, Scanning
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Mitosis
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Multienzyme Complexes / chemistry
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Multienzyme Complexes / metabolism
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Mutation
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Nuclear Proteins / metabolism
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Peptide Hydrolases / metabolism*
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Phenotype
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Plasmids / metabolism
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Proteasome Endopeptidase Complex
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Protein Structure, Quaternary
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Ribosomal Protein S9
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Ribosomal Proteins / genetics
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Ribosomal Proteins / metabolism
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / metabolism
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Securin
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Time Factors
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Ubiquitin / metabolism
Substances
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CLB2 protein, S cerevisiae
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Cell Cycle Proteins
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Cyclin B
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DNA, Complementary
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Multienzyme Complexes
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Nuclear Proteins
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PDS1 protein, S cerevisiae
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Ribosomal Protein S9
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Ribosomal Proteins
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Saccharomyces cerevisiae Proteins
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Securin
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Ubiquitin
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CDC28 Protein Kinase, S cerevisiae
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Endopeptidases
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Peptide Hydrolases
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Rpn6 protein, S cerevisiae
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex
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ATP dependent 26S protease
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Glucose
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Galactose