Abstract
The degree of cooperation and redundancy between different chaperones is an important problem in understanding how proteins fold in the cell. Here we use the yeast Saccharomyces cerevisiae as a model system to examine in vivo the chaperone requirements for assembly of the von Hippel-Lindau protein (VHL)-elongin BC (VBC) tumor suppressor complex. VHL and elongin BC expressed in yeast assembled into a correctly folded VBC complex that resembles the complex from mammalian cells. Unassembled VHL did not fold and remained associated with the cytosolic chaperones Hsp70 and TRiC/CCT, in agreement with results from mammalian cells. Analysis of the folding reaction in yeast strains carrying conditional chaperone mutants indicates that incorporation of VHL into VBC requires both functional TRiC and Hsp70. VBC assembly was defective in cells carrying either a temperature-sensitive ssa1 gene as their sole source of cytosolic Hsp70/SSA function or a temperature-sensitive mutation in CCT4, a subunit of the TRiC/CCT complex. Analysis of the VHL-chaperone interactions in these strains revealed that the cct4ts mutation decreased binding to TRiC but did not affect the interaction with Hsp70. In contrast, loss of Hsp70 function disrupted the interaction of VHL with both Hsp70 and TRiC. We conclude that, in vivo, folding of some polypeptides requires the cooperation of Hsp70 and TRiC and that Hsp70 acts to promote substrate binding to TRiC.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphatases
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Chaperonin Containing TCP-1
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Chaperonins / genetics
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Chaperonins / metabolism*
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Cytosol / metabolism
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Elongin
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Fungal Proteins / genetics
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Fungal Proteins / metabolism
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Genes, Tumor Suppressor
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HSP70 Heat-Shock Proteins / genetics
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HSP70 Heat-Shock Proteins / metabolism*
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Humans
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Intracellular Signaling Peptides and Proteins*
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Ligases / genetics
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Ligases / metabolism*
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Macromolecular Substances
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Microtubule-Associated Proteins / genetics
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Microtubule-Associated Proteins / metabolism*
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Molecular Chaperones / metabolism
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Mutation
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Protein Folding
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins
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Temperature
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Tumor Suppressor Proteins*
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Ubiquitin-Protein Ligases*
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Von Hippel-Lindau Tumor Suppressor Protein
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t-Complex Genome Region
Substances
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CCT4 protein, human
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Elongin
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Fungal Proteins
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HSP70 Heat-Shock Proteins
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Intracellular Signaling Peptides and Proteins
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Macromolecular Substances
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Microtubule-Associated Proteins
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Molecular Chaperones
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Nuclear Proteins
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Saccharomyces cerevisiae Proteins
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Transcription Factors
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Tumor Suppressor Proteins
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PPP1R11 protein, human
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Ubiquitin-Protein Ligases
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Von Hippel-Lindau Tumor Suppressor Protein
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Adenosine Triphosphatases
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Chaperonin Containing TCP-1
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Chaperonins
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SSA1 protein, S cerevisiae
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Ligases
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VHL protein, human