title: Analysis of mutationally altered forms of the Cct6 subunit of the chaperonin from Saccharomyces cerevisiae.
abstract: The Cct double-ring chaperonin complex of Saccharomyces cerevisiae is comprised of eight essential subunits, Cct1p-Cct8p, and assists the folding of substrates such as actins and tubulins. Single and multiple amino acid replacements of Cct6p were constructed by oligonucleotide-directed mutagenesis, including changes of charged to alanine residues and uncharged to charged residues. The replacements were targeted, in part, to residues corresponding to functionally critical regions identified in the published crystal structure of the Escherichia coli chaperonin, GroEL. Here, we report the critical hydrophobic residues and clusters of hydrophilic residues in regions corresponding to those from the apical domain of GroEL implicated in peptide binding and peptide release, and certain residues in the putative equatorial domain implicated in subunit-to-subunit interaction. In contrast to their homologous counterparts in Cct2p and Cct1p, the highly conserved putative ATP binding motifs of Cct6p were relatively amenable to mutations. Our data suggest that the entire Cct6p molecule might be essential for assembly of Cct complex and might participate in binding substrates. However, there appeared to exist a functional hierarchy in ATP binding/hydrolysis among Cct subunits, as suggested by the high tolerance of Cct6p to mutations within the putative ATP binding pocket.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical School, New York 14642, USA.
authors.forename: P
authors.initials: P
authors.lastname: Lin
authors.forename: T S
authors.initials: TS
authors.lastname: Cardillo
authors.forename: L M
authors.initials: LM
authors.lastname: Richard
authors.forename: G B
authors.initials: GB
authors.lastname: Segel
authors.forename: F
authors.initials: F
authors.lastname: Sherman
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism; D018833:Chaperonins / Q000235:genetics*; D002874:Chromosome Mapping; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013827:Thiabendazole / Q000494:pharmacology
chemical_list: D018834:Chaperonin 60; D004271:DNA, Fungal; D005656:Fungal Proteins; D010455:Peptides; D000242:Cyclic AMP; D018833:Chaperonins; D013827:Thiabendazole