ANALYSIS OF MUTATIONALLY ALTERED FORMS OF THE CCT6 SUBUNIT OF THE CHAPERONIN FROM SACCHAROMYCES-CEREVISIAE

The Cct double-ring chaperonin complex of Saccharomyces cerevisiae is comprised of eight essential subunits, Cct1p-Cct8p, and assists the fo lding of substrates such as actins and tubulins. Single and multiple a mino acid replacements of Cct6p were constructed by oligonucleotide-di rected mutagenesis, including changes of charged to alanine residues a nd uncharged to charged residues. The replacements were targeted, in p art, to residues corresponding to functionally critical regions identi fied in the published crystal structure of the Escherichia coli chaper onin, GroEL. Here, we report the critical hydrophobic residues and clu sters of hydrophilic residues in regions corresponding to those from t he apical domain of GroEL implicated in peptide binding and peptide re lease, and certain residues in the putative equatorial domain implicat ed in subunit-to-subunit interaction. In contrast to their homologous counterparts in Cct2p and Cct1p, the highly conserved putative ATP bin ding motifs of Cct6p were relatively amenable to mutations. Our data s uggest that the entire Cct6p molecule might be essential for assembly of Cct complex and might participate in binding substrates. However, t here appeared to exist a functional hierarchy in ATP binding/hydrolysi s among Cct subunits, as suggested by the high tolerance of Cct6p to m utations within the putative ATP binding pocket.