The unique hetero-oligomeric nature of the subunits in the catalytic cooperativity of the yeast Cct chaperonin complex

Abstract

The structural and functional organization of the Cct complex was addressed by genetic analyses of subunit interactions and catalytic cooperativity among five of the eight different essential subunits, Cct1p-Cct8p, in the yeast Saccharomyces cerevisiae. The cct1-1, cct2-3, and cct3-1 alleles, containing mutations at the conserved putative ATP-binding motif, GDGTT, are cold-sensitive, whereas single and multiple replacements of the corresponding motif in Cct6p are well tolerated by the cell. We demonstrated herein that cct6-3 (L19S), but not the parolog cct1-5 (R26I), specifically suppresses the cct1-1, cct2-3, and cct3-1 alleles, and that this suppression can be modulated by mutations in a putative phosphorylation motif, RXS, and the putative ATP-binding pocket of Cct6p. Our results suggest that the Cct ring is comprised of a single hetero-oligomer containing eight subunits of differential functional hierarchy, in which catalytic cooperativity of ATP-binding/hydrolysis takes place in a sequential manner different from the concerted cooperativity proposed for GroEL.

Figure 1

(a) Model depicting a highly sequential order of positive cooperative ATP binding within the stoichiometrical array of Cct subunits in the ring in the R state (denoted by an asterisk), and negative cooperative ATP binding in the opposite ring in the T state, based on the KNF model (see text). (b) Proposed sequence of cooperative ATP binding/hydrolysis among Cct1p, Cct2p, Cct3p, and Cct6p based on the KNF model. In the Cct complex containing normal Cct6⁺p, cooperative ATP binding occurs in the order of Cct1p, Cct3p, Cct2p, and Cct6p. Cct6p is placed the downstream-most position due to its high tolerance to mutations at the putative ATP-binding loop, GDGTT. In contrast, in Cct complex containing Cct6–3p, cooperative ATP binding can occur in the order of Cct6–3p, Cct1p, Cct3p, and Cct2p, due to an increased T to R state transition in Cct6–3p. This results from a change of the relative positions of Cct6–3p with respect to Cct1–1p, Cct2–3p, and Cct3–1p, thereby displacing these mutants further downstream in the course of sequential cooperative ATP binding/hydrolysis. As a result, mutations at the GDGTT motifs in these mutant subunits become more tolerable to the cell. Double arrows between subunits indicate unknown numbers of intervening subunits. Single arrow indicates two adjacent subunits, as is proposed between Cct2p and Cct6p.

Figure 2

The conserved putative ATP-binding/hydrolysis motifs from various chaperonins. The L19S (cct6–3) mutation of Cct6p suppresses, but does not bypass, the conditional lethal alleles, cct1–1, cct2–3, and cct3–1, all of which have mutations within the putative ATP-binding site, GDGTTT/S; however, L19S does not suppress cct1–2 and cct1–3, which have replacements in other conserved ATP-binding/hydrolysis motifs. Double mutations involving any two motifs in Cct6p result in synthetic lethality, whereas leucine-19 interacts with two but not others of these motifs, suggesting that these four motifs possess both common and distinct functions, to account for the specificity of suppression by L19S. The changes of cct2–1, cct2–2, cct2–3, cct2–4, and cct3–1 mutations were determined by direct DNA sequencing of the PCR products of the genomic DNA using the methods of Sanger et al. (17).

Figure 3

An alignment of amino acid sequences of the GroEL, TF55, and S. cerevisiae Cct1p–Cct8p subunits. Amino acid positions are indicated on the top row for Cct6p and GroEL (shown in parentheses). The leucine-19 residue of Cct6p and its parologous residues from other chaperonins, including arginine-26 of Cct1p and arginine-13 of GroEL, are denoted by a box. The putative cAMP kinase phosphorylation motif, RXS, for Cct6p is shown in reverse type.