THE CHAPERONIN CONTAINING TCP-1 (CCT) DISPLAYS A SINGLE-RING MEDIATEDDISASSEMBLY AND REASSEMBLY CYCLE

The chaperonin-containing TCP-1 (CCT) assists in the folding of actins and tubulins in eukaryotic cells. CCT is composed of 8 subunit specie s encoded by separate genes. CCT purifies as a single hetero-oligomeri c protein complex of 950 kDa through multiple chromatographic and anti body affinity procedures. The CCT 16-mer contains 7 polypeptide specie s in equimolar amounts (CCt alpha, beta, gamma, delta, epsilon, zeta, eta), together with another subunit (CCT theta) which is around half-m olar Here we show, by in vitro translation of CCT subunit mRNAs in rab bit reticulocyte lysate, that none of the CCT subunit proteins are the mselves folded by CCT. However, the newly translated CCT subunits can incorporate into the endogenous CCT complex present in the lysate via a mechanism involving a nucleotide-dependent disassembly reaction to p roduce single-rings and then a reassembly reaction whereby free CCT su bunits assemble onto these single-rings. This cycling behaviour is an inherent property of the CCT chaperonin complex and provides a powerfu l method for introducing single amino acid residue changes into this 8 578 residue protein complex.