Chaperonin-containing TCP-1 Complex Research Articles

The chaperonin containing TCP-1 (CCT) displays a single-ring mediated disassembly 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 species encoded by separate genes. CCT purifies as a single hetero-oligomeric protein complex of 950 kDa through multiple chromatographic and antibody affinity procedures. The CCT 16-mer contains 7 polypeptide species in equimolar amounts (CCTalpha, beta, gamma, delta, epsilon, zeta, eta), together with another subunit (CCTtheta) which is around half-molar. Here we show, by in vitro translation of CCT subunit mRNAs in rabbit reticulocyte lysate, that none of the CCT subunit proteins are themselves 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 produce single-rings and then a reassembly reaction whereby free CCT subunits assemble onto these single-rings. This cycling behaviour is an inherent property of the CCT chaperonin complex and provides a powerful method for introducing single amino acid residue changes into this 8578 residue protein complex.

The third member of the Tetrahymena CCT subunit gene family, TpCCT alpha, encodes a component of the hetero-oligomeric chaperonin complex.

The sequence of a third member of the Tetrahymena pyriformis chaperonin CCT ('chaperonin containing TCP1') subunit gene family is presented. This gene, designated TpCCT alpha, is the orthologue of the mouse chaperonin gene TCP1/CCT alpha. To characterize the CCT complex in this ciliate, we have produced polyclonal antibodies against synthetic peptides based on C-terminal sequences deduced from the primary sequences of the TpCCT alpha, TpCCT gamma and TpCCT eta subunits. We have also used polyclonal antibodies produced against recombinant yeast CCT alpha and CCT beta subunits. Using these antibodies, we show that Tetrahymena cells contain a hetero-oligomeric CCT chaperonin comprising at least seven distinct subunits. Three of these were assigned to specific TpCCT genes, whereas a fourth was recognized by the polyclonal antibody against yeast CCT beta, suggesting that this gene is also present in the ciliate. The CCT complex also contains other unidentified proteins that were recognized by the polyclonal antibody UM-1, raised against the putative ATP binding domain of the chaperonin proteins. TpCCT alpha gene expression was shown in exponentially growing cells and cells regenerating their cilia for different periods to have a similar pattern to the previously identified genes TpCCT gamma and TpCCT eta, and also to tubulin genes.

MAPPING OF G2/M-PHASE PREVALENCES OF CHAPERON-ENCODING TRANSCRIPTS BY MEANS OF A SENSITIVE DIFFERENTIAL HYBRIDIZATION APPROACH

The sensitivity of the differential hybridization approach is significantly increased by the application of size-selected probes. RNA from elutriated phase-synchronous Ehrlich ascites tumor (EAT) cells has previously been used to prepare cell cycle phase-specific cDNA libraries in the in-vitro transcription vector pBluescript. PCR amplification of the libraries with vector-fitting primer pairs generates amplified cDNA reflecting the mRNA complexities of cells in G1, S and G2/M phases. Probes with reduced complexities were recovered after side-by-side electrophoresis of equal amounts of PCR-amplified cDNA and elution of probes from parallel gel sections. Such size-selected probes release significant differential clones which escape their detection in the conventional differential hybridization approach. Three clones hybridizing preferentially with the G2/M phase-specific probe were further characterized. The genes were identified by their nucleotide sequences. They encode proteins known to be involved in protein folding: heatshock cognate protein,HSC70; heatshock cognate protein,HSC73; η subunit of the chaperonin containing TCP-1 complex, CCT. The G2/M phase-prevalent expression of these genes were further verified on the mRNA and on the protein level by Northern and Western blot analysis which confirms the significance of the differential hybridization approach and which indicates that the expression of this group of proteins increases with cell cycle progression. The expression of the chaperonin-containing TCP-1 complex appears to be specifically linked with the S to G2/M phase transition of the cell cycle.

Chromosomal Assignment and Imprinting Tests for the Mouse Delta Subunit of the Cytosolic Chaperonin Containing TCP-1 (Cct4) Gene to Proximal Chromosome 11

The CCT (chaperonin containing TCP-1) complex functions as a molecular chaperone in the eukaryotic cytosol. This complex consists of several species of related polypeptides. The chromosomal localization of the mouseCct4gene encoding the delta subunit of CCT was assigned in this study to proximal chromosome 11 by genetic mapping. Restriction mapping analysis using YAC and pulse-field gel electrophoresis showed thatCct4is located within a region about 300 kb from the imprinted geneU2af1-rs1. Expression ofCct4was biallelic, and thereforeCct4is not imprinted in neonatal mice. The localization of the human homologue ofCct4on chromosome 2 corresponds well with the fact that homologues of other genes in the proximal region of mouse chromosome 11 also map to the region of conserved synteny in human chromosome 2.