THE 3RD MEMBER OF THE TETRAHYMENA CCT SUBUNIT GENE FAMILY, TPCCT-ALPHA, ENCODES A COMPONENT OF THE HETEROOLIGOMERIC CHAPERONIN COMPLEX

The sequence of a third member of the Tetrahymena pyriformis chaperoni n CCT ('chaperonin containing TCP1') subunit gene family is presented. This gene, designated TpCCT alpha, is the orthologue of the mouse cha peronin gene TCP1/CCT alpha. To characterize the CCT complex in this c iliate, we have produced polyclonal antibodies against synthetic pepti des based on C-terminal sequences deduced from the primary sequences o f the TpCCT alpha, TpCCT gamma and TpCCT eta subunits. We have also us ed polyclonal antibodies produced against recombinant yeast CCT alpha and CCT beta subunits. Using these antibodies, we show that Tetrahymen a cells contain a hetero-oligomeric CCT chaperonin comprising at least seven distinct subunits. Three of these were assigned to specific TpC CT genes, whereas a fourth was recognized by the polyclonal antibody a gainst yeast CCT beta, suggesting that this gene is also present in th e ciliate. The CCT complex also contains other unidentified proteins t hat were recognized by the polyclonal antibody UM-1, raised against th e putative ATP binding domain of the chaperonin proteins. TpCCT alpha gene expression was shown in exponentially growing cells and cells reg enerating 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.