FIGURE 8.

PDCD5 interacts directly with CCTβ. A, cross-linking of PDCD5 to CCT. HEK-293T cells were transfected with the indicated FLAG-PDCD5 constructs, BzF tRNA, and BzF amino acid synthetase and treated with 1 mm BzF 3 h post-transfection. Lysates were exposed ± UV light prior to immunoprecipitation and blotting for FLAG-PDCD5. The asterisk indicates a PDCD5 cross-linked band. HC, heavy chain. B, cross-linking of PDCD5 to the CCT subunits was measured by co-immunoprecipitation from HEK-293T cells treated as in A and exposed to UV light prior to immunoprecipitation with a CCTϵ antibody and blotting for each of the CCT subunits. The asterisk indicates a CCTβ cross-linked band. The arrows mark the position of the CCT subunits. NS, nonspecific band. C, PDCD5 cross-linking to CCTβ depends on an interaction with the PDCD5 C terminus. HEK-293T cells were treated as in A and lysates were exposed ± UV light prior to immunoprecipitation with a CCTϵ antibody and blotting for CCTβ. The asterisk indicates a CCTβ cross-linked band. D, BzF catalyzes PDCD5 cross-linking to CCT without incorporation into Amber codon sites. HEK-293T cells were transfected with FLAG-PDCD5 (WT) in the absence of BzF tRNA and BzF AA synthetase. Cells were treated ± 1 mm BzF 3 h post-transfection. Lysates were exposed ± UV light prior to immunoprecipitation with CCTϵ and immunoblotting for CCTβ. The asterisk indicates a CCTβ cross-linked band.