Abstract
Purpose:
The chaperonin CCT is a large ATPase complex comprised of eight t-complex protein 1 (TCP-1) subunits, assembled as two stacked rings. This essential chaperonin remains constitutively active, serving as a folding chamber for nascent cytosolic proteins. Our goal was to develop an animal model to study the protein interactions of CCT in retinal photoreceptors.
Methods:
Epitope tags were designed to protrude outside of the assembled chaperonin complex, and genetically introduced either into an internal sequence of TCP-1α or at the C-terminus of TCP-1ε. Both proteins were overexpressed in the photoreceptors of transgenic mice. The retinal morphology was analyzed by light microscopy, and the protein expression was monitored by immunofluorescence and Western blotting. Protein-protein interactions were studied using pull down assays coupled to LC/MS/MS.
Results:
The photoreceptors of the transgenic mice were found to retain normal structure and function, and both epitope-tagged TCP-1α and TCP-1ε could be readily detected and affinity-purified from the retinas. When captured under non-denaturing conditions, TCP-1α or TCP-1ε each precipitated as a high-molecular weigh heteromeric complex, containing the entire set of CCT subunits.
Conclusions:
Overexpression of the epitope-tagged TCP-1α or TCP-1ε subunits had no adverse effect on photoreceptor structure and function, and both subunits appeared to be competent to assemble into a chaperonin complex. Therefore, the transgenic mice generated can serve as viable models for the study of CCT protein interactions.
Keywords: 450 chaperones •
648 photoreceptors •
740 transgenics/knock-outs