Purchase this article with an account.
Maxim Sokolov, Marycharmain Belcastro, Xueli Gao, Vadim Y Arshavsky, Nikolai P Skiba, Satyabrata Sinha; Studies of Protein Interaction of the Chaperonin CCT in Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6012.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
This PDF is available to Subscribers Only