Abstract
CCT is a member of the chaperonin family of molecular chaperones and consists of eight distinct subunit species which occupy fixed positions within the chaperonin rings. The activity of CCT is closely linked to the integrity of the cytoskeleton as newly synthesized actin and tubulin monomers are dependent upon CCT to reach their native conformations. Furthermore, an additional role for CCT involving interactions with assembling/assembled microfilaments and microtubules is emerging. CCT is also known to interact with other proteins, only some of which will be genuine folding substrates. Here, we identify the actin filament remodeling protein gelsolin as a CCT-binding partner, and although it does not behave as a classical folding substrate, gelsolin binds to CCT with a degree of specificity. In cultured cells, the levels of CCT monomers affect levels of gelsolin, suggesting an additional link between CCT and the actin cytoskeleton that is mediated via the actin filament severing and capping protein gelsolin.
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Acknowledgements
The authors would like to thank Drs. Anne Roobol and Martin Carden for the rabbit anti-CCTε antibody, Prof. Keith Willison for the monoclonal antibodies to CCT subunits, and Prof. Shigeomi Shimizu for the provision of gelsolin plasmids. Protein identification was carried out by the Proteomics Core Facility at the University of Gothenburg. We acknowledge grants from The Swedish Research Council, Assar Gabrielsson’s Fond, Carl Tryggers Stiftelse, and The Royal Society of Arts and Sciences in Gothenburg.
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Fig. S1
Band shift analysis of gelsolin binding to the CCT oligomer. Full-length human gelsolin (hGsn), mouse gelsolin domain 4 (mGsn domain 4), and full-length human β-actin were translated in the presence of 35S methionine at 30°C for 45 min in a rabbit reticulocyte lysate in vitro translation system. Samples (2 μl) were incubated for 30 min on ice in the presence and absence of 2.5 μg anti-CCT antibodies made up to a total volume of 10 μl with PBS. Proteins were separated by native PAGE and visualized by autoradiography. Arrow heads indicate the unshifted CCT oligomer and asterisks the antibody shifted CCT oligomer (PPT 170 kb)
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Brackley, K.I., Grantham, J. Interactions between the actin filament capping and severing protein gelsolin and the molecular chaperone CCT: evidence for nonclassical substrate interactions. Cell Stress and Chaperones 16, 173–179 (2011). https://doi.org/10.1007/s12192-010-0230-x
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DOI: https://doi.org/10.1007/s12192-010-0230-x