Over-expression analysis of all eight subunits of the molecular chaperone CCT in mammalian cells reveals a novel function for CCTdelta.

Spiess M, Echbarthi M, Svanström A, Karlsson R, Grantham J.
J Mol Biol. 2015 Jun 20. pii: S0022-2836(15)00347-2. doi: 10.1016/j.jmb.2015.06.007. [Epub ahead of print]

Abstract

Chaperonin containing TCP-1 (CCT) forms a classical chaperonin barrel structure where two rings of subunits surround a central cavity. Each ring consists of eight distinct subunits, creating a complex binding interface that makes CCT unique amongst the chaperonins. In addition to acting as a multimeric chaperonin, there is increasing evidence indicating that the CCT subunits, when monomeric, possess additional functions. Here we assess the role of the CCT subunits individually, using a GFP tagging approach to express each of the subunits in their monomeric form in cultured mammalian cells. Over-expression of CCTdelta, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface. Two point mutations, one in the apical domain and one in the ATP binding pocket of CCTdelta have been identified that abolish protrusion formation, consistent with the apical domain containing a novel interaction site that is influenced by the ATPase activity in the equatorial domain. Structured Illumination Microscopy, together with subcellular fractionation, reveals that only the wild type CCTdelta is associated with the plasma membrane, thus connecting spatial organization with surface protrusion formation. Expression of the equivalent subunit in yeast, GFP-Cct4, rescues growth of the temperature sensitive strain cct4-1 at the non-permissive temperature, indicative of conserved subunit-specific activities for CCTdelta.

A novel function of the monomeric CCTepsilon subunit connects the Serum Response Factor pathway to chaperone-mediated actin folding.

Elliott KL, Svanström A, Spiess M, Karlsson R, Grantham J.
Mol Biol Cell. 2015 Jun 10. pii: mbc.E15-01-0048. [Epub ahead of print]

Abstract

Correct protein folding is fundamental for maintaining protein homeostasis and avoiding the formation of potentially cytotoxic protein aggregates. Whilst some proteins appear to fold unaided, actin requires assistance from the oligomeric molecular chaperone CCT. Here we report an additional connection between CCT and actin by identifying one of the CCT subunits, CCTepsilon, as a component of the myocardin-related cotranscription factor-A (MRTF-A)/Serum Response Factor (SRF) pathway. The SRF pathway registers changes in G-actin levels, leading to the transcriptional up-regulation of a large number of genes following actin polymerization. These genes encode numerous actin-binding proteins as well as actin. We show that depletion of the CCTepsilon subunit by siRNA enhances SRF signaling in cultured mammalian cells, by an actin assembly-independent mechanism. Over-expression of CCTepsilon in its monomeric form revealed that CCTepsilon binds via its substrate binding domain to the C-terminal region of MRTF-A and that CCTepsilon is able to alter the nuclear accumulation of MRTF-A following stimulation by serum addition. As the levels of monomeric CCTepsilon will conversely reflect the levels of CCT oligomer, our results suggest that CCTepsilon provides a connection between the actin folding capacity of the cell and actin expression.