Eukaryotic chaperonin CCT: Prolonged interaction with cytoskeletal components

Abstract

T h e chaperonin containing TCP-1 (CCT) is composed from eight -60 k D a ATPase subunits encoded by separate, conserved genes. Unlike its bacterial GroEL relative, CCT forms hetero-oligomeric rings, with 8-fold symmetry. Moreover, CCT assists folding of archetypically eukaryotic polypeptides, especially actins a n d tubulins. Presently CCT is seen as a n essential chaperone for the maturation of actin monomer and tubulin dimer, acting after prefoldin (GimC) to receive nascent chains from ribosomes a n d before they achieve a native (polymerisable) state. In the case of tubulin dimer formation a n d assembly, post-CCT cofactors are required. Our research challenges ideas of CCT being limited to a role as a fixed 16-mer involved only in peri-(or immediately post-) translational folding. CCT subunits selectively interact with polymerised microtubules, influence actin assembly a n d bind F-actin in vitro. CCT subunits are also found a t sites of actin assembly in cells away from ribosomes and remain associated with actin over days during its slow axonal transport in vivo, a strictly post-translational process. The structural differences between bovine lens alpha-A and alphaLens alpha-A and alpha-B crystallin have been reported to act differently in their protection against non-thermal destabilisation of proteins. The nature of this difference, however, is not completely understood. Therefore we used a combination of thermally and solvent-induced structural changes to investigate the difference in the secondary, the tertiary and the quaternary structure of alpha-A and alpha-B crystallin. We demonstrated the relation between the changes in the tertiary and the quaternary structure for both polypeptides. Far-ultraviolet circular dichroism revealed that the secondary structure of alpha-B crystallin is more stable than alpha-A crystallin and the temperature-induced secondary structure changes of both polypeptides are partially reversible. Tryptophan fluorescence revealed two distinct transitions for both alpha-A and alpha-B crystallin. Compared to alpha-B crystallin, both transitions of alpha-A crystallin ocurred at high temperature. The changes in the hydrophobicity are accompanied by changes in the quaternary structure and are biphasic, as shown by bis-1-anilino-8-napthalenesulfonate fluorescence and sedimentation velocity. These phenomena explain the difference in the chaperone capacity of alpha-A and alpha-B crystallin carried out at different temperatures. The quaternary structure of alpha crystallin is more stable than alpha-A and alpha-B crystallin. The latter has a strong tendency to dissociate under thermal or solvent destabilisation. This phenomenon is related to the difference in subunit organisation of alpha-A and alpha-B crystallin where both hydrophobic and ionic interactions are involved. We find that an important subunit rearrangement of alpha-A crystallin takes place once the molecule is destabilised. This subunit rearrangement is a requisite phenomenon for maintaining alpha crystallin in its globular for...