Cofactors and co-chaperones of the chaperonin CCT: mechanistic insights and in vivo relevance

Protein folding is the essential process by which a linear chain of amino acids folds upon itself to adopt a defined three-dimensional structure. All proteins must undergo folding to be functional and while the amino acid sequence dictates the tertiary structure, in the crowded cellular environment the folding process is not always spontaneous. To circumvent this problem proteins called molecular chaperones have evolved to stabilize non-folded polypeptides and facilitate their transformation to the folded state. This thesis revolves around a molecular chaperone called CCT, which uses a barrel-shaped structure to bind non-native proteins and sequester them in a protected environment to allow folding. My work has focused on two proteins that co-operate with CCT in the cell to promote efficient folding of its substrates. The first, called prefoldin (PFD), is another molecular chaperone that binds non-native polypeptides and delivers them to CCT for completion of folding. The second is a family of proteins called phosducin-like proteins (PhLPs), which bind CCT and affect its ability to fold substrates. Here we show that PFD uses long coiled-coil tentacles to grasp substrates using interhelical-hydrophobic residues at the very tips. We find that the general properties of a coiled-coil are sufficient to confer some chaperone activity indicating the importance of this super-secondary structure to PFD function. We also find that archaeal PFD can alter its shape to accommodate substrates of different sizes, but that in most cases a large proportion of the substrate protrudes from the PFD cavity. We also show that the mechanism of PhLP-mediated CCT regulation involves PhLP binding to CCT-substrate complexes and slowing of ATP hydrolysis. In yeast the PhLP homologues Plp1p and Plp2p both affect cytoskeletal function but Plp2p, which is essential, also appears to affect the cell cycle. Finally, we use a genomic approach to suggest novel cellular roles for the chaperonin CCT in pathways such as septin ring assembly. Altogether these studies illuminate the role of CCT co-chaperones (PFD) and cofactors (PhLP) in modulating the chaperonin’s function and open up new research prospects by identifying novel genetic interactors of CCT.