Reduced ADP off-rate by the yeast CCT2 double mutation T394P/R510H which causes Leber congenital amaurosis in humans.

The CCT/TRiC chaperonin is found in the cytosol of all eukaryotic cells and assists protein folding in an ATP-dependent manner. The heterozygous double mutation T400P and R516H in subunit CCT2 is known to cause Leber congenital amaurosis (LCA), a hereditary congenital retinopathy. This double mutation also renders the function of subunit CCT2, when it is outside of the CCT/TRiC complex, to be defective in promoting autophagy. Here, we show using steady-state and transient kinetic analysis that the corresponding double mutation in subunit CCT2 from Saccharomyces cerevisiae reduces the off-rate of ADP during ATP hydrolysis by CCT/TRiC. We also report that the ATPase activity of CCT/TRiC is stimulated by a non-folded substrate. Our results suggest that the closed state of CCT/TRiC is stabilized by the double mutation owing to the slower off-rate of ADP, thereby impeding the exit of CCT2 from the complex that is required for its function in autophagy.