Using a collection of nonfunctional missense mutants to examine how molecular chaperones and proteases maintain protein homeostasis

Abstract

Protein homeostasis is crucial for optimal cellular life and affects thousands of proteins that have to reach their final folded active state to be functional. Protein folding is not trivial in the crowded cellular environment and many proteins cannot fold correctly. These incorrectly folded proteins either have to be rescued by chaperones or unfoldases, or have to be destroyed by proteases. This triage by chaperones and proteases is key to the cellular maintenance of proteins. We have studied the interaction between chaperones and proteases using a collection of 42 well-characterized non-functional missense mutants in the bacterial cytosolic enzymes, β-galactosidase and catechol 2,3-dioxygenase. In an initial analysis, we showed that the collection of missense mutants represented proteins that could not fold correctly and that the great majority of them were rescuable by high salt concentrations that are known to stabilize proteins. The two major chaperone complexes, Hsp60 (GroL/GroS) and Hsp70 /Hsp40 (DnaK/DnaJ), were either overproduced in the missense mutants or the two major proteases, Lon and Clp, were deleted. The deletion of the proteases was far more effective at stabilizing the missense mutants than overproducing the chaperones. A total of 16.67% of the missense mutants were rescued by the overproduction of GroL/S, 47.62% of the missense mutants were rescued by the overproduction of DnaK/DnaJ, 78.57% of the missense mutants were rescued by the deletion of Lon and 83.33% of the missense mutants were rescued by the deletion of Clp. Additional analysis of our results supported a number of models that have been proposed concerning protein homeostasis and how chaperone and proteases interact with misfolded proteins.