Nucleotide triphosphates inhibit the degradation of unfolded proteins by HsIV peptidase

Abstract

Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the Hs1U ATPase and Hs1V peptidase. In the reconstituted enzyme, Hs1U stimulates the proteolytic activity of Hs1V by one to two orders of magnitude, while Hs1V increases the rate of ATP hydrolysis by Hs1U several-fold. Here we show that Hs1V alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms. Furthermore, Hs1V alone cleaved a lactalbumin fragment sandwiched by two thioredoxin molecules, indicating that it can hydrolyze the internal peptide bonds of lactalbumin. Surprisingly, ATP inhibited the degradation of unfolded proteins by Hs1V. This inhibitory effect of ATP was markedly diminished by substitution of the Arg86 residue located in the apical pore of Hs1V with Gly, suggesting that interaction of ATP with the Arg residue blocks access of unfolded proteins to the proteolytic chamber of Hs1V. These results suggest that uncomplexed Hs1V is inactive under normal conditions, but may can degrade unfolded proteins when the ATP level is low, as it is during carbon starvation.