Specific regions of the SulA protein recognized and degraded by the ATP-dependent ClpYQ (HslUV) protease in Escherichia coli

In Escherichia coli, ClpYQ (HsIUV) is a two-component ATP-dependent protease, in which ClpYQ is the peptidase subunit and ClpY is the ATPase and unfoldase. CIpY functions to recognize protein substrates, and denature and translocate the unfolded polypeptides into the proteolytic site of ClpYQ for degradation. However, it is not clear how the natural substrates are recognized by the ClpYQ protease and the mechanism by which the substrates are selected, unfolded and translocated by ClpY into the interior site of ClpQ hexamers. Both Lon and ClpYQ pro teases can degrade SuIA, a cell division inhibitor, in bacterial cells. In this study, using yeast two-hybrid and in vivo degradation analyses, we first demonstrated that the C-terminal internal hydrophobic region (139th similar to 149th aa) of SuIA is necessary for binding and degradation by ClpYQ. A conserved region, GFIMRP, between 142th and 147th residues of SulA, were identified among various Gram-negative bacteria. By using MBP-SulA(F143Y) (phenylalanine substituted with tyrosine) as a substrate, our results showed that this conserved residue of SulA is necessary for recognition and degradation by ClpYQ. Supporting these data, MBP-SulA(F143Y), MBP-SulA(F143N) (phenylalanine substituted with asparagine) led to a longer half-life with ClpYQ protease in vivo. In contrast, MBP-SulA(F143D) and MBP-SulA(F143S) both have shorter half-lives. Therefore, in the E. coil ClpYQ protease complex, ClpY recognizes the C-terminal region of SuIA, and F143 of SuIA plays an important role for the recognition and degradation by ClpYQ protease.