In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease, in which ClpQ is the peptidase subunit and ClpY is the ATPase and unfoldase. ClpY functions to recognize protein substrates, and denature and translocate the unfolded polypeptides into the proteolytic site of ClpQ 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 proteases can degrade SulA, 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 (139^th∼149^th aa) of SulA is necessary for binding and degradation by ClpYQ. A conserved region, GFIMRP, between 142^th and 147^th 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. coli ClpYQ protease complex, ClpY recognizes the C-terminal region of SulA, and F143 of SulA plays an important role for the recognition and degradation by ClpYQ protease.

在大肠杆菌中，ClpYQ（HslUV）是一种两成分的ATP依赖性蛋白酶，其中ClpQ是肽酶亚基，ClpY是ATPase和解折叠酶。ClpY的功能是识别蛋白质底物，并使未折叠的多肽变性并易位到ClpQ的蛋白水解位点进行降解。但是，尚不清楚ClpYQ蛋白酶如何识别天然底物，以及由ClpY选择，展开和转移底物进入ClpQ六聚体内部位点的机制。Lon和ClpYQ蛋白酶均可降解细菌细胞中的SulA（一种细胞分裂抑制剂）。在这项研究中，使用酵母双杂交和体内降解分析，我们首先证明了C端内部疏水区（^第139^位~149^第苏拉的AA）是必要的由ClpYQ结合和降解。在各种革兰氏阴性细菌中鉴定出保守的区域GFIMRP，位于SulA的^第142^位至^第147^位之间。通过使用MBP-SulA（F143Y）（酪氨酸取代的苯丙氨酸）作为底物，我们的结果表明，SulA的这种保守残基对于ClpYQ的识别和降解是必需的。为支持这些数据，MBP-SulA（F143Y），MBP-SulA（F143N）（被天冬酰胺取代的苯丙氨酸）在体内具有更长的ClpYQ蛋白酶半衰期。相反，MBP-SulA（F143D）和MBP-SulA（F143S）的半衰期较短。因此，在大肠杆菌中 ClpYQ蛋白酶复合物，ClpY识别SulA的C端区域，而SulA的F143在ClpYQ蛋白酶的识别和降解中起重要作用。