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Microbiol Res ; 220: 21-31, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30744816

RESUMO

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 (139th∼149th aa) of SulA 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. 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.


Assuntos
Proteases Dependentes de ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de Proteína , Deleção de Sequência , Técnicas do Sistema de Duplo-Híbrido
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