title: Subunit oligomerization and substrate recognition of the Escherichia coli ClpYQ (HslUV) protease implicated by in vivo protein-protein interactions in the yeast two-hybrid system.
abstract: The Escherichia coli ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal beta-subunits of eukaryotes. Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex. The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor. Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates. Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA. No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA. However, ClpY, lacking domain I (ClpY(Delta I)) was able to interact with itself and with intact ClpY. The C-terminal region of ClpY is important for interaction with other ClpY subunits. The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding. Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY. Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.
authors.affiliation: Department of Agricultural Chemistry, National Taiwan University, Taipei 106, Taiwan.
authors.forename: Yi-Ying
authors.initials: YY
authors.lastname: Lee
authors.forename: Chiung-Fang
authors.initials: CF
authors.lastname: Chang
authors.forename: Chueh-Ling
authors.initials: CL
authors.lastname: Kuo
authors.forename: Meng-Ching
authors.initials: MC
authors.lastname: Chen
authors.forename: Chien Hung
authors.initials: CH
authors.lastname: Yu
authors.forename: Pei-I
authors.initials: PI
authors.lastname: Lin
authors.forename: Whi Fin
authors.initials: WF
authors.lastname: Wu
mesh_terms: D049069:ATP-Dependent Proteases; D001426:Bacterial Proteins / Q000378:metabolism; D020134:Catalytic Domain; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D012808:Sigma Factor / Q000378:metabolism; D020079:Terminal Repeat Sequences; D014157:Transcription Factors / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; C483566:ClpYQ protease, E coli; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; C053139:sulA protein, E coli; C067572:RcsA protein, E coli; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp