title: Purification and characterization of the heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli.
abstract: The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to beta-type subunits of the 20 S proteasomes, and HslU, a 50-kDa protein related to the ATPase ClpX. We have recently shown that HslV and HslU can function together as a novel ATP-dependent protease, the HslVU protease. We have now purified both proteins to apparent homogeneity from extracts of E. coli carrying the hslVU operon on a multicopy plasmid. HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic peptides. HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present. With a 1:4 molar ratio of HslV to HslU, approximately a 200-fold increase in peptide hydrolysis was observed. HslV stimulated the ATPase activity of HslU 2-4-fold, but had little influence on the affinity of HslU to ATP. The nonhydrolyzable ATP analog, beta,gamma-methylene-ATP, did not support peptide hydrolysis. Other nucleotides (CTP, dATP) that were slowly hydrolyzed by HslU allowed some peptide hydrolysis. Therefore, ATP cleavage appears essential for the HslV activity. Upon gel filtration on a Sephacryl S-300 column, HslV behaved as a 250-kDa oligomer (i.e. 12-14 subunits), and HslU behaved as a 100-kDa protein (i.e. a dimer) in the absence of ATP, but as a 450-kDa multimer (8-10 subunits) in its presence. Therefore ATP appears necessary for oligomerization of HslU. Thus the HslVU protease appears to be a two-component protease in which HslV harbors the peptidase activity, while HslU provides an essential ATPase activity.
authors.affiliation: Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea.
authors.forename: S J
authors.initials: SJ
authors.lastname: Yoo
authors.forename: J H
authors.initials: JH
authors.lastname: Seol
authors.forename: D H
authors.initials: DH
authors.lastname: Shin
authors.forename: M
authors.initials: M
authors.lastname: Rohrwild
authors.forename: M S
authors.initials: MS
authors.lastname: Kang
authors.forename: K
authors.initials: K
authors.lastname: Tanaka
authors.forename: A L
authors.initials: AL
authors.lastname: Goldberg
authors.forename: C H
authors.initials: CH
authors.lastname: Chung
mesh_terms: D049069:ATP-Dependent Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D009876:Operon; D016133:Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D009842:Oligopeptides; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases