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Volume 271, Number 24, Issue of June 14, 1996 pp. 14035-14040
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Purification and Characterization of the Heat Shock Proteins HslV and HslU That Form a New ATP-dependent Protease in Escherichia coli

(Received for publication, January 22, 1996, and in revised form, March 21, 1996)

Soon Ji Yoo , Jae Hong Seol , Dong Hun Shin , Markus Rohrwild ^§ , Man-Sik Kang , Keiji Tanaka ^¶ , Alfred L. Goldberg ^§ and Chin Ha Chung

From the  Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea, the ^§ Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, and ^¶ Institute for Enzyme Research, Tokushima University, Tokushima 770, Japan

The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to -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, ,-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.

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