Yoo S J, Seol J H, Shin D H, Rohrwild M, Kang M S, Tanaka K, Goldberg A L, Chung C H
Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea.
J Biol Chem. 1996 Jun 14;271(24):14035-40. doi: 10.1074/jbc.271.24.14035.
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.
大肠杆菌中的hslVU操纵子编码两种热休克蛋白，HslV（一种与20 S蛋白酶体的β型亚基同源的19 kDa蛋白）和HslU（一种与ATP酶ClpX相关的50 kDa蛋白）。我们最近发现，HslV和HslU可作为一种新型的ATP依赖性蛋白酶——HslVU蛋白酶共同发挥作用。现在，我们已从携带多拷贝质粒上的hslVU操纵子的大肠杆菌提取物中，将这两种蛋白纯化至表观均一。HslU自身可水解ATP，而纯HslV是一种可降解某些疏水肽的弱肽酶。当存在ATP时，HslU可显著刺激HslV的肽水解作用。在HslV与HslU的摩尔比为1:4时，观察到肽水解作用增加了约200倍。HslV可将HslU的ATP酶活性提高2 - 4倍，但对HslU与ATP的亲和力影响很小。不可水解的ATP类似物β,γ-亚甲基-ATP不支持肽水解。其他可被HslU缓慢水解的核苷酸（CTP、dATP）可允许一定程度的肽水解。因此，ATP的水解似乎对HslV的活性至关重要。在Sephacryl S - 300柱上进行凝胶过滤时，在无ATP的情况下，HslV表现为250 kDa的寡聚体（即12 - 14个亚基），HslU表现为100 kDa的蛋白（即二聚体），但在有ATP的情况下，HslU表现为450 kDa的多聚体（8 - 10个亚基）。因此，ATP似乎是HslU寡聚化所必需的。这样看来，HslVU蛋白酶似乎是一种双组分蛋白酶，其中HslV具有肽酶活性，而HslU提供必需的ATP酶活性。
