We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hsIVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome (3 subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coi expressing heat-shock Proteasomes are multicatalytic proteolytic complexes present in both the nucleus and cytosol of eukaryotic cells (1). The 26S form of the proteasome catalyzes the degradation of ubiquitinconjugated proteins (2-5), and thus it plays a key role in many cellular processes, including progression through the cell cycle (6, 7), removal of abnormal proteins, and antigen presentation (8). The proteolytic core of the 26S complex is the 20S (700 kDa) proteasome particle, which consists of four sevenmembered rings. The subunits of the 20S proteasome fall into two families (9, 10): the a-type forms the two outer rings, and P-type, which contain the active sites, forms the two inner rings of the complex.Proteasomes were thought to exist exclusively in eukaryotes and certain archaebacteria (11). However, 20S proteasomes were recently discovered in the actinomycete Rhodococcus (12), and in the Escherichia coli genome sequencing project, a novel heat-shock locus (hslVU) was discovered that encodes a 19-kDa protein (HslV) (13), whose sequence is similar to 3-type proteasome subunits. This discovery of proteasome-related genes was surprising, because several groups had failed to observe a structure in E. coli resembling the proteasome or proteins resembling ubiquitin. The hslV gene is cotranscribed with the adjacent hslU gene, which codes for a 50-kDa protein containing one ATP/GTP binding motif (13 For the expression of glutathione S-transferase (GST)-fusion proteins, the hslVand hslU genes were PCR amplified separately using A phage 18-126 DNA bearing the hslVU operon, kindly provided by F. Blattner (University of Wisconsin-Madison), and cloned into the vector pGEX-2T (Pharmacia). Vector pV106 (GST-HslV) and pU206 (GST-HslU) were electroporated into E. coli C600 cells. GST-fusion proteins were purified from strains C106 and C206 using the GST Purification Module (Pharmacia). To obtain antibodies, purified GST-HslV and GST-HslU proteins were injected into rabbits. Polyclonal anti-HslV and antiHslU antibodies were then affinity purified using the GST-fusions as ligands, and depleted of anti-GST antibodies using a GST column.Abbreviations: hsl, heat-shock locus; GST, glutathone S-transferase. tTo whom reprint requests should be addressed.5808

HslVU is a new two-component protease in Escherichia coli composed of the proteasome-related peptidase HslIV and the ATPase HsIU. We have used electron microscopy and image analysis to examine the structural organization of HslV and HslU homo-oligomers and the active HslVU enzyme. Electron micrographs of HslV reveal ring-shaped particles, and averaging of top views reveal six-fold rotational symmetry, in contrast to other beta-type proteasome subunits, which form rings with seven-fold symmetry. Side views of HslV show two rings stacked together, thus, HslV behaves as dodecamer. The ATPase HslU forms ring-shaped particles in the presence of ATP, AMP-PNP or ADP, suggesting that nucleotide binding, but not hydrolysis, is required for oligomerization. Subunit crosslinking, STEM mass estimation, and analysis of HslU top views indicate that HslU exists both as hexameric and heptameric rings. With AMP-PNP present, maximal proteolytic activity is observed with a molar ratio of HslU to HslV subunits of 1:1, and negative staining electron microscopy shows that HslV and HsIU form cylindrical four-ring structures in which the HsIV dodecamer is flanked at each end by a HslU ring.

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.

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