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Hslv-Hslu: a Novel ATP-Dependent Protease Complex in Escherichia Coli Related to the Eukaryotic Proteasome MARKUS ROHRWILD*, OLIVIER COUX*, H.-C

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Hslv-Hslu: a Novel ATP-Dependent Protease Complex in Escherichia Coli Related to the Eukaryotic Proteasome MARKUS ROHRWILD*, OLIVIER COUX*, H.-C Proc. Natl. Acad. Sci. USA Vol. 93, pp. 5808-5813, June 1996 Biochemistry HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome MARKUS ROHRWILD*, OLIVIER COUX*, H.-C. HUANG*, RICHARD P. MOERSCHELL*, SOON JI YOOt, JAE HONG SEOLt, CHIN HA CHUNGt, AND ALFRED L. GOLDBERG*$ *Department of Cell Biology, Harvard Medical School, Boston, MA 02115; and tDepartment of Molecular Biology and Research Center for Cell Differentiation, Seoul National University, Seoul 151-742, Korea Communicated by Marc W. Kirschner, Harvard Medical School, Boston, MA, February 8, 1996 (received for review December 27, 1995) ABSTRACT We have isolated a new type ofATP-dependent which is essential for the degradation of certain proteins by the protease from Escherichia coli. It is the product ofthe heat-shock ClpP protease (15). These findings raise the possibility that HslV locus hsIVU that encodes two proteins: HslV, a 19-kDa protein and HslU function together as an ATP-dependent protease. similar to proteasome (3 subunits, and HslU, a 50-kDa protein In E. coli, heat-shock genes are transcribed by the RNA related to the ATPase ClpX. In the presence ofATP, the protease polymerase holoenzyme containing either of the alternative hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC sigma factors, o32 or o24 (16, 17). One major group of proteins and very slowly certain other chymotrypsin substrates. This induced in E. coli during heat-shock are the molecular chap- activity increased 10-fold in E. coi expressing heat-shock pro- erones that catalyze the refolding of heat-damaged proteins; teins constitutively and 100-fold in cells expressing HslV and another group of up-regulated proteins are proteases that HslU from a high copy plasmid. Although HsIV and HslU could catalyze the selective degradation of such abnormal proteins be coimmunoprecipitated from cell extracts of both strains with (18). In E. coli, these proteases include the ATP-dependent an anti-HslV antibody, these two components were readily sep- proteases La (Lon) (19) and ClpP (Ti) (20) and the periplasmic arated by various types of chromatography. ATP stimulated protease HtrA (DegP) (21). The hslVU promoter perfectly peptidase activity up to 150-fold, whereas other nucleoside matches the consensus ofpromoters recognized by a32, and the triphosphates, a nonhydrolyzable ATP analog,ADP, orAMP had transcription of this operon increases on heat-shock (13). There- no effect. Peptidase activity was blocked by the anti-HslV anti- fore, it seemed likely that HslV and HslU may also be involved body and by several types of inhibitors of the eukaryotic protea- in the degradation of damaged polypeptides during heat-shock. some (a threonine protease) but not by inhibitors ofother classes The aim of this study was to test whether hslV encodes a ofproteases. Unlike eukaryotic proteasomes, the HsIVU protease proteolytic enzyme similar to the eukaryotic proteasome and lacked tryptic-like and peptidyl-glutamyl-peptidase activities. whether its function is regulated by the HslU ATPase. We Electron micrographs reveal ring-shaped particles similar to en demonstrate here a novel type of protease that requires ATP face images of the 20S proteasome or the ClpAP protease. Thus, for function and contains both HslV and HslV and HslU appear to form a complex in which ATP hydro- hydrolysis peptidase lysis by HslU is essential for peptide hydrolysis by the protea- HslU proteins. some-like component HslV. MATERIAL AND METHODS Proteasomes are multicatalytic proteolytic complexes present Strains, Plasmids, Media, and Culture Conditions. Strain in both the nucleus and cytosol of eukaryotic cells (1). The 26S SG12051 (C600, lonS10, clpPl::Cmr), kindly provided by S. form of the proteasome catalyzes the degradation of ubiquitin- Gottesman (National Cancer Institute, Bethesda, MD), was conjugated proteins (2-5), and thus it plays a key role in many transformed with plasmids pUHE211-1 (rpoH, Apr) and cellular processes, including progression through the cell cycle pDMI1 (lacI, Kmr) by electroporation to yield strain HSL32. (6, 7), removal of abnormal proteins, and antigen presentation cells were transformed with (8). The proteolytic core of the 26S complex is the 20S (700 BL21(DE3) (Stratagene) plasmid kDa) proteasome particle, which consists of four seven- pGEM-HSL (hslVU, Apr), which carries the hslVU operon under membered rings. The subunits of the 20S proteasome fall into its own promoter (strain B405). The hslVU operon was amplified two families (9, 10): the a-type forms the two outer rings, and by PCR using genomic DNA form E. coli K12. The PCR P-type, which contain the active sites, forms the two inner rings fragment, containing the hslVU operon and an additional 120 bp of the complex. upstream of the translational start ofhslVand 146 bp downstream Proteasomes were thought to exist exclusively in eukaryotes of the translational termination ofhslU, was cloned into pGEM-T and certain archaebacteria (11). However, 20S proteasomes vector (Promega) (pGEM-HSL). were recently discovered in the actinomycete Rhodococcus For the expression of glutathione S-transferase (GST)-fusion (12), and in the Escherichia coli genome sequencing project, a proteins, the hslVand hslU genes were PCR amplified separately novel heat-shock locus (hslVU) was discovered that encodes a using A phage 18-126 DNA bearing the hslVU operon, kindly 19-kDa protein (HslV) (13), whose sequence is similar to 3-type provided by F. Blattner (University of Wisconsin-Madison), and proteasome subunits. This discovery ofproteasome-related genes cloned into the vector pGEX-2T (Pharmacia). Vector pV106 was surprising, because several groups had failed to observe a (GST-HslV) and pU206 (GST-HslU) were electroporated intoE. structure in E. coli resembling the proteasome or proteins re- coli C600 cells. GST-fusion proteins were purified from strains sembling ubiquitin. The hslV gene is cotranscribed with the C106 and C206 using the GST Purification Module (Pharmacia). adjacent hslU gene, which codes for a 50-kDa protein containing To obtain antibodies, purified GST-HslV and GST-HslU pro- one ATP/GTP binding motif (13). Although both HslV and teins were injected into rabbits. Polyclonal anti-HslV and anti- HslU have close homologs in other bacteria (14), HslU is also HslU antibodies were then affinitypurified using the GST-fusions related to the ClpX ATPase in E. coli (about 50% identity) (13), as ligands, and depleted of anti-GST antibodies using a GST column. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: hsl, heat-shock locus; GST, glutathone S-transferase. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5808 Downloaded by guest on October 2, 2021 Biochemistry: Rohrwild et al. Proc. Natl. Acad. Sci. USA 93 (1996) 5809 All strains were grown at 37°C in Luria-Bertani broth if not grids for 1 min, after which excess buffer was blotted off with mentioned otherwise. When necessary, the medium was sup- filter paper. Specimens were stained with 1% (wt/vol) uranyl plemented with ampicillin (100 tug/ml), chloramphenicol (25 acetate for 1 min and excess stain was blotted off. Grids were ,Lg/ml), or kanamycin (25 jig/ml). For strains HSL32, C106, allowed to dry, and specimens were viewed in a JEOL 1200 EX and C206, isopropyl-P-D-thiogalactoside (IPTG) was added to or JEOL 100 CX electron microscope operating at 80 kV. a final concentration of 1 mM to induce expression of o32 or Micrographs were recorded on Kodak Electron Microscopy the GST fusion proteins during exponential growth. Film 4489 emulsion at a nominal magnification of x53,000. Immunoprecipitation. Cells were grown in minimal medium supplemented with essential amino acids, thiamine, and 0.5% glucose. At mid-log phase, 50 pzCi (1 Ci = 37 GBq) per ml RESULTS culture of TranS-Label (ICN) was added in the presence of 50 HsIV and HslU Form a Complex with Proteolytic Activity. ,uM nonradiolabeled methionine for 2 hr. Cells were harvested To demonstrate the existence of the putative HslVU protease, and sonicated in 50 mM Tris (pH 7.6), 5 mM MgCl2, and 1 mM the two ATP (bufferA). After removal of debris, immunoprecipitation we initially used E. coli strain SG12051, which lacks of the crude lysate was performed under nondenaturing major ATP-dependent proteases La and ClpP. To stimulate conditions in buffer A plus 1% Nonidet P-40. Extracts were the expression of the heat-shock proteins HslV and HslU at incubated with anti-HslV antibody for 1 hr on ice, followed by 37°C, o32 was expressed in this strain (HSL32) from an the addition of BSA-coated protein A-sepharose beads for an inducible plasmid under the control of the lac promoter. additional hour under gentle rocking at 4°C. The precipitates Induction of o32 with IPTG led to an approximately 10-fold were then washed repeatedly at room temperature for 1 hr increase in the content of HslV and HslU proteins (Fig. 1A, either in buffer A or buffer A plus 150 mM NaCI and 0.2% lanes 1 and 2). Crude lysates of this strain were assayed for SDS. Finally, the beads were boiled in SDS/PAGE sample activity against fluorogenic peptide substrates of the protea- buffer and analyzed by 15% SDS/PAGE. Bands were detected some. The peptide Z-Gly-Gly-Leu-AMC was only hydrolyzed by autoradiography. in the presence ofATP. This activity was about 10-fold greater Peptidase Assays. Enzyme samples were preincubated at after induction of a32, than in the parental strain not carrying room temperature for 30 min in 25 mM Tris (pH 7.6), 5 mM the a32 plasmid (Fig. 1B). Thus, HslV and HslU or possibly MgCl2, and 1 mM DTT in the presence or absence of 1 mM ATP before addition of the peptide substrate.
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