JOURNAL OF BACTERIOLOGY, Dec. 1997, p. 7219–7225 Vol. 179, No. 23 0021-9193/97/$04.0010 Copyright © 1997, American Society for Microbiology Synergistic Roles of HslVU and Other ATP-Dependent Proteases in Controlling In Vivo Turnover of s32 and Abnormal Proteins in Escherichia coli MASAAKI KANEMORI, KAZUYO NISHIHARA, HIDEKI YANAGI, AND TAKASHI YURA* HSP Research Institute, Kyoto Research Park, Kyoto 600, Japan Received 7 April 1997/Accepted 19 September 1997 Production of abnormal proteins during steady-state growth induces the heat shock response by stabilizing normally unstable s32 (encoded by the rpoH gene) specifically required for transcription of heat shock genes. We report here that a multicopy plasmid carrying the hslVU operon encoding a novel ATP-dependent protease inhibits the heat shock response induced by production of human prourokinase (proUK) in Escherichia coli. The overproduction of HslVU (ClpQY) protease markedly reduced the stability and accumulation of proUK and thus reduced the induction of heat shock proteins. In agreement with this finding, deletion of the chromosomal hslVU genes significantly enhanced levels of proUK and s32 without appreciably affecting cell growth. When the DhslVU deletion was combined with another protease mutation (lon, clpP,orftsH/hflB), the resulting multiple mutations caused higher stabilization of proUK and s32, enhanced synthesis of heat shock proteins, and temperature-sensitive growth. Furthermore, overproduction of HslVU protease reduced s32 levels in strains that were otherwise expected to produce enhanced levels of s32 due either to the absence of Lon-ClpXP proteases or to the limiting levels of FtsH protease. Thus, a set of ATP-dependent proteases appear to play synergistic roles in the negative control of the heat shock response by modulating in vivo turnover of s32 as well as through degradation of abnormal proteins. When Escherichia coli cells are exposed to high temperature, membrane precursor proteins, or foreign proteins, can induce synthesis of a set of heat shock proteins (HSPs) is rapidly HSPs and mimic the heat shock response (7, 15). The func- induced. The induction occurs by transient increase in s32 (the tional rpoH gene is required for such induction, and the extent rpoH gene product), which directs RNA polymerase to tran- of induction depends on the amount of unfolded proteins and scribe the heat shock genes (11, 44). The increase in s32 level not on protein degradation (27). Moreover, the increased s32 depends on both increased synthesis and stabilization of s32, level under these conditions results primarily from stabilization which is very unstable (half-life of about 1 min) (33). Whereas and not from increased synthesis of s32 (17, 39). These and induction of s32 synthesis occurs at the translation level medi- other data suggested that stabilization and enhanced synthesis ated by the rpoH mRNA secondary structure (25, 45), stabili- of s32 that occur upon exposure to high temperature involve 32 zation occurs presumably by sequestering s from DnaK and two distinct pathways or mechanisms (11, 17, 43). DnaJ chaperones (34, 35) or proteases such as FtsH/HflB (13, Because degradation of s32 and abnormal proteins appar- 37). This homeostatic response appears to be regulated by a ently requires the same set of chaperones (DnaK, DnaJ, and complex feedback circuit involving chaperones, proteases, and GrpE), it has been suggested that unfolded or misfolded pro- other components of signalling pathways that have yet to be teins generated under heat shock and other stress rapidly bind identified (2, 6, 11, 43, 44). and titrate the chaperones away from s32 and thus stabilize s32 Many HSPs are molecular chaperones or ATP-dependent and induce the heat shock response (2, 4). The facts that s32 proteases. The DnaK-DnaJ-GrpE and GroEL-GroES chaper- directly binds to DnaK and DnaJ (5, 20) and that overproduc- one teams play major roles in protein folding and assembly in tion of DnaK reduces the heat shock response at high temper- many cellular processes, perhaps including protein turnover (6, ature (35) or upon production of abnormal proteins (17) are 11, 12). The HSPs Lon/La, ClpP, ClpX, and FtsH are all ATP- consistent with such suggestions. Although little is known dependent proteases or their subunits: Lon is a single polypep- about the mechanisms involved, it is quite reasonable that tide that forms homotetramers, whereas Clp is a large complex enhanced levels of abnormal proteins trigger a chain of events that consists of a catalytic subunit (ClpP) and a regulatory leading to increased synthesis of HSPs. subunit (ClpA or ClpX) which confers substrate specificity (8, To further understand the nature of heat shock response 9, 10, 22). FtsH is a membrane-bound metalloprotease essen- s32 induced by abnormal proteins, we looked for additional genes tial for growth and can degrade in vivo and in vitro (13, 37). and factors that may affect the response following production Degradation of cellular proteins is mostly energy dependent, of human prourokinase (proUK), which is known to form since it is inhibited by 90% upon depletion of ATP. Lon and partially unfolded structures (17). We report here that over- ClpAP/Ti proteases play major roles in proteolysis: disruption production of a pair of HSPs, encoded by the hslVU operon of both lon and clpA (or clpP) inhibits degradation of cellular (3), markedly inhibits proUK-induced HSP synthesis primarily proteins by 70 to 80% (10, 22). by promoting degradation of proUK. Analysis of mutants lack- Production of abnormal proteins by addition of amino acid ing HslVU proteins, with or without simultaneous loss (or analogs or puromycin, or accumulation of mutant proteins, reduction) of other ATP-dependent protease(s), led us to pro- pose that HslVU and other proteases jointly participate in the * Corresponding author. Phone: (81)-75-315-8619. Fax: (81)-75-315- negative control of heat shock response by modulating turn- 8659. E-mail: [email protected]. over of s32 as well as of unfolded or misfolded proteins in vivo. 7219 7220 KANEMORI ET AL. J. BACTERIOL. While this work was in progress, we learned that HslVU (also called ClpQY) indeed represents a novel ATP-dependent pro- tease in E. coli (24, 29, 42). MATERIALS AND METHODS 2 Bacterial strains, plasmids, and phages. E. coli K-12 strain MC4100 [F araD D(argF-lac)U169 rpsL relA flbB deoC ptsF rbsR] and its derivatives were used in most experiments. Strain SG22094 (MC4100 rcsA166::Dkan clpP1::cat Dlon-510) was kindly provided by S. Gottesman. Strain FS1576 (C600 thy recD1009) (32) was used for construction of DhslVU and D(clpPX-lon) mutants. Strain YJ415 1 carrying DftsH3::kan on the chromosome and ftsH on a pSC101-based plasmid (pSTD401), as well as the isogenic parental strain (CU141), was donated by Y. Akiyama (1, 31). pUK752 is a pACYC184-based plasmid carrying the human q proUK gene (under controls of the tac promoter [tacp] and lacI ), derived from plasmid pUK-02pm0 described previously (17). An E. coli DNA library was constructed by ligating Sau3AI-digested chromosomal DNA with Charomid 9-36 (Nippon Gene, Tokyo, Japan) cut with BamHI. pKV1004 is a pBR322-based plasmid carrying the Esp3I-Eco47III fragment (2.3 kb) which was excised from a plasmid (reported here) containing the entire hslVU operon. pKV1007 is a pBR322-based plasmid carrying the hslVU operon under the control of trpp and trpR repressor. pKV1174 is a derivative of pKV1007 lacking the NsiI-BglII frag- ment (1.7 kb) of the hslVU operon. Phage P1vir was used for transduction experiments. l2137, a derivative of Charon 25, was used to construct l2137- 1 hslVU carrying the hslVU operon by in vivo recombination. lpF13-(groEp-lacZ) carried lacZ under the control of groE heat shock promoter (40). Media and chemicals. L broth has been described elsewhere (36); ampicillin (50 mg/ml), chloramphenicol (10 or 20 mg/ml), or tetracycline (5 mg/ml) was added when necessary. The synthetic medium used was medium E supplemented with 0.5% glucose and 2 mg of thiamine per ml (36); ampicillin was used at 10 35 mg/ml. L-[ S]methionine (29.6 TBq/mmol) was obtained from American Radio- chemicals; other chemicals were obtained from Nacalai Tesque, Kyoto, or Wako Pure Chemicals, Osaka, Japan. FIG. 1. Inhibition of proUK-induced heat shock response by overexpression Construction of DhslVU and D(clpPX-lon) mutants. A derivative of pKV1004 of the hslVU operon. (A) Strain MC4100 lysogenic for lpF13-(groEp-lacZ) and carrying the hslVU operon with extended flanking regions (1 kb for each side) harboring a pACYC184-based proUK expression plasmid (pUK752) and a com- was first constructed, and the internal portion of the hslVU genes (0.6-kb NsiI- patible pBR322 or its derivative pKV1004 carrying hslVU was grown to mid-log NruI fragment) was replaced by 1.4-kb fragment containing the tetracycline phase in L broth with or without 5 mM IPTG at 30°C to determine b-galactosi- resistance (tet) gene of pBR322. The resulting plasmid, pKV1172, retained the 59 dase activity. The data from a typical experiment are shown after normalization portion of hslV and the 39 portion of hslU, but these segments were shown to be to values for the control cells carrying pBR322 grown without IPTG. (B) The devoid of activities. This plasmid was treated with an appropriate restriction same set of cultures was used to extract whole-cell proteins, which were analyzed enzyme and then introduced into strain FS1576 (recD) by selecting for tetracy- by SDS-PAGE (10% gel) followed by staining with Coomassie blue. cline resistance. One such transformant that carried the DhslVU1172::tet deletion on the chromosome was used to transduce the deletion into MC4100 by phage P1, and the resulting deletion mutant was designated KY2039.