Effects of Lipoprotein Overproduction on the Induction of DegP (HtrA) Involved in Quality Control in the Escherichia coli Periplasm*

Recent biochemical examination has revealed the presence of at least 90 different lipoproteins in Escherichia coli . Among previously identified lipoproteins, only an outer membrane lipoprotein, NlpE, is known to induce expression of the degP gene upon its overproduction. The degP gene encodes a periplasmic protease, which is thought to be involved in the digestion of unfolded proteins, and is essential for growth at high tem-peratures. However, it is not completely clear why NlpE overproduction causes degP expression. Moreover, among newly confirmed lipoproteins, there may be oth-ers that also induce degP expression. Therefore, we overproduced each of the 90 lipoproteins and examined the level of degP expression as (cid:1) -galactosidase activity by using a degP promoter- lacZ fusion. The extent of degP expression caused by NlpE overproduction was dependent on the mode of degP - lacZ fusion. On the other hand, new inner membrane lipoprotein YafY strongly induced degP expression irrespective of the mode of fusion even though the level of overproduced YafY was lower than that of NlpE. The induction of degP expression by YafY overproduction was dependent on the Cpx two-component system. Alteration of the li-poprotein-sorting signals of NlpE and YafY did not abol-ish the degP induction. However, a YafY

Bacterial lipoproteins are synthesized as precursors in the cytoplasm and then translocated to the periplasmic side of the inner membrane where they are processed to mature forms. Mature lipoproteins have an N-terminal Cys that is modified by thioether-linked diacylglycerol and amino-linked acyl chains (1). In Escherichia coli, lipoproteins are anchored to the periplasmic leaflet of the inner or outer membrane through N-terminal lipids. When lipoproteins have an Asp at position 2 and Asp, Glu, Gln, or Asn at position 3 (2) they are retained in the inner membrane presumably through an interaction between Asp at position 2 and phospholipids (3).
The Lol system comprising five Lol proteins is required for the sorting and outer membrane localization of lipoproteins. The LolCDE complex, an ATP binding cassette transporter, releases outer membrane-directed lipoproteins from the inner membrane (4,5) leading to the formation of a water-soluble complex comprising one molecule each of a lipoprotein and LolA in the periplasm (6,7). The LolA-lipoprotein complex then interacts with the outer membrane receptor LolB, which catalyzes the anchoring of lipoproteins to the outer membrane (8,9). The crystal structures of LolA and LolB are very similar to each other despite the fact that their amino acid sequences are dissimilar (10).
More than 100 lipoproteins are predicted to be present in E. coli (11,12). We recently cloned almost all putative lipoprotein genes and examined whether or not the proteins encoded by these genes are modified with lipids. These analyses revealed that E. coli possesses at least 90 lipoproteins. 1 Although most lipoproteins have no known functions, they are expected to play important roles in various activities in the periplasm, because the protein moieties of lipoproteins are generally soluble and presumably exposed to the periplasm. Among previously confirmed lipoproteins, only NlpE is known to induce expression of the degP gene upon overproduction, which encodes a periplasmic protease (13,14). Overproduction of NlpE activates a two-component signal transduction system comprising CpxA and CpxR and then stimulates the expression of degP (13,14), which is also positively regulated by E . This stress response system involving the Cpx two-component system and E is thought to represent the quality control mechanism of the periplasm (15,16). When unfolded proteins are accumulated in the periplasm, DegP is induced to clean the periplasm. DegP has also been reported to exhibit chaperone activity (17). However, it is not clear why only NlpE among the lipoproteins induces DegP. Moreover, it is possible that some newly confirmed lipoproteins may also induce DegP. Here we report that the inner membrane lipoprotein YafY also induces DegP.
Construction of pDegP-LacZ-To construct pDegP-LacZ encoding LacZ under the control of the degP promoter, the 480-bp upstream region of degP was amplified by PCR using oligonucleotide primers 5Ј-CACACAAGCTTGCCATCCAGATGTCGAGCGGCTTGAATTGC-3Ј (the HindIII site is underlined) and 5Ј-CGCGCGGATCCCAGTCTC-GATTAACAGATAACGCAAAATTG-3Ј (the BamHI site is underlined) with MC4100 chromosomal DNA as a template. The amplified DNA was digested with HindIII and BamHI and then inserted into the same site of pCB192 (20) carrying lacZ and bla. The plasmid thus constructed was digested with ScaI and HindIII, and the resultant fragment carrying degP-lacZ was cloned at the SmaI-HindIII site of pSTV28 (Takara), which carries a chloramphenicol resistance gene.
Overproduction of Lipoproteins-Construction of plasmids (pKT-lipoprotein) carrying each lipoprotein gene under the control of the lac promoter-operator will be reported elsewhere. Lipoproteins were constructed so as to have a T7-tag followed by a hexahistidine tag at the C termini and then overproduced by adding 1 mM IPTG. 2 Construction of Lipoprotein Derivatives-To construct NlpE(DD) and YafY(SS) having Asp and Ser, respectively, as the second and third residues, pKT-NlpE and pKT-YafY were mutagenized using a QuikChange site-directed mutagenesis kit (Stratagene) with the respective pairs of oligonucleotides shown in Table I. To construct nonlipidated derivatives of NlpE (mNlpE) and YafY (mYafY), a Cys to Ala mutation was introduced at position 1 of the mature region, as described for the construction of mLolB (8). The oligonucleotide primers used are indicated in Table I. Mutations were confirmed by sequencing.
Construction of YafY-YfjS Chimeric Lipoproteins-Both pKT-YafY and pKT-YfjS have unique sites for SnaBI and AccI in the lipoproteincoding region and for MluI outside of the coding region. The two plasmids were digested with these restriction enzymes, and the resultant fragments were used to construct various chimeric lipoproteins. Twelve residues, which were not identical between YafY and YfjS, were mutagenized to the counterpart residues using the QuikChange site-directed mutagenesis kit. In some cases, the mutated pKT-YafY and pKT-YfjS were digested with the restriction enzymes described above for the exchange of regions having altered residues.

Inner Membrane Lipoprotein YafY Strongly Induces DegP-
Plasmids (pKT-lipoprotein) carrying lipoprotein genes under the control of the tac promoter-operator were transformed into MC4100 cells harboring pDegP-LacZ, which carries lacZ fused to the upstream region of degP. ␤-Galactosidase activity was measured by the reported method (24). Some lipoproteins caused a small increase in the ␤-galactosidase activity even in the absence of IPTG, whereas these lipoproteins caused significant increases in the ␤-galactosidase activity upon the addition of IPTG (Fig. 1). 6 of the 90 lipoproteins examined could not be overproduced, because their overproduction caused lysis of cells. 3 1 ␤-Galactosidase activity was examined at least twice with or without overproduction of each of the 84 lipoproteins, and typical results are shown in Fig. 1. In contrast to the observations by Silhavy and co-workers (13,14), not only NlpE (Fig. 1A, arrow) but also several other lipoproteins caused appreciable increases in the ␤-galactosidase activity. Above all, the increase caused by YafY overproduction was remarkable ( Fig. 1C, arrow). YafY is most likely an inner membrane lipoprotein, because it has the strongest inner membrane retention signal, Asp-Asp (2), at the second and third positions (see Fig. 5).
The levels of overproduced lipoproteins were examined in membranes prepared from cells grown in the presence of IPTG (Fig. 2). The overproduction levels differed depending on the lipoprotein species and were normalized as to molar amount by means of the molar amount of OmpA. The extent of degP induction determined with lipoprotein overproduction was plotted as a function of the normalized level of the lipoprotein (Fig. 3A). The extent of degP induction was found to be independent of the molar amount of a lipoprotein, for example, YegR was overproduced more than NlpE but did not induce degP. Many lipoproteins were overproduced to levels similar to that of OmpA and caused only marginal degP induction. Strikingly, YafY strongly induced degP, although the level of its overproduction was similar to the molar amount of OmpA. YafY has no known function but is predicted to be located in the inner membrane, because it has Asp at both the second and third positions. This dipeptide functions as a Lol-avoidance signal and causes the retention of lipoproteins in the inner membrane (2,3,25).
Induction of DegP by YafY Is Dependent on the Cpx Twocomponent System-It has been shown that overproduction of NlpE induces DegP through activation of the two-component system comprising CpxA and CpxR (13,14). CpxA is the sensor kinase in the inner membrane, is autophosphorylated upon detection of envelope stress, and then transfers the phosphate to the response regulator CpxR. The phosphorylated CpxR then induces the expression of genes encoding factors involved in the holding and digestion of envelope proteins. DegP induction caused by the overproduction of 11 lipoproteins, which include both DegP-induction positive and negative lipoproteins in MC4100 cells, was examined in PAD280 (cpxA ϩ ) and its derivative PAD299 (cpxA::spc). Both strains carry the degP-lacZ fusion gene, which was inserted into the chromosome by means of phage transduction (19). The levels of overproduced lipoproteins were essentially the same in PAD280 and PAD299 (Fig. 3B) and did not significantly differ from those determined in MC4100 (Fig. 3A), except for OsmB. However, the ␤-galactosidase activity determined in PAD280 was significantly higher than that determined in MC4100 harboring pDegP- LacZ. The difference in ␤-galactosidase activity between the two strains is presumably caused by differences in the location and structure of the degP-lacZ fusion genes. The lacZ gene in PAD280 was fused to a truncated degP and located on the chromosome, whereas that in MC4100 harboring pDegP-LacZ was fused to the upstream region of degP carried by a plasmid. Although the mRNA length is different, only lacZ is expressed upon induction of the degP promoter in either strain. The difference in the structures of the transcripts might have caused the difference in DegP expression. The copy numbers of the degP-lacZ fusion genes were also different between the two strains. The number of phosphorylated CpxR molecules might be insufficient for the full induction of lacZ in MC4100 harboring pDegP-LacZ. Despite the very high ␤-galactosidase activity in the PAD280 strain, the induction of DegP on the overproduction of most lipoproteins was nearly completely abolished in the cpxA null strain PAD299 (Fig. 3B). DegP induction by NlpE was remarkable in PAD280, as reported previously (15). Furthermore, the overproduction of YafY strongly induced DegP in a Cpx-dependent manner even in PAD280. Pal, OsmB, and NlpA also caused DegP induction in a Cpx-dependent manner, albeit to a lesser extent. Membrane Anchoring of NlpE Is Required for DegP Induction-Lipoproteins are sorted to either the inner or outer membrane depending on the sorting signal located at position 2 (2,(25)(26)(27). Asp at this position with Asp, Glu, Asn, or Gln at position 3 causes the strong retention of lipoproteins on the inner membrane (2). In contrast, residues other than Asp at position 2 direct lipoproteins to the outer membrane. NlpE has Ser at positions 2 and 3 and is located on the outer membrane (14), whereas YafY having Asp at positions 2 and 3 is expected to be located on the inner membrane. The second and third residues were mutated to mislocate NlpE and YafY to the inner  and outer membranes, respectively. Moreover, the N-terminal Cys was changed to Ala to express them as soluble periplasmic proteins. These derivatives were induced in MC4100 harboring pDegP-LacZ, and the levels of their overproduction and their subcellular location were examined (Fig. 4). The level of NlpE(DD) was lower than that of NlpE (Fig. 4A), whereas DegP induction was stronger with the former than with the latter (Fig. 4C). The level of overproduced YafY(SS) was also significantly lower than that of the wild-type YafY, but it still induced DegP. Both derivatives remained associated with spheroplasts (Fig. 4B). Non-lipidated mNlpE molecules were mostly found in the periplasm (Fig. 4B). Interestingly, the level of overproduced mNlpE was higher than that of NlpE(DD) (Fig. 4A), but mNlpE did not induce DegP (Fig.  4C), indicating that NlpE must be membrane-anchored for the induction of DegP. In contrast to that of mNlpE, the expression of mYafY was very low, and it remained in the spheroplasts for an unknown reason. Therefore, it remains to be clarified whether or not membrane anchoring is also essential for DegP induction by YafY.
When NlpE, YafY, and their lipidated derivatives were overproduced, inner and outer membranes could not be separated by means of sucrose density gradient centrifugation (data not shown). Therefore, membranes were prepared from cells grown in the presence of a low concentration of IPTG and then fractionated (Fig. 4D). NlpE(DD) was located in the inner membrane as expected suggesting the inner membrane location of the overproduced NlpE(DD). Smear bands appeared for the outer membrane fractions after prolonged incubation with anti-His-tag antibodies. These bands migrated slightly slower than that of NlpE(DD) and seemed to be nonspecific ones. YafY(SS) remained in the inner membrane even though it had an outer membrane-directed signal. It is likely that YafY is retained in the inner membrane through an interaction with other inner membrane components. If this is the case, the Asp residues at positions 2 and 3 of the wild-type YafY may not function as a lipoproteinsorting signal.

Residues of YafY Important for DegP Induction-
The amino acid sequence of YafY is highly homologous to that of YfjS (Fig.  5). Furthermore, the levels of overproduced YafY and YfjS were similar (Fig. 3). On the other hand, YfjS did not induce DegP in MC4100 or PAD280 (Fig. 3). To determine the critical regions or residues for DegP induction, a number of YafY-YfjS chimeras were constructed (Fig. 6). The expression levels of these derivatives did not differ significantly (data not shown). Both the yafY and yfjS genes have unique sites for SnaBI and AccI inside the coding region. In addition, plasmids carrying the lipoprotein genes have a unique site for MluI outside the coding region. By taking advantage of these restriction sites, the mature regions of the two lipoproteins were separated into three parts, i.e. the N-terminal, central, and C-terminal regions (Fig.  5). Six YafY-YfjS chimeric lipoproteins were constructed by exchanging these regions (Fig. 6, chimeras A-F). Replacement of the N-or C-terminal region of YafY by the corresponding region of YfjS significantly reduced DegP induction (Fig. 6,  chimeras A and C). In contrast, replacement of the central region of YafY by that of YfjS only slightly reduced DegP induction (Fig. 6, chimera B). The N-or C-terminal region of YafY alone did not cause the maximum induction of DegP (Fig.  6, chimeras D and F), whereas the level of induction was higher than that caused by the central region alone (chimera E). These results indicate that the N-and C-terminal regions are both important for DegP induction.
To determine the most important residues for DegP induction in the N-and C-terminal regions of YafY, eight residues present in YafY but not in YfjS were variously exchanged with those in YfjS (Fig. 6, chimeras G-N). This revealed that Asp at position 40, Tyr at position 41, and Gln at position 116 are critical residues for DegP induction. Finally, chimera N thus constructed was a derivative of YfjS possessing these three residues of YafY. Induction of DegP caused by the overproduction of chimera N was significantly higher than that by the overproduction of YfjS, although the DegP induction by chimera N was still lower that that by the wild-type YafY.

DISCUSSION
The protein moieties of most, if not all, lipoproteins of E. coli are expected to be exposed to the periplasm. Lipoproteins are therefore expected to be responsible for various functions in the periplasm. In this study, we focused on the periplasmic stress response and found that not only NlpE but also a new inner membrane lipoprotein, YafY, causes DegP induction upon overproduction. The role of NlpE in up-regulation of the Cpx twocomponent system has been extensively studied (15). It has been revealed that the Cpx system is generally required for a response to various envelope stresses and induces the expression of genes such as dsbA, degP, ppiA, and spy (13,28,29). On the other hand, NlpE is specifically required for the activation of the Cpx system caused by cell adhesion to a hydrophobic surface (30). It is speculated that NlpE is located upstream of the Cpx pathway and senses cell adhesion (30). However, it is not completely clear why overproduction of NlpE also activates the Cpx system. Overproduction of outer membrane proteins causes the accumulation of an unfolded ␤-barrel structure and then activates E , which is negatively regulated by anti-factor RseA and a periplasmic protein, RseB. Unfolded proteins are speculated to bind to the PDZ domain of a periplasmic protease DegS, which eventually causes the digestion of RseA (16). Expression of degP is under the regulation of both the Cpx pathway and E .
Among the 90 lipoproteins examined, the effect of YafY overproduction on the induction of DegP was remarkable (Figs. 1  and 3). This induction was dependent on the cpxA gene (Fig.  3B), indicating that YafY overproduction causes an induction of the expression of degP through the activation of the Cpx system. Although the function of YafY is not known, it seems likely that YafY is involved in the monitoring of certain envelope stresses as speculated for the case of NlpE. Consistent with its sorting signal, YafY was located on the inner membrane. However, alteration of its signal to an outer membranespecific one did not cause the sorting of YafY(SS) to the outer membrane (Fig. 4D). These results suggest that the location of YafY is not determined by the sorting signal. Previous examinations (2,27) revealed that the membrane location of E. coli lipoproteins is basically determined by the second residue as originally proposed (26). The reason that YafY(SS) remained on the inner membrane is not known at present. However, it has been reported that the function of sorting signals is abolished depending on the structure of the lipoprotein mature region. For example, dimerization of lipoproteins inhibits the LolCDEdependent release of outer membrane-specific lipoproteins (3), and an intramolecular disulfide bond formed in the N-terminal region abolishes the Lol avoidance function of Asp at position 2 (31). It is likely that YafY exists as a homo-or hetero-oligomer on the inner membrane thereby inhibiting the localization of YafY(SS) to the outer membrane.
In contrast to YafY, NlpE(DD) expressed at a low level was located on the inner membrane in accordance with its sorting signal (Fig. 4D). We therefore speculate that overproduced NlpE(DD) is also located on the inner membrane, although its overproduction perturbed membrane separation. We previously observed that overproduction of inner membrane-specific derivatives of the major outer membrane lipoprotein Lpp pre-C, the indicated lipoprotein derivatives either were overproduced or not in MC4100 cells harboring pDegP-LacZ. ␤-Galactosidase activity was determined as described in the legend to Fig. 1. D, membranes were prepared from cells grown in the presence of 10 M IPTG and fractionated into inner and outer membranes by means of sucrose density gradient centrifugation as reported (8). After SDS-PAGE analysis, lipoproteins were detected with anti-His-tag antibodies. OmpA and SecG were detected with the respective antibodies.

FIG. 4. DegP induction caused by overproduction of lipoprotein derivatives.
A-C, NlpE, YafY, and their derivatives were overproduced in MC4100 cells harboring pDegP-LacZ as in Fig. 1. A, the same amounts of cells overproducing the specified lipoprotein derivatives were analyzed by SDS-PAGE and immunoblotting with anti-Histag antibodies. Inner membrane protein SecG was analyzed as an internal standard with anti-SecG antibodies. B, cells in A were converted into spheroplasts as described (6) vented membrane separation (32). The induction of DegP by NlpE(DD) was stronger than that by NlpE (Fig. 4C), although the overproduction level was significantly lower with NlpE(DD) than with NlpE (Fig. 4A). This may suggest that the overproduction of NlpE generates mislocated NlpE on the inner membrane and that this species of NlpE is responsible for the activation of the Cpx system. It may be interesting to determine whether or not NlpE(DD) is also required for the response to cell adhesion.
The level of mNlpE was more than 3-fold higher that that of NlpE(DD) (Fig. 4A), whereas mNlpE did not induce DegP (Fig.  4C). Because most mNlpE molecules were present in the periplasm (Fig. 4B), the membrane anchoring of NlpE seems to be essential for the induction of DegP. Overproduction of lipoproteins possessing three fatty acyl chains may cause an alteration of the properties of membranes. However, because only a limited number of species of lipoproteins induced DegP upon overproduction (Figs. 1 and 3) changes in the membrane properties cannot be the reason for DegP induction. Instead, these results suggest that the protein moiety of NlpE should be close to the membrane for efficient induction of DegP through activation of the Cpx system. On the other hand, mYafY was expressed at a very low level (Fig. 4A) and was only detected in membrane fractions (Fig. 4B). Therefore, it is not clear at present whether or not the membrane anchoring of YafY is also required for DegP induction.
The identity of the amino acid sequences of YafY and YfjS is higher than 90% (Fig. 5), whereas DegP was strongly induced only by YafY overproduction. On the exchanging of regions and residues, only three residues of YafY were found to be important for DegP induction. However, neither YafY nor YfjS has any known function. Moreover, a motif search did not allow speculation as to their functions. To clarify the specific role of YafY in stress response mechanisms, it is essential to reveal Various YafY-YfjS chimeric lipoproteins and their derivatives were constructed as described under "Experimental Procedures" and overproduced in MC4100 cells harboring pDegP-LacZ. ␤-Galactosidase activity was determined at least twice and the averages are given on the right. the differences in structure and function between YafY and YfjS. The various YafY derivatives constructed here will be useful for the analyses of a possible interaction with the Cpx components in the inner membrane. It seems to be possible that NlpE and YafY are both components of a regulatory network, which also consists of the Cpx system, and that overproduction of one of the two lipoproteins leads to uncontrolled expression of DegP.