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YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA

2003; Springer Nature; Volume: 22; Issue: 23 Linguagem: Inglês

10.1093/emboj/cdg602

1460-2075

Kazue Kanehara, Koreaki Ito, Yoshinori Akiyama,

Evolution and Genetic Dynamics

Article1 December 2003free access YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA Kazue Kanehara Kazue Kanehara Institute for Virus Research, Kyoto University, Kyoto, 606-8507 Japan Search for more papers by this author Koreaki Ito Koreaki Ito Institute for Virus Research, Kyoto University, Kyoto, 606-8507 Japan Search for more papers by this author Yoshinori Akiyama Corresponding Author Yoshinori Akiyama Institute for Virus Research, Kyoto University, Kyoto, 606-8507 Japan Search for more papers by this author Kazue Kanehara Kazue Kanehara Institute for Virus Research, Kyoto University, Kyoto, 606-8507 Japan Search for more papers by this author Koreaki Ito Koreaki Ito Institute for Virus Research, Kyoto University, Kyoto, 606-8507 Japan Search for more papers by this author Yoshinori Akiyama Corresponding Author Yoshinori Akiyama Institute for Virus Research, Kyoto University, Kyoto, 606-8507 Japan Search for more papers by this author Author Information Kazue Kanehara1, Koreaki Ito1 and Yoshinori Akiyama 1 1Institute for Virus Research, Kyoto University, Kyoto, 606-8507 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6389-6398https://doi.org/10.1093/emboj/cdg602 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info σE is an alternative sigma factor involved in a pathway of extracytoplasmic stress responses in Escherichia coli. Under normal growth conditions, σE activity is down-regulated by the membrane-bound anti-σE protein, RseA. Extracytoplasmic stress signals induce degradation of RseA by two successive proteolytic events: DegS-catalyzed first cleavage at a periplasmic site followed by YaeL-mediated second proteolysis at an intramembrane region. Normally, the second reaction (site-2 proteolysis) only occurs after the first cleavage (site-1 cleavage). Here, we show that YaeL variants with the periplasmic PDZ domain deleted or mutated allows unregulated cleavage of RseA and consequent σE activation. It was also found that a glutamine-rich region in the periplasmic domain of RseA was required for the avoidance of the YaeL-mediated proteolysis in the absence of site-1 cleavage. These results indicate that multiple negative elements both in the enzyme (PDZ domain) and in the substrate (glutamine-rich region) determine the strict dependence of the site-2 proteolysis on the site-1 cleavage. Introduction Gene expression can be modulated in response to changes in extracytoplasmic environments. In this process, external signals are somehow transmitted across the membrane. Escherichia coli cells sense surface stresses such as accumulation of abnormal proteins in the periplasm and the outer membrane to induce expression of a set of genes encoding chaperones, foldases and proteases that function to cope with the stresses (Missiakas and Raina, 1998; Raivio and Silhavy, 1999, 2001). Two major stress-response pathways, Cpx and σE, are known in E.coli. The Cpx pathway utilizes the CpxA–CpxR two component phospho-relay mechanism whereas σE pathway utilizes this dedicated sigma factor specialized for the extracytoplasmic stress response. Under normal growth conditions, a majority of σE is sequestered by its interaction with the N-terminal cytoplasmic domain of the membrane-bound anti-σE factor, RseA (De Las Peñas et al., 1997; Missiakas et al., 1997). A periplasmic protein, RseB, may also participate in the negative regulation of the σE activity through its interaction with the periplasmic domain of RseA (De Las Peñas et al., 1997; Missiakas et al., 1997). Structural studies suggested that the cytoplasmic domain of RseA and RNA polymerase competitively bind to σE (Campbell et al., 2003). RseA is a type I membrane protein having a transmembrane segment in the middle of the molecule (Missiakas et al., 1997). Exposure of the cell to an extracytoplasmic stress results in rapid degradation of RseA, liberation of σE and expression of the stress-inducible genes (Ades et al., 1999). It has been shown that two membrane proteases, DegS and YaeL, are involved in this stress-induced degradation of RseA (Alba et al., 2002; Kanehara et al., 2002). DegS is a member of the DegP protease family (Waller and Sauer, 1996) and has an N-terminal transmembrane segment as well as a periplasmic region composed of a serine-protease domain and an evolutionarily conserved 'PDZ' domain generally thought to be involved in protein–protein interaction (Alba et al., 2001). In response to stress signals, DegS cleaves off a substantial portion of the periplasmic domain of RseA. The transient degradation product of RseA, referred to as RseAΔP in this paper, then receives the second proteolysis by YaeL (Alba et al., 2002; Kanehara et al., 2002). YaeL spans the membrane four times with its central periplasmic domain also containing a PDZ-like sequence (Kanehara et al., 2001). It is an E.coli homolog of S2P protease that participates in proteolytic activation of SREBP (sterol regulatory element binding protein) and ATF6 in mammalian cells (Brown et al., 2000; Weihofen and Martoglio, 2003). Cleavage of these substrates by S2P is also preceded by a cleavage by another membrane protease, S1P (Sakai et al., 1998; Ye et al., 2000), although there is no apparent sequence similarity between S1P and DegS. Presumably, S2P cleaves the substrate protein within the transmembrane segment (Duncan et al., 1998), and so does YaeL. Indeed, the metalloprotease active site motif (HExxH) exists close to or within a transmembrane segment in both S2P and YaeL (Brown et al., 2000; Kanehara et al., 2001). Recent studies have shown that regulation of membrane protein functions through proteolysis within the membrane, or RIP (regulated intramembrane proteolysis), plays critical roles in diverse cellular processes (Brown et al., 2000; Weihofen and Martoglio, 2003). It is likely that the cytoplasmic domain of RseA is liberated as a result of YaeL-dependent proteolysis of RseAΔP. On the other hand, in vivo and in vitro studies showed that the RseA cytoplasmic domain retains an ability to bind to and inactivate σE (De Las Peñas et al., 1997; Missiakas et al., 1997). Thus, it may further be degraded by some other proteases, including ClpXP (Flynn et al., 2003). Genes degS and yaeL are both essential for viability (Alba et al., 2001, 2002; Kanehara et al., 2002), but they can be disrupted in the presence of increased σE activity due to its overproduction or to the absence of RseA (Alba et al., 2002; Kanehara et al., 2002). Thus, the essential function of DegS and YaeL is to provide cells with a sufficient amount of active σE. In the absence of DegS, the intact RseA molecule accumulates stably, indicating that YaeL cannot act against RseA without previous cleavage by DegS (Alba et al., 2001, 2002; Kanehara et al., 2002). In contrast, a C-terminally truncated RseA construct (RseA140) that mimics RseAΔP was degraded in a YaeL-dependent manner irrespective of the presence or absence of DegS (Kanehara et al., 2002). Thus, there might be some regulatory mechanism that prevents YaeL-dependent proteolysis of RseA prior to the first cleavage by DegS. This study was aimed at identifying elements required for this regulation. We investigated the role of the PDZ domain of YaeL and the periplasmic domain of RseA in regulated two-step proteolysis of RseA. Our results suggest that these enzyme and substrate domains have important roles in preventing a premature cleavage of RseA by YaeL. Results Loss of the PDZ domain function in YaeL results in unregulated cleavage of RseA The central periplasmic domain of YaeL contains a region homologous to the PDZ domain (Kanehara et al., 2001). PDZ domains are generally considered to be involved in protein–protein interactions (Harris and Lim, 2001), and it has recently been shown that the similar domain in DegS plays an important role in sensing extracytoplasmic stresses (Walsh et al., 2003). To examine a role of the YaeL PDZ domain in the YaeL function, we constructed a YaeL derivative with its periplasmic PDZ domain deleted (YaeLΔPDZ) (Figure 1A). Figure 1.YaeL PDZ region and complementation abilities of mutant forms of YaeL. (A) The amino acid sequence of the YaeL PDZ homologous region and the mutational alterations constructed in this study. The entire region of this sequence was deleted in YaeLΔPDZ. (B) Complementation abilities of the PDZ mutants of YaeL. Strain KK31 [yaeL::kan/pKK6 (Para-yaeL)] was transformed with plasmids encoding the indicated forms of YaeL-His6-Myc under the lac promoter control. They were pKK11 (YaeL+-His6-Myc), pTWV228 (vector), pKK131 (YaeLΔPDZ-His6-Myc), pKK135 [YaeLΔPDZ(D402N)-His6-Myc], pKK138 [YaeL(G214Q)-His6-Myc], pKK139 [YaeL(A234K/A235K)-His6-Myc], pKK140 [YaeL(G243Q)-His6-Myc], pKK136 [YaeL(D244K)-His6-Myc] and pKK141 [YaeL(I246Y)-His6-Myc]. Cultures in L-arabinose (0.2%) were diluted 103-fold with 0.9% NaCl solution and 4 μl portions (containing ∼4 × 103 cells) were spotted on an L agar plate containing 0.2% arabinose (Ara) or 1 mM IPTG. The plates were incubated at 37°C for 14 h. Download figure Download PowerPoint The essential role of YaeL is to provide cells with a sufficient amount of active σE through proteolytic inactivation of RseA (Alba et al., 2002; Kanehara et al., 2002). It was found that YaeLΔPDZ, expressed from a multicopy plasmid, supported the growth of the ΔyaeL strain (Figure 1B). Introduction of the D402N amino acid alteration, which had been shown to inactivate YaeL with respect to its ability to degrade RseA (Kanehara et al., 2002), into YaeLΔPDZ abolished the complementation activity. These results suggested that YaeLΔPDZ is functional and able to proteolytically inactivate RseA. To examine effects of YaeLΔPDZ on the RseA degradation, it was expressed together with HA-RseA (RseA with an N-terminally attached hemagglutinin epitope), which was detected by anti-HA immunoblotting (Figure 2). Cell fractionation experiments as described by Kanehara et al. (2001) for YaeL revealed that HA-RseA expressed from a plasmid was membrane-bound (data not shown). Additionally, we confirmed that HA-RseA had a cytosolic N-terminus and a periplasmic C-terminus (Y.Akiyama, unpublished data). Figure 2.Effects of YaeL PDZ mutations on degradation of RseAΔP and RseA. (A) RseA accumulation in the presence of DegS. Strain KK211 (ΔyaeL ΔrseA)/pSTD691 (HA-RseA) was further transformed with pTWV228 (vector, lane 1); pKK11 (YaeL+-His6-Myc, lane 2); pKK131 (YaeLΔPDZ-His6-Myc, lane 3); pKK135 [YaeLΔPDZ(D402N)-His6-Myc, lane 4], pKK138 [YaeL(G214Q)-His6-Myc, lane 5]; pKK139 [YaeL(A234K/A235K)-His6-Myc, lane 6]; pKK140 [YaeL(G243Q)-His6-Myc, lane 7]; pKK136 [YaeL(D244K)-His6-Myc, lane 8] and pKK141 [YaeL(I246Y)His6-Myc, lane 9]. Cells were precultured in L glucose (0.4%) medium, inoculated into M9 medium supplemented with 20 amino acids, 2 μg/ml thiamine, 0.4% glucose and 1 mM IPTG, and grown at 30°C for 3.5 h. Whole cellular proteins were subjected to SDS–PAGE and anti-HA (upper panels) and anti-Myc (lower panels) immunoblotting. (B) RseA accumulation in the absence of DegS. Strain AD1840 (ΔdegS ΔyaeL ΔrseA)/pSTD691 was further transformed with the same set of plasmids and processed as described in (A). Asterisk indicates a C-terminally cleaved product of HA-RseA that was produced by the action of some unknown periplasmic proteases and this truncated product is susceptible to active YaeL. (C) Stability of RseA in the absence of DegS. Strain AD1840/pSTD691 was further transformed with pTWV228, pKK11, pKK131 or pKK135. Cells were grown in M9 medium supplemented with 18 amino acids (other than Met and Cys), 2 μg/ml thiamine and 0.4% glucose at 30°C, and induced with 1 mM IPTG and 1 mM cAMP for 2 h. Cells were then pulse-labeled with [35S]methionine for 1.5 min followed by chase with unlabeled methionine for the indicated periods. Samples were processed for anti-HA immunoprecipitation. Radioactivities associated with HA-RseA were determined after SDS–PAGE and phosphorimaging, and are reported as values relative to the 1 min chase radioactivity for each culture set as 100%. Download figure Download PowerPoint RseA is first cleaved by DegS on the periplasmic side and then by YaeL within the membrane (Alba et al., 2002; Kanehara et al., 2002). Thus, the ΔyaeL cells accumulated RseAΔP, a DegS degradation product of RseA, in which most of the periplasmic domain had been degraded (Figure 2A, lane 1). Co-expression of wild-type YaeL significantly lowered the level of RseAΔP (lane 2) as observed previously (Kanehara et al., 2002). YaeLΔPDZ exerted a stronger effect; it lowered the RseAΔP abundance to a barely detectable level (lane 3). On the other hand, co-expression of YaeLΔPDZ(D402N) did not affect the level of RseAΔP (lane 4). These results show that deletion of the PDZ domain from YaeL did not compromise the YaeL function to proteolyze RseAΔP. Instead, the activity appeared to be enhanced. Expression of wild-type YaeL only insignificantly affected the accumulation level of intact RseA in the ΔdegS ΔyaeL strain (Figure 2B, lanes 1 and 2), confirming that the YaeL-dependent proteolysis of RseA requires its prior cleavage by DegS (Alba et al., 2002; Kanehara et al., 2002). Strikingly, the expression of YaeLΔPDZ markedly reduced the abundance of RseA (lane 3). YaeLΔPDZ(D402N) was not effective in this respect (lane 4), suggesting that the observed decrease in RseA upon YaeLΔPDZ expression was due to its proteolytic function. Pulse–chase experiments showed that RseA molecules newly synthesized in the absence of DegS were degraded rapidly in the presence of YaeLΔPDZ, but not in the presence of the wild-type YaeL protein (Figure 2C). These results indicate that YaeLΔPDZ, unlike the wild-type YaeL, can degrade the intact RseA protein. Consistent with YaeL and YaeLΔPDZ being a protease directly degrading RseA, we observed physical interactions between the presumed enzyme and substrate. YaeL and YaeLΔPDZ derivatives, each having a protease motif mutation and a C-terminally attached His6-Myc, were expressed together with HA-RseA. Then membranes were either treated with a cleavable crosslinker, dithio-bis(succinimidyl propinate) (DSP), or mock-treated, solubilized with N-dodecyl-β-D-maltoside and subjected to anti-HA immunoprecipitation. As shown in Figure 3 (lanes 1–4), both YaeL(H22F)-His6-Myc and YaeLΔPDZ(D402N)-His6-Myc were pulled down with HA-RseA. The crosslinking was not essential but it increased the efficiency of co-isolation significantly. In these experiments, we detected in vivo association between these proteins, because very little co-isolation was observed when they were present in different vesicles before detergent solubilization (Figure 3, lanes 9–12). The absence of the PDZ domain did not compromise the protein association. Rather, immunoprecipitation without crosslinking suggested that YaeLΔPDZ has an enhanced ability of RseA-binding (Figure 3, lane 4). These results indicate that YaeL as well as its ΔPDZ derivative interact directly with RseA. Figure 3.Crosslinking and co-immunoprecipitation of RseA and YaeL. For lanes 1–8, membrane vesicles carrying indicated combinations of YaeL(H22F)-His6-Myc (shown as PDZ+), YaeLΔPDZ(D402N)-His6-Myc (shown as ΔPDZ), HA-RseA (shown as WT) and HA-RseA165 (shown as 165), were treated with or without DSP. For lanes 9–16, YaeL and RseA were present in separate membrane vesicles (MV1 and MV2), which were mixed and DSP-treated or mock-treated. Membrane proteins were solubilized with 1% N-dodecyl-β-D-maltoside and subjected to immunoprecipitation using immobilized mouse-monoclonal anti-HA antibodies. Proteins recovered were solubilized in SDS, reduced with β-mercaptoethanol to cleave the crosslinkages, and analyzed by SDS–PAGE/immunoblotting using anti-Myc (upper panel) or anti-HA (lower panel) rabbit antibodies. Open arrow, closed arrow, open arrowhead and closed arrowhead indicate YaeL(H22F)-His6-Myc, YaeLΔPDZ(D402N)-His6-Myc, HA-RseA and HA-RseA165, respectively. Direct SDS–PAGE and immunoblotting (without the HA immunoprecipitaiton) demonstrated that all the samples used above contained similar amounts of the YaeL derivatives (not shown). Download figure Download PowerPoint To delineate the significance of the PDZ sequence further, we constructed YaeL variants with a single or multiple amino acid substitution(s), G214Q, A234K/A235K, G243Q, D244K and I246Y, in the PDZ domain (Figure 1A). The residues selected for mutagenesis are well-conserved among PDZ domains from various species (data not shown). All the mutant forms of the yaeL gene complemented the growth defect of the yaeL disruption strain (Figure 1B). Expression of any of these mutant proteins in the ΔyaeL strain greatly lowered the accumulation level of RseAΔP (Figure 2A, lanes 5–9). As observed with YaeLΔPDZ, these missense mutant proteins [other than YaeL(G243Q)] lowered the accumulation of intact RseA in the absence of DegS (Figure 2B, lanes 5–9); among them YaeL(G214Q) showed the strongest effect, comparable to that of YaeLΔPDZ. Taken together, it was suggested strongly that the PDZ domain of YaeL exerts a specific negative effect on the YaeL activity, preventing an uncontrolled cleavage of intact RseA without imposition of proper stress signals that activate DegS. PDZ domain of YaeL is required for the down-regulation of σE activity To substantiate that the PDZ mutant forms of YaeL possess unregulated protease activity, wild-type YaeL, YaeLΔPDZ, YaeLΔPDZ(D402N) or YaeL(G214Q) was expressed in a yaeL+ strain that carried a rpoHP3-lacZ reporter for the σE-directed transcription (Figure 4). YaeLΔPDZ and YaeL(G214Q) indeed elevated the LacZ activities (up to 2.5-fold), while wild-type YaeL and YaeLΔPDZ(D402N) did not. The other PDZ missense mutants other than YaeL(G243Q) also significantly elevated the LacZ activities (data not shown). Thus, the PDZ-defective YaeL variants can dominantly activate the σE pathway gene expression. Presumably, this was brought about by active and DegS-independent proteolysis of RseA. The role of the intact PDZ domain is to prevent this unregulated activation of σE. Figure 4.Activation of σE upon expression of YaeL PDZ variants. Plasmids pKK11 (YaeL+-His6-Myc), pTWV228 (vector), pKK131(YaeLΔPDZ-His6-Myc), pKK135 [YaeLΔPDZ(D402N)-His6-Myc] and pKK138 [YaeL(G214Q)-His6-Myc] were introduced into TR71 [yaeL+ λ(rpoHP3-lacZ)]. Cells were grown in L medium containing 5 mM cAMP at 30°C, during which samples were withdrawn and assayed for β-galactosidase (LacZ) activity. Results were plotted against the turbidity of culture at each point. Download figure Download PowerPoint Periplasmic domain of RseA is required for the regulated cleavage by YaeL The above results show that the PDZ domain of YaeL is crucial for the regulated two-step proteolysis of RseA. Since the regulation is also lost in RseA140 lacking most part of the periplasmic domain (C-terminal 74 residues) and mimicking RseAΔP (Kanehara et al., 2002), the periplasmic region of RseA must have some role in the regulation. To address the role played by the periplasmic domain of RseA, we first constructed two variants of RseA, RseA190 and RseA165, by deleting C-terminal 26 and 51 residues, respectively (Figure 5). These mutant forms of RseA as well as wild-type RseA were expressed from a plasmid in ΔrseA strains additionally carrying either the ΔdegS and/or the ΔyaeL mutation (Figure 6). RseA190 (lanes 5–8) and the full-length RseA (lanes 1–4) were similar in that they were stable in the ΔdegS strain but converted to the RseAΔP species in the degS+ ΔyaeL strain. In contrast, RseA165 (lanes 9–12) and RseA140 (lanes 13–16) were similar in that they only accumulated in the ΔyaeL strain, irrespective of the degS state. Figure 5.Schematic representations of mutant forms of RseA with C-terminal or internal deletions, amino acid substitutions and/or insertions. The horizontal lines indicate the portions in RseA carried on individual derivatives (the residue numbers in the authentic RseA protein are shown). The thick parts represent the transmembrane segment (TM). Vertical lines on the top line indicate the positions of glutamine residues. Amino acid sequences of region 1 (Q1) and region 2 (Q2) are shown. YaeL avoidance in the absence of DegS is expressed as the accumulation ratio (%) of each RseA derivative in AD1839 (ΔdegS yaeL+ ΔrseA) cells and in AD1840 (ΔdegS ΔyaeL ΔrseA) cells (see Figures 6 and 8). Download figure Download PowerPoint Figure 6.YaeL-avoidance is preserved in RseA190 but lost in RseA165. Plasmids pKK55 (HA-RseA, lanes 1–4), pSTD670 (HA-RseA190, lanes 5–8), pSTD671 (HA-RseA165, lanes 9–12) and pKK58 (HA-RseA140, lanes 13–16) were introduced into a ΔrseA strain (AD1811, lanes 1, 5, 9 and 13) and its derivatives carrying ΔyaeL (KK211, lanes 2, 6, 10 and 14), ΔdegS (AD1839, lanes 3, 7, 11 and 15) and the both mutations (AD1840, lanes 4, 8, 12 and 16). Plasmid-bearing cells were grown in L-broth containing 1 mM IPTG and 1 mM cAMP at 30°C for 3.5 h. Proteins were analyzed by SDS–PAGE and anti-HA immunoblotting. Arrowhead indicates RseAΔP. Download figure Download PowerPoint In other words, YaeL can degrade RseA165 lacking C-terminal 51 residues without prior action of DegS. This was demonstrated by pulse–chase experiments (Figure 7A). RseA165 was rapidly degraded in the ΔdegS yaeL+ strain, in sharp contrast to full-length RseA, which was stable in the same strain (lanes 1–3). To confirm that this degradation was YaeL-dependent, we expressed YaeL or its protease motif variant (FEXXH) together with RseA165 in either the degS+ ΔyaeL or the ΔdegS ΔyaeL strain (Figure 7B). Accumulation of RseA165 was abolished by co-expression of YaeL (lanes 7 and 10) but not by YaeL(FEXXH) (lanes 8 and 11). YaeL only slightly lowered the accumulation level of wild-type RseA in these strains (lanes 1 and 4), while it reduced the amount of RseAΔP to an almost undetectable level (lane 1). These results show that RseA165 is subject to YaeL-dependent proteolysis in the absence of DegS. Interestingly, RseA165 was converted to RseAΔP in the degS+ΔyaeL strain (Figure 6, lane 10), indicating that it is still susceptible to DegS. Thus, RseA165 acquired the sensitivity to YaeL, while retaining the sensitivity to DegS. It is suggested then that the periplasmic RseA segment of residues 166–190 contains some element required for the YaeL-avoidance. As expected, HA-RseA165 retained the ability to interact either with YaeL(H22F)-His6-Myc (Figure 3, lanes 5 and 6) or with YaeLΔPDZ(D402N)-His6-Myc (Figure 3, lanes 7 and 8). Our results so far indicate that direct interaction between the YaeL PDZ domain and the RseA periplasmic region, if any, is not strong enough to give a positive signal in two-hybrid analyses (data not shown). Figure 7.YaeL-dependent and DegS-independent degradation of RseA165. (A) Cells of AD1839 (ΔdegS ΔrseA, lanes 1–3) and AD1840 (ΔdegS ΔyaeL ΔrseA, lanes 4–6), each carrying pKK55 (HA-RseA+, upper panels) or pSTD671 (HA-RseA165, lower panels) were grown in M9 medium supplemented with 18 amino acids (other than Met and Cys), 2 μg/ml thiamine and 0.4% glucose at 30°C, and induced with 1 mM IPTG and 1 mM cAMP. Cells were then pulse-labeled with [35S]methionine for 1 min followed by chase with unlabeled methionine for the indicated periods. Samples were processed for anti-HA immunoprecipitation, SDS–PAGE and phosphorimaging. (B) Strains KK211 (ΔyaeL ΔrseA)/pKK55 (HA-RseA) (lanes 1–3), AD1840 (ΔdegS ΔyaeL ΔrseA)/pKK55 (lanes 4–6), KK211/pSTD671 (HA-RseA165) (lanes 7–9) and AD1840/pSTD671 (lanes 10–12) were transformed further with one of the following plasmids: pSTD630 (YaeL+-His6-Myc; WT) for lanes 1, 4, 7 and 10; pSTD631 [YaeL(H22F)-His6-Myc; M] for lanes 2, 5, 8 and 11 and pMPM-T3 (vector; V) for lanes 3, 6, 9 and 12. Plasmid-bearing cells were grown at 30°C in L-broth containing 1 mM IPTG and 1 mM cAMP. Total cellular proteins were subjected to SDS–PAGE and anti-HA immunoblotting. Download figure Download PowerPoint Glutamine-rich segments are involved in the YaeL-avoidance While the mode of YaeL–RseA interaction detected in our pull-down assay should have been related to the ability of the enzyme to degrade the substrate, these two proteins should also interact for the purpose of the negative regulation, in which the periplasmic region of RseA is involved. To further identify RseA element(s) that are required for the regulation, a series of C-terminal deletion mutants of RseA were constructed (Figure 5). They were expressed in four sets of strains: degS+ yaeL+, degS+ ΔyaeL, ΔdegS yaeL+ and ΔdegS ΔyaeL (Figure 8). YaeL-avoidance in the absence of DegS was assessed by comparing accumulation levels in the ΔdegS yaeL+ strain (lanes 3) and in the ΔdegS ΔyaeL strain (lanes 4). Lower accumulation in the former strain indicates the occurrence of DegS-independent and YaeL-dependent degradation. As shown in Figure 8, YaeL-avoidance was retained in RseA185 (Figure 8A) and RseA180 (Figure 8B) but was lost in RseA170 and the smaller derivatives (Figure 8C–G). Cleavage by DegS was observed for RseA155 (Figure 8E) and the larger derivatives (Figure 8A–D), consistent with the recent observation that DegS cleaves RseA between Val148 and Ser149 in vitro (Walsh et al., 2003). Thus, N-terminal 180 residues of RseA is sufficient for the YaeL-avoidance. Figure 8.YaeL-avoidance assays for various mutant forms of RseA. Cells carrying a plasmid encoding a HA-RseA derivative as indicated were analyzed for the RseA accumulation as described in the legend to Figure 6. The host strains used were AD1811 (ΔrseA, lanes 1); KK211 (ΔyaeL ΔrseA, lanes 2); AD1839 (ΔdegS ΔrseA, lanes 3) and AD1840 (ΔdegS ΔyaeL ΔrseA, lanes 4). Download figure Download PowerPoint However, our internal deletion analysis indicated that multiple regions can contribute to the YaeL-avoidance in intact RseA. An internal deletion of the residues 156 to 180 did not abolish the YaeL-avoidance (Figure 8H–K), while larger internal deletions, Δ(156–190) (Figure 8L) and Δ(156–200) (Figure 8M), abolished it. One possible explanation for the above results would be that RseA has two or more regions that can confer the YaeL-avoidance independently. One of such regions may be around residue 170 and the other around residue 190. Consistent with this notion, degradation of RseA190 (Figure 8N and O) and RseA185 (data not shown) became DegS-independent when an internal deletion, Δ(156–165) or Δ(156–170), was introduced into them. It is remarkable that the periplasmic domain of RseA has a high content of glutamine residues (22%) as compared with its cytoplasmic domain (8.3%) or with the overall E.coli proteins (4.4%). In particular, two regions, residue 162–169 designated here as region Q1 and residue 190–200 designated as region Q2 can be noted for their high glutamine content (6/8 and 6/11, respectively, see Figure 5). These regions Q1 and Q2 roughly overlap with the YaeL-avoidance elements suggested from our deletion mapping. Although deletion of either Q1 or Q2 alone only weakly affected the stability of RseA in the absence of DegS (Figure 8J and Q), deletion of both Q1 and Q2 in RseA (Figure 8R) as well as deletion of Q1 in RseA190 (Figure 8P) resulted in the YaeL-dependent destabilization. We then mutagenized Q1 and Q2 to replace their glutamine residues by alanines. The alanine substituted version of Q1 in RseA190 (Figure 8S) and those of Q1 and Q2 in RseA (Figure 8T) had greatly reduced ability to confer the YaeL-avoidance character. These results show that the presence of either Q1 or Q2 is required for the effective negative regulation of YaeL-dependent proteolysis in the absence of DegS. Finally, we inserted eight consecutive glutamine residues into the C-terminal part of RseA190Δ(156–170) (Figure 8U). The polyglutamine insertion was found to stabilize the protein significantly in the ΔdegS yaeL+ background, while it was still degraded by the coordinated actions of DegS and YaeL. As a control, we inserted eight consecutive serine residues into the same position (Figure 8V). The polyserine insertion did not affect the stability of RseA190Δ(156–170). We propose that polyglutamine in a C-terminal region can confer the YaeL-avoidance character to RseA. Its exact position may not be crucial. Discussion Transmembrane signal transduction from the periplasmic side to the cytosolic side should accompany the extracytoplasmic stress responses that occur in E.coli. In the σE pathway, two-step proteolysis of RseA is the key mechanism of this signaling. While the site-1 cleavage of RseA is catalyzed by DegS, the second proteolysis is dependent on YaeL with intact metalloprotease sequence motifs. In vivo studies suggest strongly that the latter reaction is catalyzed by YaeL (Alba et al., 2002; Kanehara et al., 2002). In support of this notion, we showed that YaeL and RseA interact with each other (Figure 3). Although biochemical verification is still required, these two proteins should represent an enzyme (YaeL) and a substrate (RseA). While YaeL-dependent cleavage of RseA is essential for activation of σE, YaeL never acts to cleave intact RseA in the absence of DegS. On the other hand, the degradation intermediate of RseA, RseAΔP, can never be detected under any growth conditions, unless YaeL has been impaired genetically (Alba et al., 2002; Kanehara et al., 2002). Thus, the rate-limiting step of RseA degradation should be the site-1 cleavage reaction by DegS. Once this happen