Characterization of the yaeL gene product and its S2P-protease motifs in Escherichia coli

Gene 281 (2001) 71–79 www.elsevier.com/locate/gene

Characterization of the yaeL gene product and its S2P-protease motifs in Escherichia coli Kazue Kanehara, Yoshinori Akiyama, Koreaki Ito* Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan Received 22 August 2001; received in revised form 29 October 2001; accepted 10 November 2001 Received by D.L. Court

Abstract An Escherichia coli open reading frame, yaeL, encodes a predicted homolog of human site-2 protease (S2P), a putative membrane-bound zinc metalloproteinase involved in the proteolytic activation of regulatory factors for sterol biosynthesis and for stress responses. The potential importance of YaeL in processes analogous to the regulated intramembrane proteolysis in E. coli prompted us to characterize it. Cell fractionation and alkaline phosphatase fusion experiments established that YaeL has four transmembrane segments with both termini orienting toward the periplasm. A strain in which a chromosomal disruption of yaeL was combined with arabinose promoter-controlled yaeL on a plasmid enabled us to deplete this protein from the cell. The depletion was found to cause rapid loss of viability, cell elongation and growth cessation. Mutations affecting the HEXXH metalloproteinase motif and those affecting the LDG motif, conserved among S2Ps, abolished the ability of YaeL to support cell growth. These results indicate that YaeL is indispensable in E. coli, and probably functions as a metalloproteinase at the membrane. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Membrane-bound protease; Zinc metalloproteinase; Cell division; Regulated intramembrane proteolysis

1. Introduction Protein constituents of biomembranes play fundamental roles in the life of the cell, and the processes of their biogenesis are actively investigated. It is also important to understand how their functions are regulated and maintained. Recent studies show that proteolytic reactions play important roles to quality-control biochemical reaction systems as well as to induce unidirectional changes in their functionality (Mayer, 2000). However, not much is known about proteolytic regulatory events that involve membrane-integrated proteins. In Escherichia coli, the FtsH protease is known to degrade some membrane proteins in their unassembled subunit states (Kihara et al., 1999; Akiyama and Ito, 2000; Chiba et al., 2000; Akiyama and Ito, 2001). Brown et al. (2000) postulated a concept of regulated intramembrane proteolysis, in which proteolytic cleavage within or near the membrane integrated region of a membrane protein leads to the liberation of a distinct

Abbreviations: kan, kanamycin resistance determinant; PhoA, alkaline phosphatase; S2P, site-2 protease; PAGE, polyacrylamide gel electrophoresis * Corresponding author. Tel.: 181-75-751-4015; fax: 181-75-771-5699. E-mail address: [email protected] (K. Ito).

product with a biological activity. This class of reactions is thought to be catalyzed by membrane-integrated metalloproteinases. The human site-2 protease (S2P) is one of them and involved in activation of sterol regulatory element-binding protein for the expression of proteins involved in the lipid biosynthesis (Rawson et al., 1997). It is also involved in the proteolytic activation of a transcription factor ATF6 in response to the endoplasmic reticulum stresses (Ye et al., 2000). An S2P-like protein, SpoIVFB, has been described in a prokaryotic species, Bacillus subtilis. It is involved in sporulation of this bacterium (Rudner et al., 1999). Although the proteolytic activities have not been demonstrated directly, the putative S2P-like proteases are characterized by multiple transmembrane segments, by the presence of an HEXXH zinc-binding motif near the N-terminal transmembrane segment, and by a LDG motif adjacent to a more C-terminally located transmembrane segment (Lewis and Thomas, 1999). Brown et al. (2000) pointed out that an open reading frame, yaeL (Accession Number AAC73287), of 450 codons and of unknown function in E. coli has the sequence features that fulfill it as a member of the S2P family. Homology searches using the Blast program indicate that YaeL homologs are conserved in a wide variety of bacteria, including E. coli, Yersinia pestis, Vibrio cholerae, Pasteurella multocida, and Haemophilus influen-

0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00823-X

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zae. The present study was undertaken to characterize this gene and its product in E. coli. The results obtained suggest that YaeL and its conserved HEXXH and LDG sequences play crucial roles in coordinating membrane-related cellular functions.

2. Materials and methods 2.1. Plasmids Plasmid pSTD477 carried yaeL that was disrupted by insertion of kan as well as the surrounding chromosomal regions. For its construction, a chromosomal 6.1 kb XbaIEcoRI fragment derived from l clone #120 of Kohara et al. (1987) was first cloned into pUC119. A 1.2 kb PstI fragment containing kan from pUC4K (Vieira and Messing, 1982) was blunted with T4 DNA polymerase and then inserted into blunt-ended BssHII site within yaeL. In the resulting plasmid, pSTD477, yaeL was disrupted at the 75th codon by kan of the opposite direction of transcription. pSTD479 carrying yaeL as the sole chromosomal gene was constructed by subcloning the 1.8 kb PstI-StuI yaeL fragment into PstI- and HincIIdigested pSTV29, a lac promoter vector based on pACYC184 (Takara Shuzo). pKK6, carrying yaeL under the ara promoter control, was constructed as follows; a yaeL fragment was amplified from pSTD479 using primers 5 0 -GGCTGCGGTACCGGTCTTTGCTTGC-3 0 (KpnI recognition sequence underlined) and 5 0 -CACGGAAGCTTCCTAACTAACTCTCATAAC-3 0 (HindIII recognition sequence underlined) and cloned into pBAD33 (Guzman et al., 1995) after digestions with these enzymes. pKK10, pKK11 and pKK14 were derivatives of pMW118 (pSC101-based lac promoter vector), pTWV228 (pBR322based lac promoter vector), and pBAD33 (pACYC184based ara promoter vector), respectively, and encoded YaeL-His6-Myc under the lac or the ara promoter control. They were constructed as follows. A yaeL fragment was amplified from pSTD479 using primers 5 0 -TAGCTGGATCCTTGATAGCCTGAGGTACCGG-3 0 (BamHI recognition sequence underlined) and 5 0 -CGGCCGAATTCTAACCGAGAGAAATCATTGA-3 0 (EcoRI recognition sequence underlined), and cloned into pTYE007 (Yoshihisa and Ito, 1996), after digestions with these enzymes. This resulted in in-frame continuation of the yaeL reading frame by a sequence for EFIEGRHHHHHHIDEEQKLISEEDLLRKR (linker region is italicized, hexahistidine region is underlined and Myc epitope region is boldfaced). The insert was then excised from the resulting plasmid as a 1.5 kb KpnI fragment, which was then cloned into the respective vectors indicated above. pKK13 carried yaeL (1.4 Kb KpnI-HindIII fragment from pKK6) cloned into pTH18cs1 (Hashimoto-Gotoh et al., 2000), a pSC101-based lac promoter vector with a temperature-sensitive replication mutation. Plasmids encoding, under the lac promoter control,

YaeL-PhoA-His6-Myc sandwich fusion proteins with different junction points were pKK19 (for YaeL-(C1)-PhoA-His6Myc), pKK20 (for YaeL-(P2)-PhoA-His6-Myc), pKK21 (for YaeL-(C2)-PhoA-His6-Myc) and pKK22 (for YaeL(P3)-PhoA-His6-Myc). For their construction, pKK10 was first subjected to site directed mutagenesis (Sawano and Miyawaki, 2000), using mutagenic primers that were designed such that the NheI recognition sequence (GCTAGC) was inserted into one of the following positions: between A321 and A322 (for constructing the C1 domain fusion), between T1128 and G1129 (for P2), between T1278 and T1279 (for C2), and after A1350 (for P3) (nucleotide numbering according to Blattner et al., 1997). A 1.4 kb SpeI fragment derived from pKH385 (Kihara et al., 1999) was then cloned into each of the engineered NheI sites described above, resulting in in-frame insertions of the alkaline phosphatase mature sequence. Plasmids encoding YaeL-His6-Myc with an amino acid substitution, His22Phe, Glu23Gln, His26Phe or Asp402Asn, under the lac promoter control were, respectively, pKK28, pKK34, pKK29 and pKK35. For their construction, pKK14 was first subjected to site-directed mutagenesis (Sawano and Miyawaki, 2000), using the following respective primers: 5 0 CTTATCACCGTGTTTGAATTTGGTCAT-3 0 , 5 0 -ATCACCGTGCATCAATTTGGTCATTTC-3 0 , 5 0 -CATGAATTTGGTTTCTTCTGGGTTGCC-3 0 , and 5 0 -TTGCCCGTACTTAACGGGGGGCATCTG-3 0 . A 1.5 kb KpnI fragment was then prepared from each of the constructions and cloned into pTWV228. Plasmid pKK36, a pKK10 derivative encoding YaeL-His6-Myc with insertion of 31 amino acids (TDSYTQVASWTEPFPFSIQGDPRSDQETAFV) between Val261 and Met262 was constructed as described by Manoil and Bailey (1997). All the DNA segments that experienced in vitro replication reactions were confirmed by sequencing. 2.2. Bacterial strains E. coli K-12 strains used were derived either from AD16 (Dpro-lac thi/F 0 lacI q Z M15 Y 1 pro 1: Kihara et al., 1995) or W3110 (wild-type laboratory strain; Sambrook et al., 1989). Strains that allow conditional depletion of YaeL were constructed as follows. Plasmid pSTD479 (yaeL) was first introduced into strain FS1576 (recD; Stahl et al., 1986). The resulting plasmid-bearing cells were transformed further with the 7.3 kb XbaI-EcoRI fragment (containing yaeL::kan), prepared from pSTD477, for kanamycin (25 mg/ml)resistance. The resulting strain, named KK1, was used as a donor in subsequent P1 transduction of the yaeL::kan maker into AD16 that carried pSTD479, yielding strain KK9. Polymerase chain reactions confirmed that KK9 had the chromosomal yaeL::kan alteration, which was then P1 transduced into a series of strains carrying a plasmid with the ara promoter-controlled yaeL or yaeL-his6-myc. Finally, D(srl-recA)306::Tn10 (Vieira and Messing, 1982) was P1 transduced into all the complemented strains to minimize homologous recombination. The final two stages of strain

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construction were carried out in the presence of 0.2% arabinose. The final strains were named as follows: KK31, AD16 yaeL::kan D(srl-recA)306::Tn10/pKK6 (Para-yaeL); KK194, AD16 yaeL::kan D(srl-recA)306::Tn10/pKK14 (Para-yaeL-his6-myc); KK100, W3110 yaeL::kan D(srlrecA)306::Tn10/pKK6 (Para-yaeL); KK193, W3110 yaeL::kan D(srl-recA)306::Tn10/pKK14 (Para-yaeL-his6myc). In some experiments, these strains were further transformed with compatible plasmids as specified in each experiment. 2.3. Media L medium (Davis et al., 1980) and M9 medium (Miller, 1972) were used. Ampicillin (50 mg/ml), chloramphenicol (20 mg/ml), kanamycin (25 mg/ml) and/or tetracycline (25 mg/ml) was added for selecting transformants and trasductants as well as for growing plasmid-bearing strains. 2.4. Pulse-labeling, immunoprecipitation and immunoblotting Pulse-labeling of cells with [ 35S]methionine were carried out essentially as described by Taura et al. (1993).Labeled proteins were immunoprecipitated using anti-PhoA serum (5 prime 3 prime, Inc) (Akiyama and Ito, 1989), separated by 10% SDS-PAGE and visualized by BAS1800 phosphor image analyzer. Immunoblotting with anti-Myc (A-14) (Santa Cruz Biotechnology, Inc) and anti-FtsH (Akiyama et al., 1994) was carried out as described previously (Shimoike et al., 1995). Antibody-decorated proteins were visualized using ECL detection kit (Amersham Pharamacia Biotech) and Fuji LAS1000 lumino-image analyzer. 2.5. Assay of alkaline phosphatase activity Cells of AD16 carrying either pKK19, pKK20, pKK21, pKK22 and pKK10 were grown at 378C in L medium containing 0.1% glucose to a mid-log phase, induced with 1 mM IPTG and 1 mM cAMP for 1 h, and subjected to alkaline phosphatase activities assays according to Michaelis et al. (1983). 2.6. Cell fractionation Cells of KK193 were grown in 100 ml of L medium with 0.02% arabinose at 378C for 3 h. Cells were fractionated into the periplasmic, cytoplasmic and membrane fraction after sucrose-lysozyme treatment followed by disruption of spheroplasts by sonication and differential centrifugations (Shimoike et al., 1995). Total membranes were layered on a step gradient of 33% and 48% sucrose-3 mM EDTA and centrifuged at 107,000 £ g for 16 h. The pellet (outer membrane fraction) was resuspended in 10 mM Tris–HCl (pH 8.1), 16% glycerol, 1 mM dithiothreitol. The cytoplasmic membranes at the interphase of the sucrose layers were

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recovered by centrifugation and resuspended in 10 mM Tris–HCl (pH 8.1), 16% glycerol, 1 mM dithiothreitol. 3. Results 3.1. Membrane localization and topology of YaeL The yaeL open reading frame consists of 450 codons (Blattner et al., 1997). The TopPreD II program (Claros and von Heijne, 1994) predicts that YaeL has four transmembrane segments (TM1–TM4), two cytoplasmic regions (C1 and C2) and three periplasmic regions (P1–P3), with both termini facing the periplasm (Fig. 1B). To facilitate identification and characterization of this protein, we constructed a plasmid encoding YaeL with C-terminally attached His6- and Myc-tag sequences (YaeL-His6-Myc). This protein was detected by anti-Myc immunoblotting as a band of apparent molecular mass of 53 kDa. Upon cell fractionation, it was recovered mainly from the cytoplasmic membrane fraction (Fig. 1A, upper panel, lane 3) but not from the cytosolic fraction (lane 1) or the periplasmic fraction (lane 2). Its partition between the cytoplasmic membrane fraction and the outer membrane fraction was similar to that of a known cytoplasmic membrane protein, FtsH (Fig. 1A, lower panel). Thus, YaeL is a cytoplasmic membrane protein. We examined whether the predicted topology of YaeL in the membrane (Fig. 1B) is correct by constructing and examining a series of PhoA sandwich fusion proteins. Earlier studies showed that the PhoA mature sequence can serve as a faithful topology reporter, especially in sandwich configurations and with insertion points at a C-terminal region of each domain (Ehrmann et al., 1990). The PhoA mature sequence was inserted in-frame into the carboxyl ends of C1, C2, and P2, as well as into the YaeL-His6 junction region (P3) of YaeL-His6-Myc (Fig. 1B). Two fusions, YaeL-(P2)-PhoA-His6-Myc and YaeL-(P3)-PhoA-His6Myc, gave high enzymatic activities of alkaline phosphatase, 4.7 and 14.4 units, respectively, whereas the others, YaeL-(C1)-PhoA-His6-Myc and YaeL-(C2)-PhoA-His6Myc, exhibited only 1.1 and 0.8 units of the enzyme activity, respectively. Pulse-labeling and immunoprecipitation experiments confirmed that the differential synthesis rates of these proteins did not differ significantly (Fig. 1C). These results suggest that YaeL indeed assumes the transmembrane configuration as shown in Fig. 1B, although there is no direct evidence for the periplasmic orientation of the Nterminal end. 3.2. YaeL is indispensable in E. coli We constructed strains, in which yaeL on the chromosome had been disrupted by the kanamycin resistance determinant (kan), while either YaeL or YaeL-His6-Myc was supplied from a plasmid under the arabinose promoter control. The yaeL::kan strain that carried the Para-yaeL 1 plasmid exhibited clear arabinose-dependence in growth on L-agar plates at

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different temperatures (Fig. 2). Essentially identical results were obtained with another yaeL::kan strain that carried the YaeL-His6-Myc-expressing plasmid (data not shown). Growth in liquid medium was followed using the latter strain

(Fig. 3A). Using the same culture, cellular abundance of YaeL-His6-Myc was monitored by SDS-PAGE and antiMyc immunoblotting (Fig. 3C). The abundance of YaeL decreased with a half life that indicates simple dilution during

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Fig. 2. Growth of YaeL-depleted strains on rich and minimum agar plates. Culture of KK100 in L-arabinose (0.02%) was serially diluted with 0.9% NaCl solution (10-fold dilutions from left to right) and 4 ml each was spotted on L (left) or M9-amino acids-glycerol (right) agar plates with (1) or without (2) 0.2% arabinose as indicated. The M9 medium was supplemented with all amino acids (20 mg/ml) and 0.4% glycerol. The L plates were incubated for 22 h (42, 37 and 308C) or 3 days (208C), the M9 plates were for 22 h (428C), 27 h (378C), 38 h (308C) or 6 days (208C).

the residual growth. A background protein of unknown identity (indicated by asterisk) remained unchanged throughout the experiment, serving as an internal control. Cells ceased to grow at about 200 min after the removal of arabinose, followed by limited cell lysis (Fig. 3A, filled circles). The viable cell number started to decline much earlier than the growth cessation; cells were already dying after as early as two generations (80 min) of growth in the absence of arabinose (Fig. 3B, filled circles). It was also found that a significant population of the cells elongated evidently after the growth in the absence of arabinose for 120 min or longer (Fig. 3D). These results indicate that YaeL is essential for growth and division of the E. coli cell, at least on the broth medium used in the above experiments. Bacterial growth was also examined on minimum-

glycerol agar plates with and without added arabinose (Fig. 2, right). While a clear arabinose-dependence was observed at 20 and 308C, the cells grew almost equally in the presence and the absence of arabinose at 37–428C. At 378C, removal of arabinose from minimum-glycerol-amino acids liquid medium did not cause growth cessation at least for several hours, although the abundance of YaeL-His6Myc decreased to undetectable levels (data not shown). These results raised a possibility that YaeL is dispensable at high temperature in minimum salt medium. To examine this possibility, we addressed whether or not the yaeL gene can be totally eliminated. We combined the chromosomal yaeL::kan mutation with a yaeL-carrying plasmid whose replication was temperature-sensitive (Hashimoto-Gotoh et al., 2000). The strain thus constructed at 308C proved to

Fig. 1. Membrane localization and topology of YaeL. (A) Cell fractionation. Cells of KK193 expressing YaeL-His6-Myc were grown in L medium with 0.02% arabinose and fractionated into cytosolic (cyto, lane 1), periplasmic (peri, lane 2), cytoplasmic membrane (IM, lane 3) and outer membrane (OM, lane 4) fractions. Each fraction was subjected to SDS-PAGE and immunoblotting for detection of YaeL-His6-Myc using anti-Myc serum (upper panel) and for detection of FtsH using anti-FtsH serum (lower panel). (B) Topology of YaeL in the cytoplasmic membrane. YaeL is shown by thick line followed by a linker sequence (thin line) and a His6-Myc tag (box). The H 22EXXH motif, the L 402DG motif and a PDZ-like domain (D 205 to Q 279) are highlighted. The positions of the PhoA fusion junctions as well as of the 31 codon insertion (Ins 31) are indicated by arrows. (C) Expression of the PhoA fusion proteins. AD16 cells carrying pKK19 (C1-PhoA, lane 1), pKK20 (P2-PhoA, lane 2), pKK21 (C2-PhoA, lane 3), pKK22 (P3-PhoA, lane 4) and pKK10 (without PhoA insertion, lane 5) were grown at 378C in M9 medium, induced with 1 mM IPTG and 5 mM cAMP for 10 min, and pulse-labeled with [ 35S]methionine for 1 min. Labeled proteins were immunoprecipitated with anti-PhoA and separated by 10% SDS-PAGE. The alkaline phosphatase activities of the fusions determined in separate experiments are shown at the bottom.

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be unable to grow at 428C both on the broth medium and on the minimum medium (in the absence of the antibiotic that marked the plasmid). These results indicate that the yaeL gene is indispensable for cell viability at any temperature both on the broth and the minimal media, although the requirement for YaeL seems to be decreased somehow in the latter medium.

3.3. The HEXXH zinc-metalloproteinase motif, the LDG motif conserved in S2P, and a PDZ-like domain are essential for the YaeL function YaeL contains a putative zinc binding motif H 22EXXH and a L 401DG sequence conserved among the site 2 membrane-bound proteases (Brown et al., 2000). They are adjacent to membrane-spanning regions (see Fig. 1B). In zinc metalloproteinases, two histidine residues of the HEXXH motif coordinate Zn 21, whereas the glutamic acid residue promotes the nucleophilic water attack on the carbonyl of the substrate peptide bond (Rawlings and Barrett, 1995). If YaeL functions as a metalloproteinase, mutational alterations of the histidine and the glutamic acid residues will affect its functionality. We constructed plasmids carrying the lac promoter-controlled yaeL-his6myc or its mutant forms with a mutation for either His22Phe, Glu23Gln or His26Phe amino acid substitution. These plasmids were introduced into the yaeL::kan strain harboring the resident plasmid with the ara promoter-controlled yaeL; the incoming and the resident plasmids were compatible with each other. Cells were first grown in the presence of arabinose and then examined for their growth in a pair of L-agar plates, one containing arabinose and the other containing isopropyl-b-d-thiogalactoside (IPTG). None of the His22Phe, Glu23Gln and His26Phe variants of YaeLHis6-Myc was able to support bacterial growth in the absence of arabinose (Fig. 4A). We also mutated Asp402 of the LDG motif to Asn. The Asp402Asn variant of YaeLHis6-Myc did not complement the YaeL depletion (Fig. 4A). We verified, by immunoblotting experiments, that all the mutant YaeL-His6-Myc proteins accumulated significantly in IPTG-induced cells of the above strains (Fig. 4B). Thus, these amino acid alterations did not destabilize the protein. We also constructed the same YaeL variants but without the C-terminal tags, and complementation results obtained with

Fig. 3. YaeL is indispensable in E. coli. (A) Cell growth. Cells of KK194 were grown to a mid-log phase in L medium with 0.02% arabinose at 378C. At time zero, cells were washed five times by repeated centrifugation/ suspension with arabinose-free L medium, and inoculated into L-arabinose medium (open triangles) or arabinose-free L medium (filled circles). During shaking at 378C turbidity was measured using a Klett-Summerson colorimeter (filter No. 54). Dilutions with prewarmed medium were introduced after points 1, 4 and 7. (B) Cell viability upon YaeL depletion. The numbers of colony forming cells were determined in separate experiments by plating cultures, after appropriate dilution, onto L-agar containing 0.2% arabinose. The viable cell counts were divided by cell particle numbers determined directly with a phase-contrast microscope using glass chambers of 5 £ 10 25 ml. The viability value of this strain was only 0.3–0.4 even in the presence of arabinose, whereas the wild type value was close to unity. (C) Cellular contents of YaeL-His6-Myc after arabinose removal. A portion of the culture without arabinose (A, filled circles) at each of the time points 1–7 was withdrawn for SDS-PAGE and immunoblotting with anti-Myc serum. Equal cell equivalents of samples were used. Asterisk indicates a non-specific background. (D) Cell elongation upon YaeL depletion. Cells at point 7 (A) were observed by a phase-contrast microscope.

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Fig. 4. Complementation abilities of mutant forms of YaeL. (A) Complementation assays. Strain KK31 (yaeL::kan/pKK6 (Para-yaeL)) was transformed further with plasmids encoding the indicated forms of YaeL-His6-Myc under the lac promoter control. They were pKK28 (H22F), pKK29 (H26F), pKK34 (E23Q), pKK35 (D402N), pKK11 (W.T.), pKK36 (Ins31) and pKK10 (W.T.). The respective empty vectors were pTWV228 and pMW118. Cultures in L-arabinose (0.02%) was serially diluted with 0.9% NaCl solution (10-fold dilutions from left to right) and 4 ml each was spotted on L agar plate containing 0.2% arabinose (Ara) or 1 mM IPTG (IPTG). The plates were incubated at 378C for 20 h. (B) Expression of the mutant proteins. Cells of AD16 carrying either pKK28 (H22F, lanes 1–2), pKK29 (H26F, lanes 3–4), pKK34 (E23Q, lanes 5–6), pKK35 (D402N, lanes 7–8), pKK11 (W.T., lanes 9–10), pKK36 (Ins31, lanes 11–12) or pKK10 (W.T., lanes 13–14) were grown in the presence (1) or absence (2) of 1 mM IPTG plus 5 mM cAMP for 2 h. Samples were subjected to anti-Myc immunoblotting.

them were essentially the same as those obtained with the tagged versions of the YaeL mutants (data not shown). These results indicate that the residues postulated to be involved in the putative catalytic function of YaeL are indeed essential for the functioning of this protein in the cell. We noticed that the large P2 domain of YaeL contains a segment, Asp 205 to Gln 279, with significant sequence similarity to the PDZ domain (Harrison, 1996). We constructed a mutant of YaeL-His6-Myc in which 31 residues of an unrelated sequence had been inserted between Val261 and Met262. This mutant protein (Fig. 4B, lane 11) failed to complement the YaeL depletion (Fig. 4A, see Ins31).

4. Discussion We have described basic characterization of the intracellular YaeL protein. Its localization in the cytoplasmic membrane was confirmed and its disposition in the membrane determined using the PhoA sandwich fusion approach. Results of these experiments indicate that YaeL is integrated into the cytoplasmic membrane with the topographical arrangements shown in Fig. 1B. Although the membrane integration of the N-terminal region has not been demonstrated directly, the N-terminal 21 residues are sufficiently hydrophobic to span the membrane.

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Using the strain in which the YaeL synthesis is controlled by the ara regulatory system, YaeL requirement was studied in terms of viability and growth of the cell. When the YaeL content decreased only about 4-fold, the cell viability started to decline. This was well before the mass increase cessation. Cell growth during this period was accompanied by cell elongation and the commitment to death. This was then followed by growth cessation and some evident cell lysis. These results suggest that YaeL plays a crucial role in the coordination of cell growth and cell division. The absence of its sufficient activity leads to fetal unbalance in cell physiology and/or in cellular compositions. During the course of this study, Dartigalongue et al. (2001) reported the work, in which they identified the promoter for yaeL (ecfE) as one of the targets of sE, an envelope stress-activated transcription factor. They also described that this gene is essential in E. coli. We have shown that the HEXXH motif and the LDG motif, which are adjacent, respectively, to the first and the third transmembrane segment, are required for the function of YaeL. These results are consistent with the notion that YaeL functions as a protease. In addition, the phenotypic consequence of the 31 amino acids insertion into the PDZlike domain suggests that the periplasmic domain, or its PDZ-like region in particular, is important for the YaeL function. The direct target of the YaeL action remains to be determined. We observed that protein distribution between the soluble and the membrane fractions became abnormal and that an abnormal form of OmpA was produced in the YaeLdepleted cells (Kanehara et al., unpublished results). Taken together with the direct or indirect involvement of YaeL in cell division (Fig. 3D), its function may be related to the organization of the E. coli cell surfaces. Furthermore, its synthesis is envelope stress-inducible (Dartigalongue et al., 2001). It is tempting to speculate that the PDZ-like domain interacts with some regulatory molecule in the periplasm or the outer membrane and transmits a signal to the cytosolic domain with the putative proteolytic activity. This putative protease could be a key coordinator of membranerelated cellular functions. Acknowledgements We thank H. Mori, S. Chiba, N. Saikawa and N. Shimohata for discussion, and M. Yamada, M. Sano and K. Mochizuki for technical assistance. This work was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan, and from CREST, Japan Science and Technology Corporation. References Akiyama, Y., Ito, K., 1989. Export of Escherichia coli alkaline phosphatase attached to an integral membrane protein, SecY. J. Biol. Chem. 264, 437–442.

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