Membrane Topology of the Escherichia coli ToIR Protein - Journal of ...

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May 14, 1993 - Second, the accessibility of ToiR to proteinase K was determined in permeabilized cells and everted vesicles with an antibody elicited againstĀ ...
JOURNAL OF BACTERIOLOGY, Sept. 1993, p. 6059-6061 0021-9193/93/186059-03$02.00/0 Copyright X) 1993, American Society for Microbiology

Vol. 175, No. 18

Membrane Topology of the Escherichia coli ToIR Protein Required for Cell Envelope Integrity M. MICHELLE MULLER,1 ANNE VIANNEY,2 JEAN-CLAUDE LAZZARONI 2 ROBERT E. WEBSTER,* AND RAYMOND PORTALIER2 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 277101, and Laboratoire de Microbiologie et Genetique Moleculaire, UMR 106 Centre National de la Recherche Scientifique, Batiment 405, Universite Claude Bernard Lyon I F-69622, Villeurbanne Cedex, France2 Received 14 May 1993/Accepted 11 July 1993

ToiR is a 142-amino-acid protein required for the import of colicins and bacteriophage and for maintenance of cell envelope integrity. The topology of ToiR in the inner membrane was analyzed by two methods. First, bacteria expressing a series of TolR-13-galactosidase, ToiR-alkaline phosphatase, and TolR-1-lactamase fusions were assayed for the appropriate enzymatic activity. Second, the accessibility of ToiR to proteinase K was determined in permeabilized cells and everted vesicles with an antibody elicited against the carboxylterminal 70%6 of TotR. The results are consistent with TolR spanning the inner membrane once via residues 23 to 43 and with the carboxyl-terminal moiety being exposed to the periplasm. Quantitative studies with the anti-ToIR antibody indicated the presence of 2 x 103 to 3 x 103 TolR molecules per cell.

The cell envelope of gram-negative bacteria creates a formidable barrier to the uptake of macromolecules from the extracellular medium. The tolQRABpal gene cluster of Escherichia coli K-12 is utilized by specific macromolecules to gain entrance into the bacterial cell and is involved in the maintenance of cell envelope integrity. Strains carrying mutations in any one of these genes release periplasmic proteins into the extracellular medium and show an increased sensitivity to antibacterial agents (14, 15, 27). tolQ, tolR, and tolA mutants are insensitive to the effects of the group A colicins and to infection by the filamentous phages but allow these molecules or viral particles to recognize their specific receptors (2, 22, 25). tolB mutants are sensitive to these phages and to colicin El and insensitive to the other group A colicins. The cellular localization of the Tol and Pal proteins has been partially investigated. TolA, the best characterized Tol protein, is anchored in the inner membrane by a single amino-terminal membrane-spanning region (17, 18). The remaining portion of the protein resides in the periplasm, where it is predicted to interact with the peptidoglycan layer or the outer membrane (19) and possibly to interact directly with colicins A and El during their entry into the cell (1). The Pal lipoprotein is anchored to the outer membrane and associated with the peptidoglycan (16). TolQ and TolR are highly homologous to the ExbB and ExbD proteins of the Ton system, respectively (7), especially in their putative membrane-spanning regions. ExbB has been found to be an inner membrane protein with three membrane-spanning regions, and evidence has been found to show an analogous topology for TolQ (3, 13). ExbD has been found to be an inner membrane protein with an N-terminal membranespanning region and a large C-terminal periplasmic domain (12). The topology of TolR is not yet known, although preliminary evidence has shown it to be associated with the inner membrane (26). More information about the localization and the topology of the Tol and Pal proteins is required to elucidate their exact *

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role. In this paper, gene fusion analysis and protease accessibility experiments with anti-TolR antibodies as a probe were used to determine the topology of TolR in the inner membrane. Isolation and analysis of toIR-lacZ and tolR-phoA fusions. Plasmid pJC185, containing an EcoRI-BamHI insert carrying orfl-tolQRAB' (17, 26) cloned into pBR328, was subjected to TnphoA and TnlacZ in vivo mutagenesis (20, 21). Alternatively, in vitro tolR-phoA fusions were isolated after subcloning of tolR in plasmid pPHO7 (8) containing part of the phoA gene allowing in vitro generation of fusions. Unidirectional deletions were generated by digesting the open plasmid with exonuclease III. Each fusion was characterized after sequencing the junction region. 3-Galactosidase fusions to residues 16 and 19 of TolR were highly active. Cellular fractionation showed that this activity was present in the cytoplasm only. Many TolRPhoA active fusions were characterized in the residues 34 to 73 region of TolR at residues 34, 40, 43, 44, 45, 46, 48, 49, 52, 53, 54, and 73. No alkaline phosphatase fusions were obtained between residues 73 and 142 of TolR, although several out-of-framephoA insertions were characterized, indicating that the procedure used for generation of fusions was efficient in this region. Perhaps fusions with PhoA at this position in TolR are unable to dimerize and thus be active. Therefore, TolR-1-lactamase fusions were made at TolR residues 82 and 128 (5). Bacteria containing these fusions showed -lactamase activity, confirming that the carboxylterminal end of TolR is in the periplasm. The periplasmic, cytoplasmic, and membrane fractions were prepared (11, 24) and analyzed for the presence of active alkaline phosphatase. Bacteria containing active TolR-PhoA fusions had 240 to 320 U of alkaline phosphatase activity, where 1 U is the amount of activity required to hydrolyze 1 nmol ofp-nitrophenyl phosphate per min per mg of bacteria (dry weight). Approximately 80% of this activity was found in the cytoplasmic membrane. A Western blot analysis was performed to show directly that enzyme activity was essentially due to the stable TolR-PhoA hybrid proteins (Fig. 1). No band corresponding to mature alkaline phosphatase was detected in the presence of fusions to 6059

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FIG. 1. Immunoblot analysis of the TolR-PhoA hybrid proteins present in the inner membrane. Cell envelopes were fractionated into inner and outer membranes according to the method described by Schnaitman (24). The hybrid proteins were detected by Western blot analysis by using PhoA antibodies and 125I-protein A. Shown are TolR-PhoA fusion proteins with residues 34 (lane 2), 40 (lane 3), 49 (lane 4), 54 (lane 5), and 73 (lane 6). Lanes 1 and 7 correspond to the mature alkaline phosphatase of E. coli.

residues 34, 40, and 73 (Fig. 1, lanes 2, 3, and 6), while only faint bands were found in the presence of fusions to residues 49 and 54 of TolR (Fig. 1, lanes 4 and 5). These indicated that alkaline phosphatase hybrids were correctly secreted through the cytoplasmic membrane and dimerized in the periplasm while remaining attached to the inner membrane. Furthermore, with the exception of the TolR73-PhoA fusion, hybrid proteins migrated at the expected molecular weights. No fusions that contradicted the proposed topology were obtained. Protease accessibility of ToIR. Plasmid pMMM1 encodes TolR amino acids 45 to 142 along with 17 additional vectorencoded residues on the amino terminus (10). Two TolR fragments are expressed from pMMM1 in K17(DE3): the expected 115-residue peptide and a larger, 127-amino-acid fragment resulting from suppression of the amber codon after TolR codon 142. Amino-terminal sequence analysis of the two purified fragments confirmed their identification. Antibodies obtained to the TolR fragments detected a band near the predicted molecular weight of TolR in wildtype strains which was absent in TPS300, a mutant strain containing a chloramphenicol cassette in the ribosomebinding site of TolR (Fig. 2A, lanes 1 and 2). Further confirmation of the specificity of the TolR antibody can be found in the analysis of a fusion protein expressed from pJKG10, which contains regions of TolR, alkaline phosphatase, and filamentous phage gene I protein (10). This fusion protein migrated on sodium dodecyl sulfate-polyacrylamide gels as a protein with a molecular mass of 66 kDa and reacted with both anti-TolR and anti-alkaline phosphatase antibodies as determined by Western blot analysis. Comparing the amounts of anti-TolR reactive material in known concentrations of bacteria with various amounts of purified TolR fragment protein by Western blot analysis indicated the presence of 2 x 103 to 3 x 103 molecules per wild-type bacterium. The periplasmic, cytoplasmic, and inner and outer membrane fractions of wild-type bacteria were isolated as described by Guy-Caffey et al. (9). Detectable levels of TolR were observed by Western blot analysis only in the inner membrane-containing fractions (results not shown). However, the TolR fragments encoded by pMMM1 were present only in the cytoplasmic fraction, suggesting that the signal sequence for membrane insertion resides in the aminoterminal 44 amino acids of TolR. Protease accessibility experiments were performed to

TolR

FIG. 2. Protease accessibility studies. (A) Western blot analysis of bacterial TolR with antibody against the carboxyl-terminal 70% of TolR. GM1 wild-type bacteria (lane 1) and TPS300 toiR mutant bacteria (lane 2) were grown to a concentration of 2 x 108, and samples of equal size were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (12% polyacrylamide, 0.32% bis) and Western blot analysis with anti-TolR antibody. K17(DE3) bacteria (lanes 3 to 4) and K17(DE3)/pMMM1 bacteria (lanes 5 to 8) were grown to a concentration of 2 x 108, harvested, and permeabilized with lysozyme and EDTA in the presence of 20% sucrose. After incubation in the presence (lanes 4, 6, and 8) or absence (lanes 3, 5, and 7) of proteinase K for 1 h, the bacteria were subjected to sodium dodecyl sulfate-gel electrophoresis and Western blot analysis with anti-TolR antibody and 1 I-protein A. In the case of lanes 7 and 8, the cells were broken prior to the incubation in the presence or absence of proteinase K. (B) Western blot analysis of everted vesicles treated with proteinase K by using anti-TolR and anti-TolA antibodies. Vesicles were prepared as described in Chang et al. (6) and were incubated on ice for 30 min with proteinase K. Lanes: 1, untreated everted vesicles; 2, 3, and 4, everted vesicles treated with 20, 100, and 250 mg of proteinase K per ml, respectively; 5, everted vesicles treated with 100 mg of proteinase K per ml and 1% Triton X-100 to permeabilize the vesicles.

determine the topology of TolR in the inner membrane. K17(DE3) and K17(DE3)/pMMM1 bacteria were exposed to proteinase K after the outer membrane had been permeabilized or the bacteria had been completely lysed. The resulting material was analyzed for the presence of anti-TolRreactive material by Western blot analysis (Fig. 2A, lanes 3 to 8). Proteinase K completely digested the C-terminal 70% of wild-type TolR recognized by the antibody in both the permeabilized and the lysed cells (lanes 4, 6, and 8). However, the cytoplasmically localized TolR fragments expressed in K17(DE3)/pMMM1 could only be hydrolyzed by proteinase K in the lysed bacterial sample (Fig. 2A; compare lanes 6 and 8). As a control, protease accessibility experiments were performed on everted vesicles (6). Proteinase K treatment of these vesicles had no effect on the size of TolR or of TolA (Fig. 2B). The antibody to TolA recognizes only the region exposed to the periplasm, confirming the nature of the everted vesicles (17, 18). When the everted vesicles were treated with 1% Triton X-100 prior to protease addition, both TolR and TolA became susceptible to hydrolysis by the protease.

In conclusion, the cell fractionation and protease accessibility experiments identify TolR as an inner membrane protein which has 70% of its carboxyl-terminal amino acids exposed in the periplasm. This orientation is further confirmed by the observations that bacteria containing fusions of P-galactosidase to ToIR residues 16 and 19 are ,B-galacto-

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sidase positive while PhoA fusions at tolR codons 34 to 73 show alkaline phosphatase activity and TolR-bla fusions at tolR codons 82 and 128 show 3-lactamase activity. These data are most consistent with the hypothesis that TolR contains a single membrane-spanning region at residues 24 to 43 oriented with the carboxyl terminus in the periplasm. The observation that the 23 amino-terminal residues proposed to be exposed in the cytoplasm do not appear to be accessible to proteinase K in everted vesicles suggests some type of steric hindrance of the protease for cleavage of residues close to the membrane surface. This topology of TolR in the inner membrane is analogous to the topology for the ExbD protein (12). ExbD and ExbB serve as auxiliary proteins to TonB in the energy-coupled transport of ferric siderophores and vitamin B12 across the outer membrane of E. coli (4, 23). Sequence analysis has shown strong homologies between the ExbB and ExbD proteins and the TolQ and TolR proteins, respectively (7), especially in the hydrophobic regions. However, the homology is not great enough between the carboxyl-terminal 70% of ExbD and TolR to be detected by cross-reactivity with the anti-TolR antibody in TolR null mutants (Fig. 2A, lane 2). The exact role of the TolQRAB and Pal proteins in the physiology of E. coli is unclear. Given that TolA and now TolR have been localized to the inner membrane and that the Pal lipoprotein has been localized in the outer membrane, the possibility exists that this group of proteins may somehow form a structure that bridges the cell envelope or is otherwise involved in the transport of structural components of the outer membrane from the inner membrane or cytoplasm. Although these structures may consist of multimers of Tol and Pal proteins, the number of such structures would be small, since there are fewer than 1,000 molecules of TolA and 3,000 molecules of TolR per bacterium. We thank C. Manoil, C. Gutierrez, and B. Bachmann for providing us with phage and strains and J. Broome-Smith for the pJBS633 plasmid. We also thank Gerda Vergara for assistance with the figures and Wayne Beyer for sequencing. This work was supported by research funds from the Centre National de la Recherche Scientifique (UMR 106) and the Universite Claude Bernard. A.V. was supported by an MRT fellowship. This work was also supported by Public Health Service grant GM18305 from the National Institute of General Medical Sciences. REFERENCES 1. Benedetti, H., C. Lazdunski, and R. Uoubes. 1991. Protein import into Escherichia coli: colicins A and El interact with a component of their translocation system. EMBO J. 10:19891995. 2. Bernstein, A., B. Rolfe, and K. Onodera. 1972. Pleiotropic properties and genetic organization of the toU, B locus of Escherichia coli K-12. J. Bacteriol. 112:74-83. 3. Bourdineaud, J. P., S. P. Howard, and C. Lazdunski. 1989. Localization and assembly into the Eschenichia coli cell envelope of a protein required for entry of colicin A. J. Bacteriol. 171:2458-2465. 4. Braun, V., K. Gunter, and K. Onodera. 1991. Transport of iron across the outer membrane. Biol. Metals 4:14-24. 5. Broome-Smith, J. K., M. Tadayyon, and Y. Zhang. 1990. ,-Lactamase as a probe of membrane protein assembly and protein export. Mol. Microbiol. 4:1637-1644. 6. Chang, N. C., G. Blobel, and P. Model. 1978. Detection of prokaryotic signal peptidase in an Eschenichia coli membrane fraction: endoproteolytic cleavage of nascent fl pre-coat protein. J. Bacteriol. 75:361-365.

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