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. 1993 Jul;175(14):4485–4491. doi: 10.1128/jb.175.14.4485-4491.1993

Membrane topologies of the TolQ and TolR proteins of Escherichia coli: inactivation of TolQ by a missense mutation in the proposed first transmembrane segment.

K Kampfenkel 1, V Braun 1
PMCID: PMC204889  PMID: 8331075

Abstract

The TolQ and TolR proteins of Escherichia coli are required for the uptake of group A colicins and for infection by filamentous phages. Their topology in the cytoplasmic membrane was determined by cleavage with aminopeptidase K, proteinase K, and trypsin in spheroplasts and cell lysates. From the results obtained, it is proposed that the N terminus of TolQ is located in the periplasm and that it contains three transmembrane segments (residues 9 to 36, 127 to 159, and 162 to 191), a small periplasmic loop, and two large portions in the cytoplasm. The N terminus of TolR is located in the cytoplasm and is followed by a transmembrane segment (residues 21 to 40), and the remainder of the protein is located in the periplasm. A tolQ mutant, which rendered cells resistant to group A colicins and sensitive to cholate, had alanine 13 replaced by glycine and was lacking serine 14 in the first transmembrane segment. The membrane topologies of TolQ and TolR are similar to those proposed for ExbB and ExbD, respectively, which is consistent with the partial functional substitution between ExbB and TolQ and between ExbD and TolR. The amino acid sequences of these proteins display the highest homology in the transmembrane segments, which indicates that the membrane-spanning regions play an important role in the activities of the proteins.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benedetti H., Lazdunski C., Lloubès R. Protein import into Escherichia coli: colicins A and E1 interact with a component of their translocation system. EMBO J. 1991 Aug;10(8):1989–1995. doi: 10.1002/j.1460-2075.1991.tb07728.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bitter W., Tommassen J., Weisbeek P. J. Identification and characterization of the exbB, exbD and tonB genes of Pseudomonas putida WCS358: their involvement in ferric-pseudobactin transport. Mol Microbiol. 1993 Jan;7(1):117–130. doi: 10.1111/j.1365-2958.1993.tb01103.x. [DOI] [PubMed] [Google Scholar]
  3. Bourdineaud J. P., Howard S. P., Lazdunski C. Localization and assembly into the Escherichia coli envelope of a protein required for entry of colicin A. J Bacteriol. 1989 May;171(5):2458–2465. doi: 10.1128/jb.171.5.2458-2465.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Braun V., Günter K., Hantke K. Transport of iron across the outer membrane. Biol Met. 1991;4(1):14–22. doi: 10.1007/BF01135552. [DOI] [PubMed] [Google Scholar]
  5. Braun V., Herrmann C. Evolutionary relationship of uptake systems for biopolymers in Escherichia coli: cross-complementation between the TonB-ExbB-ExbD and the TolA-TolQ-TolR proteins. Mol Microbiol. 1993 Apr;8(2):261–268. doi: 10.1111/j.1365-2958.1993.tb01570.x. [DOI] [PubMed] [Google Scholar]
  6. Braun V. The structurally related exbB and tolQ genes are interchangeable in conferring tonB-dependent colicin, bacteriophage, and albomycin sensitivity. J Bacteriol. 1989 Nov;171(11):6387–6390. doi: 10.1128/jb.171.11.6387-6390.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eick-Helmerich K., Braun V. Import of biopolymers into Escherichia coli: nucleotide sequences of the exbB and exbD genes are homologous to those of the tolQ and tolR genes, respectively. J Bacteriol. 1989 Sep;171(9):5117–5126. doi: 10.1128/jb.171.9.5117-5126.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fischer E., Günter K., Braun V. Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB. J Bacteriol. 1989 Sep;171(9):5127–5134. doi: 10.1128/jb.171.9.5127-5134.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hancock R. W., Braun V. Nature of the energy requirement for the irreversible adsorption of bacteriophages T1 and phi80 to Escherichia coli. J Bacteriol. 1976 Feb;125(2):409–415. doi: 10.1128/jb.125.2.409-415.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hannavy K., Barr G. C., Dorman C. J., Adamson J., Mazengera L. R., Gallagher M. P., Evans J. S., Levine B. A., Trayer I. P., Higgins C. F. TonB protein of Salmonella typhimurium. A model for signal transduction between membranes. J Mol Biol. 1990 Dec 20;216(4):897–910. doi: 10.1016/S0022-2836(99)80009-6. [DOI] [PubMed] [Google Scholar]
  11. Kadner R. J. Vitamin B12 transport in Escherichia coli: energy coupling between membranes. Mol Microbiol. 1990 Dec;4(12):2027–2033. doi: 10.1111/j.1365-2958.1990.tb00562.x. [DOI] [PubMed] [Google Scholar]
  12. Kampfenkel K., Braun V. Membrane topology of the Escherichia coli ExbD protein. J Bacteriol. 1992 Aug;174(16):5485–5487. doi: 10.1128/jb.174.16.5485-5487.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kampfenkel K., Braun V. Topology of the ExbB protein in the cytoplasmic membrane of Escherichia coli. J Biol Chem. 1993 Mar 15;268(8):6050–6057. [PubMed] [Google Scholar]
  14. Kampfenkel K. Limited proteolysis of NADP-malate dehydrogenase from pea chloroplast by aminopeptidase K yields monomers. Evidence of proteolytic degradation of NADP-malate dehydrogenase during purification from pea. Biochim Biophys Acta. 1992 Dec 8;1156(1):71–77. doi: 10.1016/0304-4165(92)90098-f. [DOI] [PubMed] [Google Scholar]
  15. Levengood-Freyermuth S. K., Click E. M., Webster R. E. Role of the carboxyl-terminal domain of TolA in protein import and integrity of the outer membrane. J Bacteriol. 1993 Jan;175(1):222–228. doi: 10.1128/jb.175.1.222-228.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Levengood S. K., Beyer W. F., Jr, Webster R. E. TolA: a membrane protein involved in colicin uptake contains an extended helical region. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):5939–5943. doi: 10.1073/pnas.88.14.5939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
  18. Mohana Rao J. K., Argos P. A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta. 1986 Jan 30;869(2):197–214. doi: 10.1016/0167-4838(86)90295-5. [DOI] [PubMed] [Google Scholar]
  19. Park Y. M., Stauffer G. V. DNA sequence of the metC gene and its flanking regions from Salmonella typhimurium LT2 and homology with the corresponding sequence of Escherichia coli. Mol Gen Genet. 1989 Mar;216(1):164–169. doi: 10.1007/BF00332246. [DOI] [PubMed] [Google Scholar]
  20. Postle K. TonB and the gram-negative dilemma. Mol Microbiol. 1990 Dec;4(12):2019–2025. doi: 10.1111/j.1365-2958.1990.tb00561.x. [DOI] [PubMed] [Google Scholar]
  21. Roof S. K., Allard J. D., Bertrand K. P., Postle K. Analysis of Escherichia coli TonB membrane topology by use of PhoA fusions. J Bacteriol. 1991 Sep;173(17):5554–5557. doi: 10.1128/jb.173.17.5554-5557.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  24. Sun T. P., Webster R. E. Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli. J Bacteriol. 1987 Jun;169(6):2667–2674. doi: 10.1128/jb.169.6.2667-2674.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sun T. P., Webster R. E. fii, a bacterial locus required for filamentous phage infection and its relation to colicin-tolerant tolA and tolB. J Bacteriol. 1986 Jan;165(1):107–115. doi: 10.1128/jb.165.1.107-115.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Traub I., Gaisser S., Braun V. Activity domains of the TonB protein. Mol Microbiol. 1993 Apr;8(2):409–423. doi: 10.1111/j.1365-2958.1993.tb01584.x. [DOI] [PubMed] [Google Scholar]
  28. Webster R. E. The tol gene products and the import of macromolecules into Escherichia coli. Mol Microbiol. 1991 May;5(5):1005–1011. doi: 10.1111/j.1365-2958.1991.tb01873.x. [DOI] [PubMed] [Google Scholar]
  29. von Heijne G. Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol. 1992 May 20;225(2):487–494. doi: 10.1016/0022-2836(92)90934-c. [DOI] [PubMed] [Google Scholar]
  30. von Heijne G. The signal peptide. J Membr Biol. 1990 May;115(3):195–201. doi: 10.1007/BF01868635. [DOI] [PubMed] [Google Scholar]

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