Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Sep;176(17):5459–5465. doi: 10.1128/jb.176.17.5459-5465.1994

Membrane topology of Escherichia coli diacylglycerol kinase.

R L Smith 1, J F O'Toole 1, M E Maguire 1, C R Sanders 2nd 1
PMCID: PMC196734  PMID: 8071224

Abstract

The topology of Escherichia coli diacylglycerol kinase (DAGK) within the cytoplasmic membrane was elucidated by a combined approach involving both multiple aligned sequence analysis and fusion protein experiments. Hydropathy plots of the five prokaryotic DAGK sequences available were uniform in their prediction of three transmembrane segments. The hydropathy predictions were experimentally tested genetically by fusing C-terminal deletion derivatives of DAGK to beta-lactamase and beta-galactosidase. Following expression, the enzymatic activities of the chimeric proteins were measured and used to determine the cellular location of the fusion junction. These studies confirmed the hydropathy predictions for DAGK with respect to the number and approximate sequence locations of the transmembrane segments. Further analysis of the aligned DAGK sequences detected probable alpha-helical N-terminal capping motifs and two amphipathic alpha-helices within the enzyme. The combined fusion and sequence data indicate that DAGK is a polytopic integral membrane protein with three transmembrane segments with the N terminus of the protein in the cytoplasm, the C terminus in the periplasmic space, and two amphipathic helices near the cytoplasmic surface.

Full text

PDF
5459

Images in this article

5462Image
on p.5462
5464Image
on p.5464

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Boyd D., Manoil C., Beckwith J. Determinants of membrane protein topology. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8525–8529. doi: 10.1073/pnas.84.23.8525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Broome-Smith J. K., Tadayyon M., Zhang Y. Beta-lactamase as a probe of membrane protein assembly and protein export. Mol Microbiol. 1990 Oct;4(10):1637–1644. doi: 10.1111/j.1365-2958.1990.tb00540.x. [DOI] [PubMed] [Google Scholar]
  4. Calamia J., Manoil C. lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4937–4941. doi: 10.1073/pnas.87.13.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cameron B., Blanche F., Rouyez M. C., Bisch D., Famechon A., Couder M., Cauchois L., Thibaut D., Debussche L., Crouzet J. Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5'-phosphate) synthase. J Bacteriol. 1991 Oct;173(19):6066–6073. doi: 10.1128/jb.173.19.6066-6073.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cornette J. L., Cease K. B., Margalit H., Spouge J. L., Berzofsky J. A., DeLisi C. Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins. J Mol Biol. 1987 Jun 5;195(3):659–685. doi: 10.1016/0022-2836(87)90189-6. [DOI] [PubMed] [Google Scholar]
  7. Eisenberg D., Weiss R. M., Terwilliger T. C. The hydrophobic moment detects periodicity in protein hydrophobicity. Proc Natl Acad Sci U S A. 1984 Jan;81(1):140–144. doi: 10.1073/pnas.81.1.140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gibson M. M., Bagga D. A., Miller C. G., Maguire M. E. Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system. Mol Microbiol. 1991 Nov;5(11):2753–2762. doi: 10.1111/j.1365-2958.1991.tb01984.x. [DOI] [PubMed] [Google Scholar]
  9. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  10. Harayama S., Bollinger J., Iino T., Hazelbauer G. L. Characterization of the mgl operon of Escherichia coli by transposon mutagenesis and molecular cloning. J Bacteriol. 1983 Jan;153(1):408–415. doi: 10.1128/jb.153.1.408-415.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harper E. T., Rose G. D. Helix stop signals in proteins and peptides: the capping box. Biochemistry. 1993 Aug 3;32(30):7605–7609. doi: 10.1021/bi00081a001. [DOI] [PubMed] [Google Scholar]
  12. Knighton D. R., Zheng J. H., Ten Eyck L. F., Ashford V. A., Xuong N. H., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. doi: 10.1126/science.1862342. [DOI] [PubMed] [Google Scholar]
  13. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lightner V. A., Bell R. M., Modrich P. The DNA sequences encoding plsB and dgk loci of Escherichia coli. J Biol Chem. 1983 Sep 25;258(18):10856–10861. [PubMed] [Google Scholar]
  16. Loomis C. R., Walsh J. P., Bell R. M. sn-1,2-Diacylglycerol kinase of Escherichia coli. Purification, reconstitution, and partial amino- and carboxyl-terminal analysis. J Biol Chem. 1985 Apr 10;260(7):4091–4097. [PubMed] [Google Scholar]
  17. Manoil C. Analysis of protein localization by use of gene fusions with complementary properties. J Bacteriol. 1990 Feb;172(2):1035–1042. doi: 10.1128/jb.172.2.1035-1042.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Manoil C., Boyd D., Beckwith J. Molecular genetic analysis of membrane protein topology. Trends Genet. 1988 Aug;4(8):223–226. doi: 10.1016/0168-9525(88)90154-0. [DOI] [PubMed] [Google Scholar]
  19. Miller K. J., McKinstry M. W., Hunt W. P., Nixon B. T. Identification of the diacylglycerol kinase structural gene of Rhizobium meliloti 1021. Mol Plant Microbe Interact. 1992 Sep-Oct;5(5):363–371. doi: 10.1094/mpmi-5-363. [DOI] [PubMed] [Google Scholar]
  20. Nilsson I., von Heijne G. Fine-tuning the topology of a polytopic membrane protein: role of positively and negatively charged amino acids. Cell. 1990 Sep 21;62(6):1135–1141. doi: 10.1016/0092-8674(90)90390-z. [DOI] [PubMed] [Google Scholar]
  21. Picot D., Loll P. J., Garavito R. M. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature. 1994 Jan 20;367(6460):243–249. doi: 10.1038/367243a0. [DOI] [PubMed] [Google Scholar]
  22. Sakane F., Yamada K., Kanoh H., Yokoyama C., Tanabe T. Porcine diacylglycerol kinase sequence has zinc finger and E-F hand motifs. Nature. 1990 Mar 22;344(6264):345–348. doi: 10.1038/344345a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Schaap D., de Widt J., van der Wal J., Vandekerckhove J., van Damme J., Gussow D., Ploegh H. L., van Blitterswijk W. J., van der Bend R. L. Purification, cDNA-cloning and expression of human diacylglycerol kinase. FEBS Lett. 1990 Nov 26;275(1-2):151–158. doi: 10.1016/0014-5793(90)81461-v. [DOI] [PubMed] [Google Scholar]
  25. Smith R. L., Banks J. L., Snavely M. D., Maguire M. E. Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coli. Identification of a new class of transport protein. J Biol Chem. 1993 Jul 5;268(19):14071–14080. [PubMed] [Google Scholar]
  26. Song B. H., Neuhard J. Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase. Mol Gen Genet. 1989 Apr;216(2-3):462–468. doi: 10.1007/BF00334391. [DOI] [PubMed] [Google Scholar]
  27. Tabor S., Richardson C. C. Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4076–4080. doi: 10.1073/pnas.86.11.4076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Traxler B., Lee C., Boyd D., Beckwith J. The dynamics of assembly of a cytoplasmic membrane protein in Escherichia coli. J Biol Chem. 1992 Mar 15;267(8):5339–5345. [PubMed] [Google Scholar]
  29. Walsh J. P., Bell R. M. Diacylglycerol kinase from Escherichia coli. Methods Enzymol. 1992;209:153–162. doi: 10.1016/0076-6879(92)09019-y. [DOI] [PubMed] [Google Scholar]
  30. Wu J., Tisa L. S., Rosen B. P. Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase. J Biol Chem. 1992 Jun 25;267(18):12570–12576. [PubMed] [Google Scholar]
  31. Yamashita Y., Takehara T., Kuramitsu H. K. Molecular characterization of a STreptococcus mutans mutant altered in environmental stress responses. J Bacteriol. 1993 Oct;175(19):6220–6228. doi: 10.1128/jb.175.19.6220-6228.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yun C. H., Van Doren S. R., Crofts A. R., Gennis R. B. The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides. J Biol Chem. 1991 Jun 15;266(17):10967–10973. [PubMed] [Google Scholar]
  33. von Heijne G. Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature. 1989 Oct 5;341(6241):456–458. doi: 10.1038/341456a0. [DOI] [PubMed] [Google Scholar]
  34. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES