Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Jan;176(1):44–49. doi: 10.1128/jb.176.1.44-49.1994

GTPase-dependent signaling in bacteria: characterization of a membrane-binding site for era in Escherichia coli.

Y P Lin 1, J D Sharer 1, P E March 1
PMCID: PMC205012  PMID: 8282709

Abstract

Era is an Escherichia coli GTPase that is essential for cell viability and is peripherally associated with the cytoplasmic membrane. Both immunoelectron microscopy and subcellular-fractionation experiments have shown that Era is present in cytoplasmic as well as membrane-associated pools. These data led to speculation that the mechanism of action of Era may require cycling between membrane and cytoplasmic sites. In order to investigate this possibility, an in vitro binding assay was developed to characterize the binding of Era to membrane fractions. Competition and saturation binding experiments suggest that a site that is specific for Era and capable of binding up to 5 ng of Era per microgram of membrane protein is present in membrane preparations. The binding curve is complex, indicating that multiple equilibria describe the interaction. The binding of Era to this putative receptor is dependent on guanine nucleotides; binding cannot be measured in the absence of nucleotide, and neither ATP nor UTP can substitute. Subfractionation of cell walls showed that the guanine nucleotide-dependent binding site was present in fractions enriched in cytoplasmic membrane. These data provide evidence that Era may be involved in a GTPase-receptor-coupled membrane-signaling pathway that is essential for growth in E. coli.

Full text

PDF
44

Images in this article

46Image
on p.46
47Image
on p.47

Selected References

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

  1. Ahnn J., March P. E., Takiff H. E., Inouye M. A GTP-binding protein of Escherichia coli has homology to yeast RAS proteins. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8849–8853. doi: 10.1073/pnas.83.23.8849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Albright C. F., Giddings B. W., Liu J., Vito M., Weinberg R. A. Characterization of a guanine nucleotide dissociation stimulator for a ras-related GTPase. EMBO J. 1993 Jan;12(1):339–347. doi: 10.1002/j.1460-2075.1993.tb05662.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  4. Beitel G. J., Clark S. G., Horvitz H. R. Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature. 1990 Dec 6;348(6301):503–509. doi: 10.1038/348503a0. [DOI] [PubMed] [Google Scholar]
  5. Birnbaumer L. Receptor-to-effector signaling through G proteins: roles for beta gamma dimers as well as alpha subunits. Cell. 1992 Dec 24;71(7):1069–1072. doi: 10.1016/s0092-8674(05)80056-x. [DOI] [PubMed] [Google Scholar]
  6. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  7. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  9. Chavrier P., Parton R. G., Hauri H. P., Simons K., Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990 Jul 27;62(2):317–329. doi: 10.1016/0092-8674(90)90369-p. [DOI] [PubMed] [Google Scholar]
  10. Chen S. M., Takiff H. E., Barber A. M., Dubois G. C., Bardwell J. C., Court D. L. Expression and characterization of RNase III and Era proteins. Products of the rnc operon of Escherichia coli. J Biol Chem. 1990 Feb 15;265(5):2888–2895. [PubMed] [Google Scholar]
  11. Diekmann D., Brill S., Garrett M. D., Totty N., Hsuan J., Monfries C., Hall C., Lim L., Hall A. Bcr encodes a GTPase-activating protein for p21rac. Nature. 1991 May 30;351(6325):400–402. doi: 10.1038/351400a0. [DOI] [PubMed] [Google Scholar]
  12. Donaldson J. G., Kahn R. A., Lippincott-Schwartz J., Klausner R. D. Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein. Science. 1991 Nov 22;254(5035):1197–1199. doi: 10.1126/science.1957170. [DOI] [PubMed] [Google Scholar]
  13. Emkey R., Freedman S., Feig L. A. Characterization of a GTPase-activating protein for the Ras-related Ral protein. J Biol Chem. 1991 May 25;266(15):9703–9706. [PubMed] [Google Scholar]
  14. Englard S., Seifter S. Precipitation techniques. Methods Enzymol. 1990;182:285–300. doi: 10.1016/0076-6879(90)82024-v. [DOI] [PubMed] [Google Scholar]
  15. Gollop N., March P. E. A GTP-binding protein (Era) has an essential role in growth rate and cell cycle control in Escherichia coli. J Bacteriol. 1991 Apr;173(7):2265–2270. doi: 10.1128/jb.173.7.2265-2270.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gollop N., March P. E. Localization of the membrane binding sites of Era in Escherichia coli. Res Microbiol. 1991 Feb-Apr;142(2-3):301–307. doi: 10.1016/0923-2508(91)90045-c. [DOI] [PubMed] [Google Scholar]
  17. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  18. Han M., Sternberg P. W. let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein. Cell. 1990 Nov 30;63(5):921–931. doi: 10.1016/0092-8674(90)90495-z. [DOI] [PubMed] [Google Scholar]
  19. Hart M. J., Shinjo K., Hall A., Evans T., Cerione R. A. Identification of the human platelet GTPase activating protein for the CDC42Hs protein. J Biol Chem. 1991 Nov 5;266(31):20840–20848. [PubMed] [Google Scholar]
  20. Inada T., Kawakami K., Chen S. M., Takiff H. E., Court D. L., Nakamura Y. Temperature-sensitive lethal mutant of era, a G protein in Escherichia coli. J Bacteriol. 1989 Sep;171(9):5017–5024. doi: 10.1128/jb.171.9.5017-5024.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones S., Vignais M. L., Broach J. R. The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras. Mol Cell Biol. 1991 May;11(5):2641–2646. doi: 10.1128/mcb.11.5.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Leonard D., Hart M. J., Platko J. V., Eva A., Henzel W., Evans T., Cerione R. A. The identification and characterization of a GDP-dissociation inhibitor (GDI) for the CDC42Hs protein. J Biol Chem. 1992 Nov 15;267(32):22860–22868. [PubMed] [Google Scholar]
  23. Lerner C. G., Inouye M. Pleiotropic changes resulting from depletion of Era, an essential GTP-binding protein in Escherichia coli. Mol Microbiol. 1991 Apr;5(4):951–957. doi: 10.1111/j.1365-2958.1991.tb00770.x. [DOI] [PubMed] [Google Scholar]
  24. Lochrie M. A., Simon M. I. G protein multiplicity in eukaryotic signal transduction systems. Biochemistry. 1988 Jul 12;27(14):4957–4965. doi: 10.1021/bi00414a001. [DOI] [PubMed] [Google Scholar]
  25. March P. E., Inouye M. Characterization of the lep operon of Escherichia coli. Identification of the promoter and the gene upstream of the signal peptidase I gene. J Biol Chem. 1985 Jun 25;260(12):7206–7213. [PubMed] [Google Scholar]
  26. March P. E., Lerner C. G., Ahnn J., Cui X., Inouye M. The Escherichia coli Ras-like protein (Era) has GTPase activity and is essential for cell growth. Oncogene. 1988 Jun;2(6):539–544. [PubMed] [Google Scholar]
  27. Mizuno T., Kaibuchi K., Yamamoto T., Kawamura M., Sakoda T., Fujioka H., Matsuura Y., Takai Y. A stimulatory GDP/GTP exchange protein for smg p21 is active on the post-translationally processed form of c-Ki-ras p21 and rhoA p21. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6442–6446. doi: 10.1073/pnas.88.15.6442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morii N., Kawano K., Sekine A., Yamada T., Narumiya S. Purification of GTPase-activating protein specific for the rho gene products. J Biol Chem. 1991 Apr 25;266(12):7646–7650. [PubMed] [Google Scholar]
  29. Rubinfeld B., Munemitsu S., Clark R., Conroy L., Watt K., Crosier W. J., McCormick F., Polakis P. Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1. Cell. 1991 Jun 14;65(6):1033–1042. doi: 10.1016/0092-8674(91)90555-d. [DOI] [PubMed] [Google Scholar]
  30. Salminen A., Novick P. J. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. doi: 10.1016/0092-8674(87)90455-7. [DOI] [PubMed] [Google Scholar]
  31. Schlessinger J. How receptor tyrosine kinases activate Ras. Trends Biochem Sci. 1993 Aug;18(8):273–275. doi: 10.1016/0968-0004(93)90031-h. [DOI] [PubMed] [Google Scholar]
  32. Schnaitman C. A. Protein composition of the cell wall and cytoplasmic membrane of Escherichia coli. J Bacteriol. 1970 Nov;104(2):890–901. doi: 10.1128/jb.104.2.890-901.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Segev N., Mulholland J., Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
  34. Serafini T., Orci L., Amherdt M., Brunner M., Kahn R. A., Rothman J. E. ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a novel role for a GTP-binding protein. Cell. 1991 Oct 18;67(2):239–253. doi: 10.1016/0092-8674(91)90176-y. [DOI] [PubMed] [Google Scholar]
  35. Simon M. A., Bowtell D. D., Dodson G. S., Laverty T. R., Rubin G. M. Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell. 1991 Nov 15;67(4):701–716. doi: 10.1016/0092-8674(91)90065-7. [DOI] [PubMed] [Google Scholar]
  36. Stock J. B., Surette M. G., McCleary W. R., Stock A. M. Signal transduction in bacterial chemotaxis. J Biol Chem. 1992 Oct 5;267(28):19753–19756. [PubMed] [Google Scholar]
  37. Strom M., Vollmer P., Tan T. J., Gallwitz D. A yeast GTPase-activating protein that interacts specifically with a member of the Ypt/Rab family. Nature. 1993 Feb 25;361(6414):736–739. doi: 10.1038/361736a0. [DOI] [PubMed] [Google Scholar]
  38. Takiff H. E., Chen S. M., Court D. L. Genetic analysis of the rnc operon of Escherichia coli. J Bacteriol. 1989 May;171(5):2581–2590. doi: 10.1128/jb.171.5.2581-2590.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  40. Ullrich O., Stenmark H., Alexandrov K., Huber L. A., Kaibuchi K., Sasaki T., Takai Y., Zerial M. Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins. J Biol Chem. 1993 Aug 25;268(24):18143–18150. [PubMed] [Google Scholar]

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

RESOURCES