Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Sep;179(17):5458–5464. doi: 10.1128/jb.179.17.5458-5464.1997

Mutation of a single MalK subunit severely impairs maltose transport activity in Escherichia coli.

A L Davidson 1, S Sharma 1
PMCID: PMC179417  PMID: 9287001

Abstract

The maltose transport system of Escherichia coli, a member of the ABC transport superfamily of proteins, consists of a periplasmic maltose binding protein and a membrane-associated translocation complex that contains two copies of the ATP-binding protein MalK. To examine the need for two nucleotide-binding domains in this transport complex, one of the two MalK subunits was inactivated by site-directed mutagenesis. Complexes with mutations in a single subunit were obtained by attaching a polyhistidine tag to the mutagenized version of MalK and by coexpressing both wild-type MalK and mutant (His)6MalK in the same cell. Hybrid complexes containing one mutant (His)6MalK subunit and one wild-type MalK subunit were separated from those containing two mutant (His)6MalK proteins based on differential affinities for a metal chelate column. Purified transport complexes were reconstituted into proteoliposome vesicles and assayed for maltose transport and ATPase activities. When a conserved lysine residue at position 42 that is involved in ATP binding was replaced with asparagine in both MalK subunits, maltose transport and ATPase activities were reduced to 1% of those of the wild type. When the mutation was present in only one of the two subunits, the complex had 6% of the wild-type activities. Replacement of a conserved histidine residue at position 192 in MalK with arginine generated similar results. It is clear from these results that two functional MalK proteins are required for transport activity and that the two nucleotide-binding domains do not function independently to catalyze transport.

Full Text

The Full Text of this article is available as a PDF (328.4 KB).

Selected References

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

  1. Anderson M. P., Berger H. A., Rich D. P., Gregory R. J., Smith A. E., Welsh M. J. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell. 1991 Nov 15;67(4):775–784. doi: 10.1016/0092-8674(91)90072-7. [DOI] [PubMed] [Google Scholar]
  2. Azzaria M., Schurr E., Gros P. Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance. Mol Cell Biol. 1989 Dec;9(12):5289–5297. doi: 10.1128/mcb.9.12.5289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berkower C., Michaelis S. Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily. EMBO J. 1991 Dec;10(12):3777–3785. doi: 10.1002/j.1460-2075.1991.tb04947.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bild G. S., Janson C. A., Boyer P. D. Subunit interaction during catalysis. ATP modulation of catalytic steps in the succinyl-CoA synthetase reaction. J Biol Chem. 1980 Sep 10;255(17):8109–8115. [PubMed] [Google Scholar]
  5. Boyer P. D. A perspective of the binding change mechanism for ATP synthesis. FASEB J. 1989 Aug;3(10):2164–2178. doi: 10.1096/fasebj.3.10.2526771. [DOI] [PubMed] [Google Scholar]
  6. Carson M. R., Travis S. M., Welsh M. J. The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity. J Biol Chem. 1995 Jan 27;270(4):1711–1717. doi: 10.1074/jbc.270.4.1711. [DOI] [PubMed] [Google Scholar]
  7. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cross R. L., Grubmeyer C., Penefsky H. S. Mechanism of ATP hydrolysis by beef heart mitochondrial ATPase. Rate enhancements resulting from cooperative interactions between multiple catalytic sites. J Biol Chem. 1982 Oct 25;257(20):12101–12105. [PubMed] [Google Scholar]
  9. Crowe J., Masone B. S., Ribbe J. One-step purification of recombinant proteins with the 6xHis tag and Ni-NTA resin. Mol Biotechnol. 1995 Dec;4(3):247–258. doi: 10.1007/BF02779018. [DOI] [PubMed] [Google Scholar]
  10. Davidson A. L., Laghaeian S. S., Mannering D. E. The maltose transport system of Escherichia coli displays positive cooperativity in ATP hydrolysis. J Biol Chem. 1996 Mar 1;271(9):4858–4863. [PubMed] [Google Scholar]
  11. Davidson A. L., Nikaido H. Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli. J Biol Chem. 1990 Mar 15;265(8):4254–4260. [PubMed] [Google Scholar]
  12. Davidson A. L., Nikaido H. Purification and characterization of the membrane-associated components of the maltose transport system from Escherichia coli. J Biol Chem. 1991 May 15;266(14):8946–8951. [PubMed] [Google Scholar]
  13. Davidson A. L., Shuman H. A., Nikaido H. Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2360–2364. doi: 10.1073/pnas.89.6.2360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dean D. A., Hor L. I., Shuman H. A., Nikaido H. Interaction between maltose-binding protein and the membrane-associated maltose transporter complex in Escherichia coli. Mol Microbiol. 1992 Aug;6(15):2033–2040. doi: 10.1111/j.1365-2958.1992.tb01376.x. [DOI] [PubMed] [Google Scholar]
  15. Gibson A. L., Wagner L. M., Collins F. S., Oxender D. L. A bacterial system for investigating transport effects of cystic fibrosis--associated mutations. Science. 1991 Oct 4;254(5028):109–111. doi: 10.1126/science.1718037. [DOI] [PubMed] [Google Scholar]
  16. Graña D., Gardella T., Susskind M. M. The effects of mutations in the ant promoter of phage P22 depend on context. Genetics. 1988 Oct;120(2):319–327. doi: 10.1093/genetics/120.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heukeshoven J., Dernick R. Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis. 1988 Jan;9(1):28–32. doi: 10.1002/elps.1150090106. [DOI] [PubMed] [Google Scholar]
  18. Higgins C. F., Hyde S. C., Mimmack M. M., Gileadi U., Gill D. R., Gallagher M. P. Binding protein-dependent transport systems. J Bioenerg Biomembr. 1990 Aug;22(4):571–592. doi: 10.1007/BF00762962. [DOI] [PubMed] [Google Scholar]
  19. Hogue B. G., Nayak D. P. Deletion mutation in the signal anchor domain activates cleavage of the influenza virus neuraminidase, a type II transmembrane protein. J Gen Virol. 1994 May;75(Pt 5):1015–1022. doi: 10.1099/0022-1317-75-5-1015. [DOI] [PubMed] [Google Scholar]
  20. Kayalar C., Rosing J., Boyer P. D. An alternating site sequence for oxidative phosphorylation suggested by measurement of substrate binding patterns and exchange reaction inhibitions. J Biol Chem. 1977 Apr 25;252(8):2486–2491. [PubMed] [Google Scholar]
  21. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kühnau S., Reyes M., Sievertsen A., Shuman H. A., Boos W. The activities of the Escherichia coli MalK protein in maltose transport, regulation, and inducer exclusion can be separated by mutations. J Bacteriol. 1991 Apr;173(7):2180–2186. doi: 10.1128/jb.173.7.2180-2186.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Loo T. W., Clarke D. M. Rapid purification of human P-glycoprotein mutants expressed transiently in HEK 293 cells by nickel-chelate chromatography and characterization of their drug-stimulated ATPase activities. J Biol Chem. 1995 Sep 15;270(37):21449–21452. doi: 10.1074/jbc.270.37.21449. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Panagiotidis C. H., Reyes M., Sievertsen A., Boos W., Shuman H. A. Characterization of the structural requirements for assembly and nucleotide binding of an ATP-binding cassette transporter. The maltose transport system of Escherichia coli. J Biol Chem. 1993 Nov 5;268(31):23685–23696. [PubMed] [Google Scholar]
  27. Reyes M., Shuman H. A. Overproduction of MalK protein prevents expression of the Escherichia coli mal regulon. J Bacteriol. 1988 Oct;170(10):4598–4602. doi: 10.1128/jb.170.10.4598-4602.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Russel M., Model P. Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J Bacteriol. 1984 Sep;159(3):1034–1039. doi: 10.1128/jb.159.3.1034-1039.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  30. Senior A. E., al-Shawi M. K., Urbatsch I. L. The catalytic cycle of P-glycoprotein. FEBS Lett. 1995 Dec 27;377(3):285–289. doi: 10.1016/0014-5793(95)01345-8. [DOI] [PubMed] [Google Scholar]
  31. Shyamala V., Baichwal V., Beall E., Ames G. F. Structure-function analysis of the histidine permease and comparison with cystic fibrosis mutations. J Biol Chem. 1991 Oct 5;266(28):18714–18719. [PubMed] [Google Scholar]
  32. 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]
  33. Traxler B., Beckwith J. Assembly of a hetero-oligomeric membrane protein complex. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10852–10856. doi: 10.1073/pnas.89.22.10852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Treptow N. A., Shuman H. A. Genetic evidence for substrate and periplasmic-binding-protein recognition by the MalF and MalG proteins, cytoplasmic membrane components of the Escherichia coli maltose transport system. J Bacteriol. 1985 Aug;163(2):654–660. doi: 10.1128/jb.163.2.654-660.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Walter C., Höner zu Bentrup K., Schneider E. Large scale purification, nucleotide binding properties, and ATPase activity of the MalK subunit of Salmonella typhimurium maltose transport complex. J Biol Chem. 1992 May 5;267(13):8863–8869. [PubMed] [Google Scholar]
  37. Walter C., Wilken S., Schneider E. Characterization of site-directed mutations in conserved domains of MalK, a bacterial member of the ATP-binding cassette (ABC) family [corrected]. FEBS Lett. 1992 May 25;303(1):41–44. doi: 10.1016/0014-5793(92)80473-t. [DOI] [PubMed] [Google Scholar]

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

RESOURCES