Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1984 Apr;158(1):202–207. doi: 10.1128/jb.158.1.202-207.1984

Structural and functional properties of the folate transport protein from a methotrexate-resistant subline of Lactobacillus casei.

M Ananthanarayanan, J M Kojima, G B Henderson
PMCID: PMC215399  PMID: 6425261

Abstract

A methotrexate-resistant subline of Lactobacillus casei has been isolated which transports folate at a reduced rate and contains a binding protein whose affinity for folate (Kd = 280 nM) is considerably lower than that of the corresponding protein of wild-type cells (Kd = 0.6 nM). After the addition of mercaptoethanol, however, this same protein exhibits a high affinity for folate (Kd = 1.2 nM) and transports the substrate at a normal rate. Subsequent removal of mercaptoethanol causes a rapid reversal of the activation process. Binding protein labeled covalently with carbodiimide-activated [3H]folate, solubilized with Triton X-100, and subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate had an apparent molecular weight which was approximately twofold higher than that of the corresponding protein of wild-type cells, but it could be reduced to the parental size (Mr = 20,000) by prior treatment with mercaptoethanol. Purified binding protein also exhibited a similarly elevated molecular weight, and its amino acid composition was indistinguishable from that of the wild-type counterpart, except for the presence of a single cysteine residue. These findings indicate that the mutant binding protein exists in a low-affinity form due to disulfide bridge formation between two homologous protein subunits and that cleavage of this bond by mercaptoethanol generates the high-affinity state. The rapid and specific interconversion of these binding forms suggests further that the high-affinity form of the binding protein also resides in the membrane as a dimer, held together by noncovalent interactions.

Full text

PDF
202

Images in this article

206Image
on p.206
206Image
on p.206

Selected References

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

  1. Goldkorn T., Rimon G., Kaback H. R. Topology of the lac carrier protein in the membrane of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3322–3326. doi: 10.1073/pnas.80.11.3322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Henderson G. B., Huennekens F. M. Transport of folate compounds into Lactobacillus Casei. Arch Biochem Biophys. 1974 Oct;164(2):722–728. doi: 10.1016/0003-9861(74)90085-x. [DOI] [PubMed] [Google Scholar]
  3. Henderson G. B., Potuznik S. Cation-dependent binding of substrate to the folate transport protein of Lactobacillus casei. J Bacteriol. 1982 Jun;150(3):1098–1102. doi: 10.1128/jb.150.3.1098-1102.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Henderson G. B., Potuznik S. Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate. Arch Biochem Biophys. 1982 Jun;216(1):27–33. doi: 10.1016/0003-9861(82)90184-9. [DOI] [PubMed] [Google Scholar]
  5. Henderson G. B., Zevely E. M. Binding and transport of thiamine by Lactobacillus casei. J Bacteriol. 1978 Mar;133(3):1190–1196. doi: 10.1128/jb.133.3.1190-1196.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Henderson G. B., Zevely E. M., Huennekens F. M. Coupling of energy to folate transport in Lactobacillus casei. J Bacteriol. 1979 Aug;139(2):552–559. doi: 10.1128/jb.139.2.552-559.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Henderson G. B., Zevely E. M., Huennekens F. M. Folate transport in Lactobacillus casei: solubilization and general properties of the binding protein. Biochem Biophys Res Commun. 1976 Feb 9;68(3):712–717. doi: 10.1016/0006-291x(76)91203-1. [DOI] [PubMed] [Google Scholar]
  8. Henderson G. B., Zevely E. M., Huennekens F. M. Irreversible inactivation of the methotrexate transport system of L1210 cells by carbodiimide-activated substrates. J Biol Chem. 1980 May 25;255(10):4829–4833. [PubMed] [Google Scholar]
  9. Henderson G. B., Zevely E. M., Huennekens F. M. Mechanism of folate transport in Lactobacillus casei: evidence for a component shared with the thiamine and biotin transport systems. J Bacteriol. 1979 Mar;137(3):1308–1314. doi: 10.1128/jb.137.3.1308-1314.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Henderson G. B., Zevely E. M., Huennekens F. M. Purification and properties of a membrane-associated, folate-binding protein from Lactobacillus casei. J Biol Chem. 1977 Jun 10;252(11):3760–3765. [PubMed] [Google Scholar]
  11. Henderson G. B., Zevely E. M., Kadner R. J., Huennekens F. M. The folate and thiamine transport proteins of Lactobacillus casei. J Supramol Struct. 1977;6(2):239–247. doi: 10.1002/jss.400060209. [DOI] [PubMed] [Google Scholar]
  12. Huennekens F. M., Vitols K. S., Henderson G. B. Transport of folate compounds in bacterial and mammalian cells. Adv Enzymol Relat Areas Mol Biol. 1978;47:313–346. doi: 10.1002/9780470122921.ch5. [DOI] [PubMed] [Google Scholar]
  13. Klingenberg M. Membrane protein oligomeric structure and transport function. Nature. 1981 Apr 9;290(5806):449–454. doi: 10.1038/290449a0. [DOI] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. 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]
  16. Shane B., Stokstad E. L. Transport and metabolism of folates by bacteria. J Biol Chem. 1975 Mar 25;250(6):2243–2253. [PubMed] [Google Scholar]

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

RESOURCES