Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Jun;80(11):3322–3326. doi: 10.1073/pnas.80.11.3322

Topology of the lac carrier protein in the membrane of Escherichia coli.

T Goldkorn, G Rimon, H R Kaback
PMCID: PMC394034  PMID: 6344081

Abstract

Proteolysis of topologically sealed right-side-out and inside-out membrane vesicles from Escherichia coli with chymotrypsin, trypsin, or papain inactivates lac carrier function in a symmetrical manner. Concomitantly, the electrophoretic mobility of lac carrier protein photoaffinity labeled in situ with p-nitro[2-3H]phenyl-alpha-D-galactopyranoside is altered from a relative Mr of 33,000 to 20,000, and the time course of proteolysis is almost identical in vesicles of opposite polarities. In contrast, solubilization of the vesicles in NaDodSO4 followed by proteolysis causes fragmentation of the Mr 33,000 band into material that electrophoreses at the solvent front. Notably, proteolysis has no effect whatsoever on the ability of the lac carrier protein to bind substrate, as judged by photoaffinity-labeling experiments. Furthermore, the electrophoretic patterns of samples proteolyzed prior to photoaffinity labeling are the same as those observed when the procedures are reversed. These results show that the lac carrier protein spans the membrane and indicate that the binding site resides within a segment that is embedded in the bilayer.

Full text

PDF
3322

Images in this article

3324Image
on p.3324
3324Image
on p.3324

Selected References

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

  1. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  2. Büchel D. E., Gronenborn B., Müller-Hill B. Sequence of the lactose permease gene. Nature. 1980 Feb 7;283(5747):541–545. doi: 10.1038/283541a0. [DOI] [PubMed] [Google Scholar]
  3. Carrasco N., Tahara S. M., Patel L., Goldkorn T., Kaback H. R. Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6894–6898. doi: 10.1073/pnas.79.22.6894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ehring R., Beyreuther K., Wright J. K., Overath P. In vitro and in vivo products of E. coli lactose permease gene are identical. Nature. 1980 Feb 7;283(5747):537–540. doi: 10.1038/283537a0. [DOI] [PubMed] [Google Scholar]
  5. Engelman D. M., Henderson R., McLachlan A. D., Wallace B. A. Path of the polypeptide in bacteriorhodopsin. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2023–2027. doi: 10.1073/pnas.77.4.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Foster D. L., Boublik M., Kaback H. R. Structure of the lac carrier protein of Escherichia coli. J Biol Chem. 1983 Jan 10;258(1):31–34. [PubMed] [Google Scholar]
  7. Foster D. L., Garcia M. L., Newman M. J., Patel L., Kaback H. R. Lactose-proton symport by purified lac carrier protein. Biochemistry. 1982 Oct 26;21(22):5634–5638. doi: 10.1021/bi00265a038. [DOI] [PubMed] [Google Scholar]
  8. Henderson R., Unwin P. N. Three-dimensional model of purple membrane obtained by electron microscopy. Nature. 1975 Sep 4;257(5521):28–32. doi: 10.1038/257028a0. [DOI] [PubMed] [Google Scholar]
  9. Hertzberg E. L., Hinkle P. C. Oxidative phosphorylation and proton translocation in membrane vesicles prepared from Escherichia coli. Biochem Biophys Res Commun. 1974 May 7;58(1):178–184. doi: 10.1016/0006-291x(74)90908-5. [DOI] [PubMed] [Google Scholar]
  10. Kaback H. R. Transport in isolated bacterial membrane vesicles. Methods Enzymol. 1974;31:698–709. doi: 10.1016/0076-6879(74)31075-0. [DOI] [PubMed] [Google Scholar]
  11. Kaback H. R. Transport studies in bacterial membrane vesicles. Science. 1974 Dec 6;186(4167):882–892. doi: 10.1126/science.186.4167.882. [DOI] [PubMed] [Google Scholar]
  12. Kaczorowski G. J., LeBlanc G., Kaback H. R. Specific labeling of the lac carrier protein in membrane vesicles of Escherichia coli by a photoaffinity reagent. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6319–6323. doi: 10.1073/pnas.77.11.6319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  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. Lancaster J. R., Jr, Hinkle P. C. Studies of the beta-galactoside transporter in inverted membrane vesicles of Escherichia coli. II. Symmetrical binding of a dansylgalactoside induced by an electrochemical proton gradient and by lactose efflux. J Biol Chem. 1977 Nov 10;252(21):7662–7666. [PubMed] [Google Scholar]
  16. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  17. Newman M. J., Foster D. L., Wilson T. H., Kaback H. R. Purification and reconstitution of functional lactose carrier from Escherichia coli. J Biol Chem. 1981 Nov 25;256(22):11804–11808. [PubMed] [Google Scholar]
  18. Newman M. J., Wilson T. H. Solubilization and reconstitution of the lactose transport system from Escherichia coli. J Biol Chem. 1980 Nov 25;255(22):10583–10586. [PubMed] [Google Scholar]
  19. Owen P., Kaback H. R. Antigenic architecture of membrane vesicles from Escherichia coli. Biochemistry. 1979 Apr 17;18(8):1422–1426. doi: 10.1021/bi00575a005. [DOI] [PubMed] [Google Scholar]
  20. Owen P., Kaback H. R. Immunochemical analysis of membrane vesicles from Escherichia coli. Biochemistry. 1979 Apr 17;18(8):1413–1422. doi: 10.1021/bi00575a004. [DOI] [PubMed] [Google Scholar]
  21. Owen P., Kaback H. R. Molecular structure of membrane vesicles from Escherichia coli. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3148–3152. doi: 10.1073/pnas.75.7.3148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Patel L., Garcia M. L., Kaback H. R. Direct measurement of lactose/proton symport in Escherichia coli membrane vesicles: further evidence for the involvement of histidine residue(s). Biochemistry. 1982 Nov 9;21(23):5805–5810. doi: 10.1021/bi00266a013. [DOI] [PubMed] [Google Scholar]
  23. Ramos S., Schuldiner S., Kaback H. R. The use of flow dialysis for determinations of deltapH and active transport. Methods Enzymol. 1979;55:680–688. doi: 10.1016/0076-6879(79)55076-9. [DOI] [PubMed] [Google Scholar]
  24. Reenstra W. W., Patel L., Rottenberg H., Kaback H. R. Electrochemical proton gradient in inverted membrane vesicles from Escherichia coli. Biochemistry. 1980 Jan 8;19(1):1–9. doi: 10.1021/bi00542a001. [DOI] [PubMed] [Google Scholar]
  25. Rosen B. P., McClees J. S. Active transport of calcium in inverted membrane vesicles of Escherichia coli. Proc Natl Acad Sci U S A. 1974 Dec;71(12):5042–5046. doi: 10.1073/pnas.71.12.5042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schuldiner S., Kerwar G. K., Kaback H. R., Weil R. Energy-dependent binding of dansylgalactosides to the beta-galactoside carrier protein. J Biol Chem. 1975 Feb 25;250(4):1361–1370. [PubMed] [Google Scholar]
  27. Short S. A., Kaback H. R., Kohn L. D. Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles. J Biol Chem. 1975 Jun 10;250(11):4291–4296. [PubMed] [Google Scholar]
  28. Teather R. M., Bramhall J., Riede I., Wright J. K., Fürst M., Aichele G., Wilhelm U., Overath P. Lactose carrier protein of Escherichia coli. Structure and expression of plasmids carrying the Y gene of the lac operon. Eur J Biochem. 1980;108(1):223–231. doi: 10.1111/j.1432-1033.1980.tb04715.x. [DOI] [PubMed] [Google Scholar]
  29. Teather R. M., Müller-Hill B., Abrutsch U., Aichele G., Overath P. Amplification of the lactose carrier protein in Escherichia coli using a plasmid vector. Mol Gen Genet. 1978 Feb 27;159(3):239–248. doi: 10.1007/BF00268260. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES