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
. 1990 Jun;87(11):4325–4329. doi: 10.1073/pnas.87.11.4325

In vivo expression of the lacY gene in two segments leads to functional lac permease.

E Bibi 1, H R Kaback 1
PMCID: PMC54102  PMID: 2190220

Abstract

The lacY gene of Escherichia coli was cut into two approximately equal-size fragments with Afl II and subcloned individually or together under separate lac operator/promoters in plasmid pT7-5. Under these conditions, lac permease is expressed in two portions: (i) the N-terminal portion (the N terminus, the first six putative transmembrane helices, and most of putative loop 7) and (ii) the C-terminal portion (the last six putative transmembrane helices and the C terminus). Cells harboring pT7-5 encoding both fragments transport lactose at about 30% the rate of cells expressing intact permease to a comparable steady-state level of accumulation. In contrast, cells expressing either half of the permease independently do not transport lactose. As judged by [35S]methionine labeling and immunoblotting, intact permease is completely absent from the membrane of cells expressing lacY fragments either individually or together. Thus, transport activity must result from an association between independently synthesized pieces of lac permease. When the gene fragments are expressed individually, the N-terminal portion of the permease is observed inconsistently, and the C-terminal portion is not observed. When the gene fragments are expressed together, polypeptides identified as the N- and C-terminal moieties of the permease are found in the membrane. It is concluded that the N- or C-terminal halves of lac permease are proteolyzed when synthesized independently and that association between the two complementing polypeptides leads to a more stable, catalytically active complex.

Full text

PDF
4325

Images in this article

4328Image
on p.4328
4328Image
on p.4328

Selected References

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

  1. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [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., Herzlinger D., Mitchell R., DeChiara S., Danho W., Gabriel T. F., Kaback H. R. Intramolecular dislocation of the COOH terminus of the lac carrier protein in reconstituted proteoliposomes. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4672–4676. doi: 10.1073/pnas.81.15.4672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Carrasco N., Viitanen P., Herzlinger D., Kaback H. R. Monoclonal antibodies against the lac carrier protein from Escherichia coli. 1. Functional studies. Biochemistry. 1984 Jul 31;23(16):3681–3687. doi: 10.1021/bi00311a017. [DOI] [PubMed] [Google Scholar]
  6. Costello M. J., Escaig J., Matsushita K., Viitanen P. V., Menick D. R., Kaback H. R. Purified lac permease and cytochrome o oxidase are functional as monomers. J Biol Chem. 1987 Dec 15;262(35):17072–17082. [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Herzlinger D., Carrasco N., Kaback H. R. Functional and immunochemical characterization of a mutant of Escherichia coli energy uncoupled for lactose transport. Biochemistry. 1985 Jan 1;24(1):221–229. doi: 10.1021/bi00322a032. [DOI] [PubMed] [Google Scholar]
  10. Herzlinger D., Viitanen P., Carrasco N., Kaback H. R. Monoclonal antibodies against the lac carrier protein from Escherichia coli. 2. Binding studies with membrane vesicles and proteoliposomes reconstituted with purified lac carrier protein. Biochemistry. 1984 Jul 31;23(16):3688–3693. doi: 10.1021/bi00311a018. [DOI] [PubMed] [Google Scholar]
  11. Kaback H. R. Molecular biology of active transport: from membrane to molecule to mechanism. Harvey Lect. 1987;83:77–105. [PubMed] [Google Scholar]
  12. Kobilka B. K., Kobilka T. S., Daniel K., Regan J. W., Caron M. G., Lefkowitz R. J. Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity. Science. 1988 Jun 3;240(4857):1310–1316. doi: 10.1126/science.2836950. [DOI] [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. Liao M. J., Huang K. S., Khorana H. G. Regeneration of native bacteriorhodopsin structure from fragments. J Biol Chem. 1984 Apr 10;259(7):4200–4204. [PubMed] [Google Scholar]
  15. 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]
  16. Page M. G., Rosenbusch J. P. Topography of lactose permease from Escherichia coli. J Biol Chem. 1988 Nov 5;263(31):15906–15914. [PubMed] [Google Scholar]
  17. Pollitt S., Zalkin H. Role of primary structure and disulfide bond formation in beta-lactamase secretion. J Bacteriol. 1983 Jan;153(1):27–32. doi: 10.1128/jb.153.1.27-32.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Roepe P. D., Kaback H. R. Characterization and functional reconstitution of a soluble form of the hydrophobic membrane protein lac permease from Escherichia coli. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6087–6091. doi: 10.1073/pnas.86.16.6087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Roepe P. D., Zbar R. I., Sarkar H. K., Kaback H. R. A five-residue sequence near the carboxyl terminus of the polytopic membrane protein lac permease is required for stability within the membrane. Proc Natl Acad Sci U S A. 1989 Jun;86(11):3992–3996. doi: 10.1073/pnas.86.11.3992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Seckler R., Möröy T., Wright J. K., Overath P. Anti-peptide antibodies and proteases as structural probes for the lactose/H+ transporter of Escherichia coli: a loop around amino acid residue 130 faces the cytoplasmic side of the membrane. Biochemistry. 1986 May 6;25(9):2403–2409. doi: 10.1021/bi00357a016. [DOI] [PubMed] [Google Scholar]
  21. Seckler R., Wright J. K., Overath P. Peptide-specific antibody locates the COOH terminus of the lactose carrier of Escherichia coli on the cytoplasmic side of the plasma membrane. J Biol Chem. 1983 Sep 25;258(18):10817–10820. [PubMed] [Google Scholar]
  22. Seckler R., Wright J. K. Sidedness of native membrane vesicles of Escherichia coli and orientation of the reconstituted lactose: H+ carrier. Eur J Biochem. 1984 Jul 16;142(2):269–279. doi: 10.1111/j.1432-1033.1984.tb08281.x. [DOI] [PubMed] [Google Scholar]
  23. Stochaj U., Bieseler B., Ehring R. Limited proteolysis of lactose permease from Escherichia coli. Eur J Biochem. 1986 Jul 15;158(2):423–428. doi: 10.1111/j.1432-1033.1986.tb09770.x. [DOI] [PubMed] [Google Scholar]
  24. Stochaj U., Fritz H. J., Heibach C., Markgraf M., von Schaewen A., Sonnewald U., Ehring R. Truncated forms of Escherichia coli lactose permease: models for study of biosynthesis and membrane insertion. J Bacteriol. 1988 Jun;170(6):2639–2645. doi: 10.1128/jb.170.6.2639-2645.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stühmer W., Conti F., Suzuki H., Wang X. D., Noda M., Yahagi N., Kubo H., Numa S. Structural parts involved in activation and inactivation of the sodium channel. Nature. 1989 Jun 22;339(6226):597–603. doi: 10.1038/339597a0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Trumble W. R., Viitanen P. V., Sarkar H. K., Poonian M. S., Kaback H. R. Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli. Biochem Biophys Res Commun. 1984 Mar 30;119(3):860–867. doi: 10.1016/0006-291x(84)90853-2. [DOI] [PubMed] [Google Scholar]
  29. Vogel H., Wright J. K., Jähnig F. The structure of the lactose permease derived from Raman spectroscopy and prediction methods. EMBO J. 1985 Dec 16;4(13A):3625–3631. doi: 10.1002/j.1460-2075.1985.tb04126.x. [DOI] [PMC free article] [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