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
. 1993 Dec 15;90(24):11588–11592. doi: 10.1073/pnas.90.24.11588

Functional expression of P-glycoprotein encoded by the mouse mdr3 gene in yeast cells.

S Ruetz 1, M Raymond 1, P Gros 1
PMCID: PMC48029  PMID: 7903452

Abstract

We have expressed P-glycoprotein (P-gp) encoded by the mouse mdr3 gene in the yeast Saccharomyces cerevisiae and have developed an experimental protocol to isolate and purify inside-out plasma membrane vesicles (IOVs) from these cells. Biochemical characterization of IOVs from control and P-gp-expressing cells isolated by this procedure show that they are greatly enriched for plasma membrane markers, are tightly sealed, and are competent for D-glucose transport. P-gp expression in these vesicles results in the appearance of a specific ATP-dependent and temperature-sensitive transport of the drugs colchicine and vinblastine that is osmotically sensitive. P-gp-mediated drug transport into these IOVs is inhibited by a known P-gp modulator, verapamil, and can be abrogated by prior incubation of the IOVs with an anti-P-gp antibody. A Ser-939-->Phe mutation within the predicted transmembrane domain 11 of P-gp, which is known to modulate its function in mammalian cells, drastically reduces drug transport in IOVs obtained from yeast cells expressing the mutant protein. The successful demonstration of active drug transport into IOVs from P-gp-expressing yeast cells indicates that P-gp can mediate both chemotherapeutic drugs and a-pheromone transport in yeast cells.

Full text

PDF
11589

Images in this article

11590Fig. 1
on p.11590

Selected References

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

  1. Abraham E. H., Prat A. G., Gerweck L., Seneveratne T., Arceci R. J., Kramer R., Guidotti G., Cantiello H. F. The multidrug resistance (mdr1) gene product functions as an ATP channel. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):312–316. doi: 10.1073/pnas.90.1.312. [DOI] [PMC free article] [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. Beck W. T. Multidrug resistance and its circumvention. Eur J Cancer. 1990 Apr;26(4):513–515. doi: 10.1016/0277-5379(90)90028-r. [DOI] [PubMed] [Google Scholar]
  4. Chen C. J., Chin J. E., Ueda K., Clark D. P., Pastan I., Gottesman M. M., Roninson I. B. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell. 1986 Nov 7;47(3):381–389. doi: 10.1016/0092-8674(86)90595-7. [DOI] [PubMed] [Google Scholar]
  5. Devault A., Gros P. Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities. Mol Cell Biol. 1990 Apr;10(4):1652–1663. doi: 10.1128/mcb.10.4.1652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Doige C. A., Yu X., Sharom F. J. ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. Biochim Biophys Acta. 1992 Aug 24;1109(2):149–160. doi: 10.1016/0005-2736(92)90078-z. [DOI] [PubMed] [Google Scholar]
  7. Endicott J. A., Ling V. The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem. 1989;58:137–171. doi: 10.1146/annurev.bi.58.070189.001033. [DOI] [PubMed] [Google Scholar]
  8. Franzusoff A. J., Cirillo V. P. Glucose transport activity in isolated plasma membrane vesicles from Saccharomyces cerevisiae. J Biol Chem. 1983 Mar 25;258(6):3608–3614. [PubMed] [Google Scholar]
  9. Georges E., Bradley G., Gariepy J., Ling V. Detection of P-glycoprotein isoforms by gene-specific monoclonal antibodies. Proc Natl Acad Sci U S A. 1990 Jan;87(1):152–156. doi: 10.1073/pnas.87.1.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Germann U. A., Willingham M. C., Pastan I., Gottesman M. M. Expression of the human multidrug transporter in insect cells by a recombinant baculovirus. Biochemistry. 1990 Mar 6;29(9):2295–2303. doi: 10.1021/bi00461a013. [DOI] [PubMed] [Google Scholar]
  11. Gros P., Ben Neriah Y. B., Croop J. M., Housman D. E. Isolation and expression of a complementary DNA that confers multidrug resistance. Nature. 1986 Oct 23;323(6090):728–731. doi: 10.1038/323728a0. [DOI] [PubMed] [Google Scholar]
  12. Gros P., Buschman E. The mouse multidrug resistance gene family: structural and functional analysis. Int Rev Cytol. 1993;137C:169–197. [PubMed] [Google Scholar]
  13. Gros P., Croop J., Housman D. Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins. Cell. 1986 Nov 7;47(3):371–380. doi: 10.1016/0092-8674(86)90594-5. [DOI] [PubMed] [Google Scholar]
  14. Gros P., Dhir R., Croop J., Talbot F. A single amino acid substitution strongly modulates the activity and substrate specificity of the mouse mdr1 and mdr3 drug efflux pumps. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7289–7293. doi: 10.1073/pnas.88.16.7289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gros P., Raymond M., Bell J., Housman D. Cloning and characterization of a second member of the mouse mdr gene family. Mol Cell Biol. 1988 Jul;8(7):2770–2778. doi: 10.1128/mcb.8.7.2770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hamada H., Tsuruo T. Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. 170- to 180-kilodalton membrane glycoprotein is an ATPase. J Biol Chem. 1988 Jan 25;263(3):1454–1458. [PubMed] [Google Scholar]
  17. Higgins C. F., Gottesman M. M. Is the multidrug transporter a flippase? Trends Biochem Sci. 1992 Jan;17(1):18–21. doi: 10.1016/0968-0004(92)90419-a. [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. Kajiji S., Talbot F., Grizzuti K., Van Dyke-Phillips V., Agresti M., Safa A. R., Gros P. Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site. Biochemistry. 1993 Apr 27;32(16):4185–4194. doi: 10.1021/bi00067a005. [DOI] [PubMed] [Google Scholar]
  20. Kuchler K., Thorner J. Functional expression of human mdr1 in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2302–2306. doi: 10.1073/pnas.89.6.2302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Raymond M., Gros P., Whiteway M., Thomas D. Y. Functional complementation of yeast ste6 by a mammalian multidrug resistance mdr gene. Science. 1992 Apr 10;256(5054):232–234. doi: 10.1126/science.1348873. [DOI] [PubMed] [Google Scholar]
  22. Riordan J. R., Rommens J. M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J. L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989 Sep 8;245(4922):1066–1073. doi: 10.1126/science.2475911. [DOI] [PubMed] [Google Scholar]
  23. Roepe P. D. Analysis of the steady-state and initial rate of doxorubicin efflux from a series of multidrug-resistant cells expressing different levels of P-glycoprotein. Biochemistry. 1992 Dec 22;31(50):12555–12564. doi: 10.1021/bi00165a003. [DOI] [PubMed] [Google Scholar]
  24. Saeki T., Shimabuku A. M., Azuma Y., Shibano Y., Komano T., Ueda K. Expression of human P-glycoprotein in yeast cells--effects of membrane component sterols on the activity of P-glycoprotein. Agric Biol Chem. 1991 Jul;55(7):1859–1865. [PubMed] [Google Scholar]
  25. Safa A. R., Stern R. K., Choi K., Agresti M., Tamai I., Mehta N. D., Roninson I. B. Molecular basis of preferential resistance to colchicine in multidrug-resistant human cells conferred by Gly-185----Val-185 substitution in P-glycoprotein. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7225–7229. doi: 10.1073/pnas.87.18.7225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sarkadi B., Price E. M., Boucher R. C., Germann U. A., Scarborough G. A. Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase. J Biol Chem. 1992 Mar 5;267(7):4854–4858. [PubMed] [Google Scholar]
  27. Sarkadi B., Szász I., Gerlóczy A., Gárdos G. Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells. Biochim Biophys Acta. 1977 Jan 4;464(1):93–107. doi: 10.1016/0005-2736(77)90373-x. [DOI] [PubMed] [Google Scholar]
  28. Scarborough G. A. Isolation and characterization of Neurospora crassa plasma membranes. J Biol Chem. 1975 Feb 10;250(3):1106–1111. [PubMed] [Google Scholar]
  29. Scarborough G. A. Proton translocation catalyzed by the electrogenic ATPase in the plasma membrane of Neurospora. Biochemistry. 1980 Jun 24;19(13):2925–2931. doi: 10.1021/bi00554a017. [DOI] [PubMed] [Google Scholar]
  30. Schurr E., Raymond M., Bell J. C., Gros P. Characterization of the multidrug resistance protein expressed in cell clones stably transfected with the mouse mdr1 cDNA. Cancer Res. 1989 May 15;49(10):2729–2733. [PubMed] [Google Scholar]
  31. Spies T., DeMars R. Restored expression of major histocompatibility class I molecules by gene transfer of a putative peptide transporter. Nature. 1991 May 23;351(6324):323–324. doi: 10.1038/351323a0. [DOI] [PubMed] [Google Scholar]
  32. Tschopp J., Schekman R. Two distinct subfractions in isolated Saccharomyces cerevisiae plasma membranes. J Bacteriol. 1983 Oct;156(1):222–229. doi: 10.1128/jb.156.1.222-229.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Valle D., Gärtner J. Human genetics. Penetrating the peroxisome. Nature. 1993 Feb 25;361(6414):682–683. doi: 10.1038/361682a0. [DOI] [PubMed] [Google Scholar]
  34. Valverde M. A., Díaz M., Sepúlveda F. V., Gill D. R., Hyde S. C., Higgins C. F. Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein. Nature. 1992 Feb 27;355(6363):830–833. doi: 10.1038/355830a0. [DOI] [PubMed] [Google Scholar]
  35. Willsky G. R. Characterization of the plasma membrane Mg2+-ATPase from the yeast, Saccharomyces cerevisiae. J Biol Chem. 1979 May 10;254(9):3326–3332. [PubMed] [Google Scholar]
  36. Zamora J. M., Pearce H. L., Beck W. T. Physical-chemical properties shared by compounds that modulate multidrug resistance in human leukemic cells. Mol Pharmacol. 1988 Apr;33(4):454–462. [PubMed] [Google Scholar]
  37. van der Bliek A. M., Kooiman P. M., Schneider C., Borst P. Sequence of mdr3 cDNA encoding a human P-glycoprotein. Gene. 1988 Nov 30;71(2):401–411. doi: 10.1016/0378-1119(88)90057-1. [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