Skip to main content
SpringerLink
Log in

Cell surface biotinylation in the determination of epithelial membrane polarity

  • Published:
Journal of tissue culture methods

Summary

The plasma membranes of polarized epithelial cells are divided into two distinct domains separated by tight junctions and characterized by distinct polypeptide compositions. Study of the cellular mechanisms involved in generating this anisotropy has recently been facilitated through the introduction of a surface labeling technique employing NHS-biotin (29). Incorporation of the biotin marker allows proteins present at one or the other cell surface domain to be identified and isolated. This methodology is applicable to investigations of both the steady state and the dynamic aspects of epithelial polarity. We discuss protocols through which this technique can be applied to various experimental situations. Methodologic details and special considerations are discussed. The utility of surface biotinylation for studies of epithelial polarity depends on this technique's capacity to report quantitatively on the surface distributions of plasmalemmal proteins. We find that under certain circumstances, surface biotinylation is extremely inefficient. Furthermore, in some cases the efficiency of surface biotinylation is dramatically different at the two plasmalemmal domains of epithelial cells grown on permeable filter supports. Thus, surface biotinylation does not always provide an accurate picture of plasma membrane protein distributions. These situations are discussed and methods to assess the efficiency and accuracy of surface biotinylation are proposed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. The uses of esters ofN-hydroxysuccinimide in peptide synthesis. J. Am. Chem. Soc. 86:1839–1842; 1964.

    Google Scholar 

  2. Arzamazova, N. M.; Arystarkhova, E. A.; Gevondyan, N. M., et al. Sequence analysis of exposed domains of membrane-bound Na,K-ATP-ase: model of transmembrane arrangement. In: Skou, J. C.; Norby, J. G.; Maunsbach, A. B., et al., eds. The Na,K-pump. New York: Alan R. Liss; 1988:57–64.

    Google Scholar 

  3. Berridge, M. J.; Oschman, J. L. Transporting epithelia. New York: Academic Press; 1972.

    Google Scholar 

  4. Boll, W.; Partin, J. S.; Katz, A. I., et al. Distinct pathways for basolateral targetting of membrane and secretory proteins in polarized epithelial cells. Proc. Natl. Acad. Sci. USA 88:8592–8596; 1991.

    PubMed  Google Scholar 

  5. Caplan, M. J. Biogenesis and sorting of plasma membrane proteins. Curr. Top. Membr. Transp. 39:37–86; 1991.

    Google Scholar 

  6. Caplan, M. J. Biosynthesis and sorting of the sodium, potassium-ATPase. In: Reuss, L.; Russell, J. M.; Szabo, G., eds. Regulation of potassium transport across biological membranes. Austin: University of Texas Press; 1990:77–101.

    Google Scholar 

  7. Caplan, M. J.; Anderson, H. C.; Palade, G. E., et al. Intracellular sorting and polarized cell surface delivery of Na,K-ATPase, an endogenous component of MDCK cell basolateral plasma membranes. Cell 46:623–631; 1986.

    PubMed  Google Scholar 

  8. Caplan, M. J.; Matlin, K. S. Sorting of membrane and secretory proteins in polarized epithelial cells. In: Matlin, K. S.; Valentich, J. D., eds. Functional epithelial cells in culture. New York: Alan R. Liss; 1989:71–127.

    Google Scholar 

  9. Fuller, S. D.; Simons, K. Transferrin receptor polarity and recycling accuracy in “tight” and “leaky” strains of Madin-Darby canine kidney cells. J. Cell Biol. 103:1767–1779; 1986.

    PubMed  Google Scholar 

  10. Green, N. M. The avidin-biotin interaction. Adv. Protein Chem. 29:85–133; 1975.

    PubMed  Google Scholar 

  11. Hammerton, R. W.; Krzeminski, K. A.; Mays, R. W., et al. Mechanism for regulating cell surface distribution of Na,K-ATPase in polarized epithelial cells. Science 254:847–850; 1991.

    PubMed  Google Scholar 

  12. Hull, R. N.; Cherry, W. R.; Weaver, G. W. The origin and characteristics of a pig kidney cell strain, LLC-PK1. In Vitro 12:670–677; 1976.

    PubMed  Google Scholar 

  13. Hunziker, W.; Harter, C.; Matter, K., et al. Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant. Cell 66:907–920; 1991.

    PubMed  Google Scholar 

  14. Hunziker, W.; Mellman, I. Expression of macrophage-lymphocyte Fc receptors in Madin-Darby canine kidney cells: polarity and transcytosis differ for isoforms with or without coated pit localization domains. J. Cell Biol. 109:3291–3302; 1989.

    PubMed  Google Scholar 

  15. Jorgensen, P. L. Mechanism of the Na,K pump: protein structure and conformations of the pure Na,K-ATPase. Biochim. Biophys. Acta 694:27–68; 1982.

    PubMed  Google Scholar 

  16. Kriz, W.; Kaissling, B. Structural organization of the mammalian kidney. In: Seldin, D. W.; Gicbisch, G., eds. The kidney: physiology and pathophysiology. New York: Raven Press, 1992:707–772.

    Google Scholar 

  17. Le Bivic, A.; Real, F. X.; Rodriguez-Boulan, E. J. Vectorial targeting of apical and basolateral plasma membrane proteins in a human adenocarcinoma epithelial cell line. Proc. Natl. Acad. Sci. USA 86:9313–9317; 1989.

    PubMed  Google Scholar 

  18. Le Bivic, A.; Sambuy, Y.; Mostov, K., et al. Vectorial targeting of an endogenous apical sialoglycoprotein and uvomorulin in MDCK cells. J. Cell Biol. 110:1533–1539; 1990.

    PubMed  Google Scholar 

  19. Lehninger, A. L. Biochemistry. New York: Worth; 1975:79.

    Google Scholar 

  20. Lisanti, M. P.; Caras, I. W.; Davitz, M. A., et al. A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J. Cell Biol. 109:2145–2156; 1989.

    PubMed  Google Scholar 

  21. Lisanti, M. P.; Le Bivic, A.; Saltiel, A. R., et al. Preferred apical distribution of glycosyl-phosphatidylinositol (GPI) anchored proteins: a highly conserved feature of the polarized epithelial cell phenotype. J. Membr. Biol. 113:155–167; 1990.

    PubMed  Google Scholar 

  22. Lisanti, M. P.; Le Bivic, A.; Sargiacomo, M., et al. Steady-state distribution and biogenesis of endogenous MDCK glycoproteins: evidence for intracellular sorting and polarized cell surface delivery. J. Cell Biol. 109:2117–2127; 1989.

    PubMed  Google Scholar 

  23. Lisanti, M. P.; Sargiacomo, M.; Graeve, L., et al. Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a rental epithelial cell line. Proc. Natl. Acad. Sci. USA 85:9557–9561; 1988.

    PubMed  Google Scholar 

  24. Louvard, D. Apical membrane amino peptidase appears at the site of cell-cell contact in cultured kidney epithelial cells. Proc. Natl. Acad. Sci. USA 77:4132–4136; 1980.

    PubMed  Google Scholar 

  25. Madin, S. H.; Darby, N. B. In: American Type Culture Collection catalogue of strains II, ed. 1. Rockville, MD: American Type Culture Collection; 1975:47.

    Google Scholar 

  26. Matter, K.; Brauchbar, M.; Bucher, K., et al. Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2). Cell 60:429–437; 1990.

    PubMed  Google Scholar 

  27. Mostov, K., Apodaca, G., Aroeti, B., et al. Plasma membrane protein sorting in polarized epithelial cells. J. Cell Biol. 116:577–583; 1992.

    PubMed  Google Scholar 

  28. Rodriguez-Boulan, E.; Nelson, W. J. Morphogenesis of the polarized epithelial cell phenotype. Science 245:718–725; 1990.

    Google Scholar 

  29. Sargiacomo, M.; Lisanti, M.; Graeve, L., et al. Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells. J. Membr. Biol. 107:277–286; 1989.

    PubMed  Google Scholar 

  30. Shull, G. E.; Lingrel, I. B. Molecular cloning of the rat stomach H,K-ATPase. J. Biol. Chem. 261:16788–16791; 1986.

    PubMed  Google Scholar 

  31. Simons, K.; Fuller, S. D. Cell surface polarity in epithelia. Ann. Rev. Cell Biol. 1:295–340; 1985.

    Google Scholar 

  32. Simons, K.; Wandinger-Ness, A. Polarized sorting in epithelia. Cell 62:207–210; 1990.

    PubMed  Google Scholar 

  33. Smolka, A.; Helander, H. F.; Sachs, G. Monoclonal antibodies against gastric H,K-ATPase. Am. J. Physiol. 245:G589-G596; 1983.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, 06510, New Haven, Connecticut

    Cara J. Gottardi & Michael J. Caplan

Authors
  1. Cara J. Gottardi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael J. Caplan
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gottardi, C.J., Caplan, M.J. Cell surface biotinylation in the determination of epithelial membrane polarity. Journal of Tissue Culture Methods 14, 173–180 (1992). https://doi.org/10.1007/BF01409008

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01409008

Key words

Search

Navigation