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. 2001 Jul 1;357(Pt 1):11–15. doi: 10.1042/0264-6021:3570011

Cholesterol depletion reduces apical transport capacity in epithelial Madin-Darby canine kidney cells.

K Prydz 1, K Simons 1
PMCID: PMC1221922  PMID: 11415430

Abstract

Reduction of the cholesterol level in membranes of epithelial Madin-Darby canine kidney (MDCK) cells reverses the apical-to-basolateral transport ratio of the apical membrane marker protein influenza virus haemagglutinin and the secreted glycoprotein gp80. At the same time, basolateral transport of the vesicular stomatitis virus G protein is unaffected [Keller and Simons (1998) J. Cell Biol. 140, 1357-1367]. To investigate whether cholesterol depletion influences apical sorting mechanisms specifically, or apical transport capacity more generally, we studied the effect of cholesterol depletion on the secretion of three different classes of molecules from the apical and basolateral surfaces of MDCK cell layers: glycoprotein gp80, sulphated proteoglycans and proteins, and non-glycosylated rat growth hormone. In each case, cholesterol depletion reduced the fraction secreted to the apical medium and increased the fraction secreted basolaterally. The fact that this was observed for all sulphated proteins and proteoglycans and for the non-glycosylated rat growth hormone, which is randomly secreted in untreated cells, indicates that cholesterol depletion reduces the apical transport capacity, rather than interfering with specific recognition and sorting processes.

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Selected References

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  1. Bagnat M., Keränen S., Shevchenko A., Shevchenko A., Simons K. Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3254–3259. doi: 10.1073/pnas.060034697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett M. K., Wandinger-Ness A., Simons K. Release of putative exocytic transport vesicles from perforated MDCK cells. EMBO J. 1988 Dec 20;7(13):4075–4085. doi: 10.1002/j.1460-2075.1988.tb03301.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benting J. H., Rietveld A. G., Simons K. N-Glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells. J Cell Biol. 1999 Jul 26;146(2):313–320. doi: 10.1083/jcb.146.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown D. A., Crise B., Rose J. K. Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science. 1989 Sep 29;245(4925):1499–1501. doi: 10.1126/science.2571189. [DOI] [PubMed] [Google Scholar]
  5. Brown D. A., London E. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol. 1998;14:111–136. doi: 10.1146/annurev.cellbio.14.1.111. [DOI] [PubMed] [Google Scholar]
  6. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  7. Cheong K. H., Zacchetti D., Schneeberger E. E., Simons K. VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells. Proc Natl Acad Sci U S A. 1999 May 25;96(11):6241–6248. doi: 10.1073/pnas.96.11.6241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chuang J. Z., Sung C. H. The cytoplasmic tail of rhodopsin acts as a novel apical sorting signal in polarized MDCK cells. J Cell Biol. 1998 Sep 7;142(5):1245–1256. doi: 10.1083/jcb.142.5.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fenton R. G., Kung H. F., Longo D. L., Smith M. R. Regulation of intracellular actin polymerization by prenylated cellular proteins. J Cell Biol. 1992 Apr;117(2):347–356. doi: 10.1083/jcb.117.2.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Friedrichson T., Kurzchalia T. V. Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature. 1998 Aug 20;394(6695):802–805. doi: 10.1038/29570. [DOI] [PubMed] [Google Scholar]
  11. Gottlieb T. A., Beaudry G., Rizzolo L., Colman A., Rindler M., Adesnik M., Sabatini D. D. Secretion of endogenous and exogenous proteins from polarized MDCK cell monolayers. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2100–2104. doi: 10.1073/pnas.83.7.2100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gut A., Kappeler F., Hyka N., Balda M. S., Hauri H. P., Matter K. Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins. EMBO J. 1998 Apr 1;17(7):1919–1929. doi: 10.1093/emboj/17.7.1919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hansen G. H., Niels-Christiansen L. L., Thorsen E., Immerdal L., Danielsen E. M. Cholesterol depletion of enterocytes. Effect on the Golgi complex and apical membrane trafficking. J Biol Chem. 2000 Feb 18;275(7):5136–5142. doi: 10.1074/jbc.275.7.5136. [DOI] [PubMed] [Google Scholar]
  14. Jacob R., Alfalah M., Grünberg J., Obendorf M., Naim H. Y. Structural determinants required for apical sorting of an intestinal brush-border membrane protein. J Biol Chem. 2000 Mar 3;275(9):6566–6572. doi: 10.1074/jbc.275.9.6566. [DOI] [PubMed] [Google Scholar]
  15. Keller P., Simons K. Cholesterol is required for surface transport of influenza virus hemagglutinin. J Cell Biol. 1998 Mar 23;140(6):1357–1367. doi: 10.1083/jcb.140.6.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Keller P., Simons K. Post-Golgi biosynthetic trafficking. J Cell Sci. 1997 Dec;110(Pt 24):3001–3009. doi: 10.1242/jcs.110.24.3001. [DOI] [PubMed] [Google Scholar]
  17. Kolset S. O., Vuong T. T., Prydz K. Apical secretion of chondroitin sulphate in polarized Madin-Darby canine kidney (MDCK) cells. J Cell Sci. 1999 Jun;112(Pt 11):1797–1801. doi: 10.1242/jcs.112.11.1797. [DOI] [PubMed] [Google Scholar]
  18. Lafont F., Lecat S., Verkade P., Simons K. Annexin XIIIb associates with lipid microdomains to function in apical delivery. J Cell Biol. 1998 Sep 21;142(6):1413–1427. doi: 10.1083/jcb.142.6.1413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lisanti M. P., Caras I. W., Davitz M. A., Rodriguez-Boulan E. A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J Cell Biol. 1989 Nov;109(5):2145–2156. doi: 10.1083/jcb.109.5.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lisanti M. P., Field M. C., Caras I. W., Menon A. K., Rodriguez-Boulan E. Mannosamine, a novel inhibitor of glycosylphosphatidylinositol incorporation into proteins. EMBO J. 1991 Aug;10(8):1969–1977. doi: 10.1002/j.1460-2075.1991.tb07726.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marmorstein A. D., Csaky K. G., Baffi J., Lam L., Rahaal F., Rodriguez-Boulan E. Saturation of, and competition for entry into, the apical secretory pathway. Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3248–3253. doi: 10.1073/pnas.070049497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maxfield F. R., Mayor S. Cell surface dynamics of GPI-anchored proteins. Adv Exp Med Biol. 1997;419:355–364. doi: 10.1007/978-1-4419-8632-0_47. [DOI] [PubMed] [Google Scholar]
  23. Mayor S., Sabharanjak S., Maxfield F. R. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J. 1998 Aug 17;17(16):4626–4638. doi: 10.1093/emboj/17.16.4626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Melkonian K. A., Ostermeyer A. G., Chen J. Z., Roth M. G., Brown D. A. Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. Many raft proteins are acylated, while few are prenylated. J Biol Chem. 1999 Feb 5;274(6):3910–3917. doi: 10.1074/jbc.274.6.3910. [DOI] [PubMed] [Google Scholar]
  25. Mora R., Bonilha V. L., Marmorstein A., Scherer P. E., Brown D., Lisanti M. P., Rodriguez-Boulan E. Caveolin-2 localizes to the golgi complex but redistributes to plasma membrane, caveolae, and rafts when co-expressed with caveolin-1. J Biol Chem. 1999 Sep 3;274(36):25708–25717. doi: 10.1074/jbc.274.36.25708. [DOI] [PubMed] [Google Scholar]
  26. Murata M., Peränen J., Schreiner R., Wieland F., Kurzchalia T. V., Simons K. VIP21/caveolin is a cholesterol-binding protein. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10339–10343. doi: 10.1073/pnas.92.22.10339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Powell S. K., Lisanti M. P., Rodriguez-Boulan E. J. Thy-1 expresses two signals for apical localization in epithelial cells. Am J Physiol. 1991 Apr;260(4 Pt 1):C715–C720. doi: 10.1152/ajpcell.1991.260.4.C715. [DOI] [PubMed] [Google Scholar]
  28. Pralle A., Keller P., Florin E. L., Simons K., Hörber J. K. Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J Cell Biol. 2000 Mar 6;148(5):997–1008. doi: 10.1083/jcb.148.5.997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Puertollano R., Alonso M. A. A short peptide motif at the carboxyl terminus is required for incorporation of the integral membrane MAL protein to glycolipid-enriched membranes. J Biol Chem. 1998 May 22;273(21):12740–12745. doi: 10.1074/jbc.273.21.12740. [DOI] [PubMed] [Google Scholar]
  30. Rodriguez-Boulan E., Gonzalez A. Glycans in post-Golgi apical targeting: sorting signals or structural props? Trends Cell Biol. 1999 Aug;9(8):291–294. doi: 10.1016/s0962-8924(99)01595-0. [DOI] [PubMed] [Google Scholar]
  31. Scheiffele P., Peränen J., Simons K. N-glycans as apical sorting signals in epithelial cells. Nature. 1995 Nov 2;378(6552):96–98. doi: 10.1038/378096a0. [DOI] [PubMed] [Google Scholar]
  32. Scheiffele P., Roth M. G., Simons K. Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain. EMBO J. 1997 Sep 15;16(18):5501–5508. doi: 10.1093/emboj/16.18.5501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Scheiffele P., Verkade P., Fra A. M., Virta H., Simons K., Ikonen E. Caveolin-1 and -2 in the exocytic pathway of MDCK cells. J Cell Biol. 1998 Feb 23;140(4):795–806. doi: 10.1083/jcb.140.4.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simons K., Ikonen E. Functional rafts in cell membranes. Nature. 1997 Jun 5;387(6633):569–572. doi: 10.1038/42408. [DOI] [PubMed] [Google Scholar]
  35. Simons K., van Meer G. Lipid sorting in epithelial cells. Biochemistry. 1988 Aug 23;27(17):6197–6202. doi: 10.1021/bi00417a001. [DOI] [PubMed] [Google Scholar]
  36. Svennevig K., Prydz K., Kolset S. O. Proteoglycans in polarized epithelial Madin-Darby canine kidney cells. Biochem J. 1995 Nov 1;311(Pt 3):881–888. doi: 10.1042/bj3110881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Urban J., Parczyk K., Leutz A., Kayne M., Kondor-Koch C. Constitutive apical secretion of an 80-kD sulfated glycoprotein complex in the polarized epithelial Madin-Darby canine kidney cell line. J Cell Biol. 1987 Dec;105(6 Pt 1):2735–2743. doi: 10.1083/jcb.105.6.2735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Varma R., Mayor S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature. 1998 Aug 20;394(6695):798–801. doi: 10.1038/29563. [DOI] [PubMed] [Google Scholar]
  39. Wandinger-Ness A., Bennett M. K., Antony C., Simons K. Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells. J Cell Biol. 1990 Sep;111(3):987–1000. doi: 10.1083/jcb.111.3.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yeaman C., Le Gall A. H., Baldwin A. N., Monlauzeur L., Le Bivic A., Rodriguez-Boulan E. The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells. J Cell Biol. 1997 Nov 17;139(4):929–940. doi: 10.1083/jcb.139.4.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. van't Hof W., Rodriguez-Boulan E., Menon A. K. Nonpolarized distribution of glycosylphosphatidylinositols in the plasma membrane of polarized Madin-Darby canine kidney cells. J Biol Chem. 1995 Oct 13;270(41):24150–24155. doi: 10.1074/jbc.270.41.24150. [DOI] [PubMed] [Google Scholar]

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