Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Aug 15;310(Pt 1):271–278. doi: 10.1042/bj3100271

Brefeldin A inhibits the endocytosis of plasma-membrane-associated heparan sulphate proteoglycans of cultured rat ovarian granulosa cells.

L Uhlin-Hansen 1, M Yanagishita 1
PMCID: PMC1135883  PMID: 7646455

Abstract

Rat ovarian granulosa cells were labelled with [35S]sulphate for 0.5-20 h and chased in the presence or absence of 1-2 micrograms/ml of brefeldin A (BFA) for up to 21 h. Heparan [35S]sulphate (HS) proteoglycans from the culture medium, plasma membrane and intracellular fractions were then analysed by gel chromatography. In the absence of BFA, about 85% of the plasma membrane-associated HS proteoglycans were endocytosed and subsequently degraded intracellularly. Recirculation of the HS proteoglycans between the intracellular pool and the cell surface was not observed. Exposing the cells to BFA for less than 1 h did not influence the turnover of the HS proteoglycans, whereas the effect of the drug on the Golgi functions reached a maximum in approx. 10 min. When the cells were treated with BFA for more than 1-2 h, the rate of endocytosis of HS proteoglycans was reduced to about 50% of the control. The delivery of endocytosed HS proteoglycans to lysosomes were not affected by the drug. Cycloheximide also reduced the endocytosis of HS proteoglycans, but not as much as BFA, indicating that the inhibitory effect of BFA can be only partly accounted for by a block of protein transport from the endoplasmic reticulum to the plasma membrane. In contrast with the endocytosis of HS proteoglycans, neither that of 125I-transferrin, known to be mediated by clathrin-coated vesicles, nor that of 125I-ricin, a marker molecule for bulk endocytosis, was affected by BFA. The half-life of 125I-transferrin and 125I-ricin in the plasma membrane was about 10 and 25 min respectively compared with about 5 h for the HS proteoglycans. Altogether, these results indicate that the endocytosis of plasma-membrane-associated HS proteoglycans is mediated by different mechanisms than the endocytosis of most other cell-surface proteins. Further, the mechanisms involved in the endocytosis of HS proteoglycans are sensitive to BFA.

Full text

PDF
275

Selected References

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

  1. Anderson R. G. Caveolae: where incoming and outgoing messengers meet. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10909–10913. doi: 10.1073/pnas.90.23.10909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bretscher M. S. Circulating integrins: alpha 5 beta 1, alpha 6 beta 4 and Mac-1, but not alpha 3 beta 1, alpha 4 beta 1 or LFA-1. EMBO J. 1992 Feb;11(2):405–410. doi: 10.1002/j.1460-2075.1992.tb05068.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chege N. W., Pfeffer S. R. Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network. J Cell Biol. 1990 Sep;111(3):893–899. doi: 10.1083/jcb.111.3.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. David G. Integral membrane heparan sulfate proteoglycans. FASEB J. 1993 Aug;7(11):1023–1030. doi: 10.1096/fasebj.7.11.8370471. [DOI] [PubMed] [Google Scholar]
  5. Davis C. G., van Driel I. R., Russell D. W., Brown M. S., Goldstein J. L. The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis. J Biol Chem. 1987 Mar 25;262(9):4075–4082. [PubMed] [Google Scholar]
  6. Donaldson J. G., Kahn R. A., Lippincott-Schwartz J., Klausner R. D. Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein. Science. 1991 Nov 22;254(5035):1197–1199. doi: 10.1126/science.1957170. [DOI] [PubMed] [Google Scholar]
  7. Donaldson J. G., Lippincott-Schwartz J., Bloom G. S., Kreis T. E., Klausner R. D. Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action. J Cell Biol. 1990 Dec;111(6 Pt 1):2295–2306. doi: 10.1083/jcb.111.6.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doxsey S. J., Brodsky F. M., Blank G. S., Helenius A. Inhibition of endocytosis by anti-clathrin antibodies. Cell. 1987 Jul 31;50(3):453–463. doi: 10.1016/0092-8674(87)90499-5. [DOI] [PubMed] [Google Scholar]
  9. Fujiwara T., Oda K., Yokota S., Takatsuki A., Ikehara Y. Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J Biol Chem. 1988 Dec 5;263(34):18545–18552. [PubMed] [Google Scholar]
  10. Heuser J. E., Anderson R. G. Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol. 1989 Feb;108(2):389–400. doi: 10.1083/jcb.108.2.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hopkins C. R. Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma A431 cells. Cell. 1983 Nov;35(1):321–330. doi: 10.1016/0092-8674(83)90235-0. [DOI] [PubMed] [Google Scholar]
  12. Hunziker W., Whitney J. A., Mellman I. Selective inhibition of transcytosis by brefeldin A in MDCK cells. Cell. 1991 Nov 1;67(3):617–627. doi: 10.1016/0092-8674(91)90535-7. [DOI] [PubMed] [Google Scholar]
  13. Iacopetta B. J., Rothenberger S., Kühn L. C. A role for the cytoplasmic domain in transferrin receptor sorting and coated pit formation during endocytosis. Cell. 1988 Aug 12;54(4):485–489. doi: 10.1016/0092-8674(88)90069-4. [DOI] [PubMed] [Google Scholar]
  14. Keller G. A., Siegel M. W., Caras I. W. Endocytosis of glycophospholipid-anchored and transmembrane forms of CD4 by different endocytic pathways. EMBO J. 1992 Mar;11(3):863–874. doi: 10.1002/j.1460-2075.1992.tb05124.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  16. Lippincott-Schwartz J., Yuan L., Tipper C., Amherdt M., Orci L., Klausner R. D. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell. 1991 Nov 1;67(3):601–616. doi: 10.1016/0092-8674(91)90534-6. [DOI] [PubMed] [Google Scholar]
  17. Metz C. N., Brunner G., Choi-Muira N. H., Nguyen H., Gabrilove J., Caras I. W., Altszuler N., Rifkin D. B., Wilson E. L., Davitz M. A. Release of GPI-anchored membrane proteins by a cell-associated GPI-specific phospholipase D. EMBO J. 1994 Apr 1;13(7):1741–1751. doi: 10.1002/j.1460-2075.1994.tb06438.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miettinen H. M., Matter K., Hunziker W., Rose J. K., Mellman I. Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization. J Cell Biol. 1992 Feb;116(4):875–888. doi: 10.1083/jcb.116.4.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Misumi Y., Misumi Y., Miki K., Takatsuki A., Tamura G., Ikehara Y. Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J Biol Chem. 1986 Aug 25;261(24):11398–11403. [PubMed] [Google Scholar]
  20. Oda K., Hirose S., Takami N., Misumi Y., Takatsuki A., Ikehara Y. Brefeldin A arrests the intracellular transport of a precursor of complement C3 before its conversion site in rat hepatocytes. FEBS Lett. 1987 Apr 6;214(1):135–138. doi: 10.1016/0014-5793(87)80028-5. [DOI] [PubMed] [Google Scholar]
  21. Olsnes S., Refsnes K., Pihl A. Mechanism of action of the toxic lectins abrin and ricin. Nature. 1974 Jun 14;249(458):627–631. doi: 10.1038/249627a0. [DOI] [PubMed] [Google Scholar]
  22. Olsnes S., Saltvedt E., Pihl A. Isolation and comparison of galactose-binding lectins from Abrus precatorius and Ricinus communis. J Biol Chem. 1974 Feb 10;249(3):803–810. [PubMed] [Google Scholar]
  23. Pelchen-Matthews A., Boulet I., Littman D. R., Fagard R., Marsh M. The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits. J Cell Biol. 1992 Apr;117(2):279–290. doi: 10.1083/jcb.117.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Prydz K., Hansen S. H., Sandvig K., van Deurs B. Effects of brefeldin A on endocytosis, transcytosis and transport to the Golgi complex in polarized MDCK cells. J Cell Biol. 1992 Oct;119(2):259–272. doi: 10.1083/jcb.119.2.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Refsnes K., Olsnes S., Pihl A. On the toxic proteins abrin and ricin. Studies of their binding to and entry into Ehrlich ascites cells. J Biol Chem. 1974 Jun 10;249(11):3557–3562. [PubMed] [Google Scholar]
  26. Robinson M. S., Kreis T. E. Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators. Cell. 1992 Apr 3;69(1):129–138. doi: 10.1016/0092-8674(92)90124-u. [DOI] [PubMed] [Google Scholar]
  27. Roghani M., Moscatelli D. Basic fibroblast growth factor is internalized through both receptor-mediated and heparan sulfate-mediated mechanisms. J Biol Chem. 1992 Nov 5;267(31):22156–22162. [PubMed] [Google Scholar]
  28. Salzman N. H., Maxfield F. R. Intracellular fusion of sequentially formed endocytic compartments. J Cell Biol. 1988 Apr;106(4):1083–1091. doi: 10.1083/jcb.106.4.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sandvig K., Olsnes S. Effect of temperature on the uptake, excretion and degradation of abrin and ricin by HeLa cells. Exp Cell Res. 1979 Jun;121(1):15–25. doi: 10.1016/0014-4827(79)90439-7. [DOI] [PubMed] [Google Scholar]
  30. Sandvig K., Olsnes S., Petersen O. W., van Deurs B. Acidification of the cytosol inhibits endocytosis from coated pits. J Cell Biol. 1987 Aug;105(2):679–689. doi: 10.1083/jcb.105.2.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sandvig K., Olsnes S., Petersen O. W., van Deurs B. Inhibition of endocytosis from coated pits by acidification of the cytosol. J Cell Biochem. 1988 Jan;36(1):73–81. doi: 10.1002/jcb.240360108. [DOI] [PubMed] [Google Scholar]
  32. Sandvig K., van Deurs B. Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis. J Biol Chem. 1990 Apr 15;265(11):6382–6388. [PubMed] [Google Scholar]
  33. Uhlin-Hansen L., Yanagishita M. Differential effect of brefeldin A on the biosynthesis of heparan sulfate and chondroitin/dermatan sulfate proteoglycans in rat ovarian granulosa cells in culture. J Biol Chem. 1993 Aug 15;268(23):17370–17376. [PubMed] [Google Scholar]
  34. Wan J., Taub M. E., Shah D., Shen W. C. Brefeldin A enhances receptor-mediated transcytosis of transferrin in filter-grown Madin-Darby canine kidney cells. J Biol Chem. 1992 Jul 5;267(19):13446–13450. [PubMed] [Google Scholar]
  35. Wood S. A., Brown W. J. The morphology but not the function of endosomes and lysosomes is altered by brefeldin A. J Cell Biol. 1992 Oct;119(2):273–285. doi: 10.1083/jcb.119.2.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wood S. A., Park J. E., Brown W. J. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell. 1991 Nov 1;67(3):591–600. doi: 10.1016/0092-8674(91)90533-5. [DOI] [PubMed] [Google Scholar]
  37. Yanagishita M. Glycosylphosphatidylinositol-anchored and core protein-intercalated heparan sulfate proteoglycans in rat ovarian granulosa cells have distinct secretory, endocytotic, and intracellular degradative pathways. J Biol Chem. 1992 May 15;267(14):9505–9511. [PubMed] [Google Scholar]
  38. Yanagishita M., Hascall V. C. Biosynthesis of proteoglycans by rat granulosa cells cultured in vitro. J Biol Chem. 1979 Dec 25;254(24):12355–12364. [PubMed] [Google Scholar]
  39. Yanagishita M., Hascall V. C. Cell surface heparan sulfate proteoglycans. J Biol Chem. 1992 May 15;267(14):9451–9454. [PubMed] [Google Scholar]
  40. Yanagishita M., Hascall V. C. Effects of monensin on the synthesis, transport, and intracellular degradation of proteoglycans in rat ovarian granulosa cells in culture. J Biol Chem. 1985 May 10;260(9):5445–5455. [PubMed] [Google Scholar]
  41. Yanagishita M., Hascall V. C. Metabolism of proteoglycans in rat ovarian granulosa cell culture. Multiple intracellular degradative pathways and the effect of chloroquine. J Biol Chem. 1984 Aug 25;259(16):10270–10283. [PubMed] [Google Scholar]
  42. Yanagishita M., Hascall V. C. Proteoglycans synthesized by rat ovarian granulosa cells in culture. Isolation, fractionation, and characterization of proteoglycans associated with the cell layer. J Biol Chem. 1984 Aug 25;259(16):10260–10269. [PubMed] [Google Scholar]
  43. Yanagishita M. Inhibition of intracellular degradation of proteoglycans by leupeptin in rat ovarian granulosa cells. J Biol Chem. 1985 Sep 15;260(20):11075–11082. [PubMed] [Google Scholar]
  44. Yanagishita M., Midura R. J., Hascall V. C. Proteoglycans: isolation and purification from tissue cultures. Methods Enzymol. 1987;138:279–289. doi: 10.1016/0076-6879(87)38023-1. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES