Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Jun 1;125(5):1025–1035. doi: 10.1083/jcb.125.5.1025

Basolateral protein transport in streptolysin O-permeabilized MDCK cells

PMCID: PMC2120047  PMID: 8195286

Abstract

We have reconstituted polarized protein transport in streptolysin O- permeabilized MDCK cells from the TGN to the basolateral surface and to the apical surface. These transport steps are dependent on temperature, energy and exogenously supplied cytosol. Using this in vitro system we show that a whole tail peptide (WT peptide) corresponding to the cytoplasmic tail of a basolaterally sorted protein, the vesicular stomatitis virus glycoprotein (VSV G) inhibits the TGN to basolateral transport but does not affect any other transport step. Inhibition of VSV G transport to basolateral surface by WT peptide did not result in missorting of the protein to the apical surface. Mutation of the single tyrosine residue in the WT peptide reduced its inhibitory potency four- to fivefold. These results suggest that the VSV G tail physically interacts with a component of the sorting machinery. Using a cross- linking approach, we have identified proteins that associate with the cytoplasmic tail domain of VSV G. One of these polypeptides, Tin-2 (Tail interacting protein-2), associates with VSV G in the TGN, the site of protein sorting, but not in the ER nor at the cell surface. Tin- 2 does not associate with apically targeted hemagglutinin. WT peptide that inhibited the basolateral transport of VSV G also inhibited the association of Tin-2 with VSV G. Together, these properties make Tin-2 a candidate basolateral sorter. The results demonstrate the usefulness of the SLO-permeabilized cell system in dissecting the sorting machinery.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

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

  1. Apodaca G., Aroeti B., Tang K., Mostov K. E. Brefeldin-A inhibits the delivery of the polymeric immunoglobulin receptor to the basolateral surface of MDCK cells. J Biol Chem. 1993 Sep 25;268(27):20380–20385. [PubMed] [Google Scholar]
  2. Balch W. E., Dunphy W. G., Braell W. A., Rothman J. E. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell. 1984 Dec;39(2 Pt 1):405–416. doi: 10.1016/0092-8674(84)90019-9. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bhakdi S., Roth M., Sziegoleit A., Tranum-Jensen J. Isolation and identification of two hemolytic forms of streptolysin-O. Infect Immun. 1984 Nov;46(2):394–400. doi: 10.1128/iai.46.2.394-400.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bhakdi S., Tranum-Jensen J. Damage to mammalian cells by proteins that form transmembrane pores. Rev Physiol Biochem Pharmacol. 1987;107:147–223. doi: 10.1007/BFb0027646. [DOI] [PubMed] [Google Scholar]
  6. Casanova J. E., Apodaca G., Mostov K. E. An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor. Cell. 1991 Jul 12;66(1):65–75. doi: 10.1016/0092-8674(91)90139-p. [DOI] [PubMed] [Google Scholar]
  7. Doms R. W., Ruusala A., Machamer C., Helenius J., Helenius A., Rose J. K. Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein. J Cell Biol. 1988 Jul;107(1):89–99. doi: 10.1083/jcb.107.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fuller S. D., Bravo R., Simons K. An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments. EMBO J. 1985 Feb;4(2):297–307. doi: 10.1002/j.1460-2075.1985.tb03629.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gomperts B. D. GE: a GTP-binding protein mediating exocytosis. Annu Rev Physiol. 1990;52:591–606. doi: 10.1146/annurev.ph.52.030190.003111. [DOI] [PubMed] [Google Scholar]
  10. Gravotta D., Adesnik M., Sabatini D. D. Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins. J Cell Biol. 1990 Dec;111(6 Pt 2):2893–2908. doi: 10.1083/jcb.111.6.2893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  12. Gruenberg J., Howell K. E. Membrane traffic in endocytosis: insights from cell-free assays. Annu Rev Cell Biol. 1989;5:453–481. doi: 10.1146/annurev.cb.05.110189.002321. [DOI] [PubMed] [Google Scholar]
  13. Hunziker W., Harter C., Matter K., Mellman I. Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant. Cell. 1991 Sep 6;66(5):907–920. doi: 10.1016/0092-8674(91)90437-4. [DOI] [PubMed] [Google Scholar]
  14. Kobayashi T., Pimplikar S. W., Parton R. G., Bhakdi S., Simons K. Sphingolipid transport from the trans-Golgi network to the apical surface in permeabilized MDCK cells. FEBS Lett. 1992 Apr 6;300(3):227–231. doi: 10.1016/0014-5793(92)80851-7. [DOI] [PubMed] [Google Scholar]
  15. Kooy J., Toh B. H., Pettitt J. M., Erlich R., Gleeson P. A. Human autoantibodies as reagents to conserved Golgi components. Characterization of a peripheral, 230-kDa compartment-specific Golgi protein. J Biol Chem. 1992 Oct 5;267(28):20255–20263. [PubMed] [Google Scholar]
  16. Kreis T. E., Lodish H. F. Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. Cell. 1986 Sep 12;46(6):929–937. doi: 10.1016/0092-8674(86)90075-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Le Bivic A., Sambuy Y., Patzak A., Patil N., Chao M., Rodriguez-Boulan E. An internal deletion in the cytoplasmic tail reverses the apical localization of human NGF receptor in transfected MDCK cells. J Cell Biol. 1991 Nov;115(3):607–618. doi: 10.1083/jcb.115.3.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lisanti M. P., Sargiacomo M., Graeve L., Saltiel A. R., Rodriguez-Boulan E. Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9557–9561. doi: 10.1073/pnas.85.24.9557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matlin K. S., Reggio H., Helenius A., Simons K. Infectious entry pathway of influenza virus in a canine kidney cell line. J Cell Biol. 1981 Dec;91(3 Pt 1):601–613. doi: 10.1083/jcb.91.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Matlin K. S., Simons K. Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell. 1983 Aug;34(1):233–243. doi: 10.1016/0092-8674(83)90154-x. [DOI] [PubMed] [Google Scholar]
  22. Matlin K. S., Simons K. Sorting of an apical plasma membrane glycoprotein occurs before it reaches the cell surface in cultured epithelial cells. J Cell Biol. 1984 Dec;99(6):2131–2139. doi: 10.1083/jcb.99.6.2131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matter K., Hunziker W., Mellman I. Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell. 1992 Nov 27;71(5):741–753. doi: 10.1016/0092-8674(92)90551-m. [DOI] [PubMed] [Google Scholar]
  24. Mostov K., Apodaca G., Aroeti B., Okamoto C. Plasma membrane protein sorting in polarized epithelial cells. J Cell Biol. 1992 Feb;116(3):577–583. doi: 10.1083/jcb.116.3.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Narula N., McMorrow I., Plopper G., Doherty J., Matlin K. S., Burke B., Stow J. L. Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes. J Cell Biol. 1992 Apr;117(1):27–38. doi: 10.1083/jcb.117.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pfeiffer S., Fuller S. D., Simons K. Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells. J Cell Biol. 1985 Aug;101(2):470–476. doi: 10.1083/jcb.101.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pimplikar S. W., Simons K. Regulation of apical transport in epithelial cells by a Gs class of heterotrimeric G protein. Nature. 1993 Apr 1;362(6419):456–458. doi: 10.1038/362456a0. [DOI] [PubMed] [Google Scholar]
  28. Prill V., Lehmann L., von Figura K., Peters C. The cytoplasmic tail of lysosomal acid phosphatase contains overlapping but distinct signals for basolateral sorting and rapid internalization in polarized MDCK cells. EMBO J. 1993 May;12(5):2181–2193. doi: 10.1002/j.1460-2075.1993.tb05866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pryer N. K., Wuestehube L. J., Schekman R. Vesicle-mediated protein sorting. Annu Rev Biochem. 1992;61:471–516. doi: 10.1146/annurev.bi.61.070192.002351. [DOI] [PubMed] [Google Scholar]
  30. Rindler M. J., Ivanov I. E., Plesken H., Rodriguez-Boulan E., Sabatini D. D. Viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same Golgi apparatus during their intracellular transport in doubly infected Madin-Darby canine kidney cells. J Cell Biol. 1984 Apr;98(4):1304–1319. doi: 10.1083/jcb.98.4.1304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rodriguez-Boulan E., Powell S. K. Polarity of epithelial and neuronal cells. Annu Rev Cell Biol. 1992;8:395–427. doi: 10.1146/annurev.cb.08.110192.002143. [DOI] [PubMed] [Google Scholar]
  32. Rothman J. E., Orci L. Molecular dissection of the secretory pathway. Nature. 1992 Jan 30;355(6359):409–415. doi: 10.1038/355409a0. [DOI] [PubMed] [Google Scholar]
  33. Simons K., Virta H. Perforated MDCK cells support intracellular transport. EMBO J. 1987 Aug;6(8):2241–2247. doi: 10.1002/j.1460-2075.1987.tb02496.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simons K., Wandinger-Ness A. Polarized sorting in epithelia. Cell. 1990 Jul 27;62(2):207–210. doi: 10.1016/0092-8674(90)90357-k. [DOI] [PubMed] [Google Scholar]
  35. Staros J. V. N-hydroxysulfosuccinimide active esters: bis(N-hydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers. Biochemistry. 1982 Aug 17;21(17):3950–3955. doi: 10.1021/bi00260a008. [DOI] [PubMed] [Google Scholar]
  36. Tan A., Bolscher J., Feltkamp C., Ploegh H. Retrograde transport from the Golgi region to the endoplasmic reticulum is sensitive to GTP gamma S. J Cell Biol. 1992 Mar;116(6):1357–1367. doi: 10.1083/jcb.116.6.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Thomas D. C., Brewer C. B., Roth M. G. Vesicular stomatitis virus glycoprotein contains a dominant cytoplasmic basolateral sorting signal critically dependent upon a tyrosine. J Biol Chem. 1993 Feb 15;268(5):3313–3320. [PubMed] [Google Scholar]
  38. 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]
  39. Yokode M., Pathak R. K., Hammer R. E., Brown M. S., Goldstein J. L., Anderson R. G. Cytoplasmic sequence required for basolateral targeting of LDL receptor in livers of transgenic mice. J Cell Biol. 1992 Apr;117(1):39–46. doi: 10.1083/jcb.117.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES