Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Dec 1;115(5):1357–1374. doi: 10.1083/jcb.115.5.1357

Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques

PMCID: PMC2289231  PMID: 1659577

Abstract

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43- NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.

Full Text

The Full Text of this article is available as a PDF (3.5 MB).

Selected References

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

  1. Beyer E. C., Kistler J., Paul D. L., Goodenough D. A. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol. 1989 Feb;108(2):595–605. doi: 10.1083/jcb.108.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beyer E. C., Paul D. L., Goodenough D. A. Connexin family of gap junction proteins. J Membr Biol. 1990 Jul;116(3):187–194. doi: 10.1007/BF01868459. [DOI] [PubMed] [Google Scholar]
  3. Beyer E. C., Paul D. L., Goodenough D. A. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol. 1987 Dec;105(6 Pt 1):2621–2629. doi: 10.1083/jcb.105.6.2621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bruzzone R., Trimble E. R., Gjinovci A., Traub O., Willecke K., Meda P. Regulation of pancreatic exocrine function: a role for cell-to-cell communication? Pancreas. 1987;2(3):262–271. doi: 10.1097/00006676-198705000-00004. [DOI] [PubMed] [Google Scholar]
  5. Chanson M., Bruzzone R., Bosco D., Meda P. Effects of n-alcohols on junctional coupling and amylase secretion of pancreatic acinar cells. J Cell Physiol. 1989 Apr;139(1):147–156. doi: 10.1002/jcp.1041390121. [DOI] [PubMed] [Google Scholar]
  6. Crow D. S., Beyer E. C., Paul D. L., Kobe S. S., Lau A. F. Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts. Mol Cell Biol. 1990 Apr;10(4):1754–1763. doi: 10.1128/mcb.10.4.1754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Filson A. J., Azarnia R., Beyer E. C., Loewenstein W. R., Brugge J. S. Tyrosine phosphorylation of a gap junction protein correlates with inhibition of cell-to-cell communication. Cell Growth Differ. 1990 Dec;1(12):661–668. [PubMed] [Google Scholar]
  8. Furshpan E. J., Potter D. D. Low-resistance junctions between cells in embryos and tissue culture. Curr Top Dev Biol. 1968;3:95–127. doi: 10.1016/s0070-2153(08)60352-x. [DOI] [PubMed] [Google Scholar]
  9. Gilula N. B., Reeves O. R., Steinbach A. Metabolic coupling, ionic coupling and cell contacts. Nature. 1972 Feb 4;235(5336):262–265. doi: 10.1038/235262a0. [DOI] [PubMed] [Google Scholar]
  10. Goodenough D. A., Stoeckenius W. The isolation of mouse hepatocyte gap junctions. Preliminary chemical characterization and x-ray diffraction. J Cell Biol. 1972 Sep;54(3):646–656. doi: 10.1083/jcb.54.3.646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guthrie S. C., Gilula N. B. Gap junctional communication and development. Trends Neurosci. 1989 Jan;12(1):12–16. doi: 10.1016/0166-2236(89)90150-1. [DOI] [PubMed] [Google Scholar]
  12. Hertzberg E. L. A detergent-independent procedure for the isolation of gap junctions from rat liver. J Biol Chem. 1984 Aug 10;259(15):9936–9943. [PubMed] [Google Scholar]
  13. Hertzberg E. L., Gilula N. B. Isolation and characterization of gap junctions from rat liver. J Biol Chem. 1979 Mar 25;254(6):2138–2147. [PubMed] [Google Scholar]
  14. Hirokawa N., Heuser J. The inside and outside of gap-junction membranes visualized by deep etching. Cell. 1982 Sep;30(2):395–406. doi: 10.1016/0092-8674(82)90237-9. [DOI] [PubMed] [Google Scholar]
  15. Kensler R. W., Goodenough D. A. Isolation of mouse myocardial gap junctions. J Cell Biol. 1980 Sep;86(3):755–764. doi: 10.1083/jcb.86.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kistler J., Bullivant S. Dissociation of lens fibre gap junctions releases MP70. J Cell Sci. 1988 Nov;91(Pt 3):415–421. doi: 10.1242/jcs.91.3.415. [DOI] [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. Laird D. W., Puranam K. L., Revel J. P. Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes. Biochem J. 1991 Jan 1;273(Pt 1):67–72. doi: 10.1042/bj2730067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Larson D. M., Haudenschild C. C., Beyer E. C. Gap junction messenger RNA expression by vascular wall cells. Circ Res. 1990 Apr;66(4):1074–1080. doi: 10.1161/01.res.66.4.1074. [DOI] [PubMed] [Google Scholar]
  20. Le Bivic A., Quaroni A., Nichols B., Rodriguez-Boulan E. Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line. J Cell Biol. 1990 Oct;111(4):1351–1361. doi: 10.1083/jcb.111.4.1351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Le Bivic A., Real F. X., Rodriguez-Boulan E. Vectorial targeting of apical and basolateral plasma membrane proteins in a human adenocarcinoma epithelial cell line. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9313–9317. doi: 10.1073/pnas.86.23.9313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Le Bivic A., Sambuy Y., Mostov K., Rodriguez-Boulan E. Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK cells. J Cell Biol. 1990 May;110(5):1533–1539. doi: 10.1083/jcb.110.5.1533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Loewenstein W. R. Junctional intercellular communication and the control of growth. Biochim Biophys Acta. 1979 Feb 4;560(1):1–65. doi: 10.1016/0304-419x(79)90002-7. [DOI] [PubMed] [Google Scholar]
  24. Loewenstein W. R. Junctional intercellular communication: the cell-to-cell membrane channel. Physiol Rev. 1981 Oct;61(4):829–913. doi: 10.1152/physrev.1981.61.4.829. [DOI] [PubMed] [Google Scholar]
  25. Lowenstein W. R. Regulation of cell-to-cell communication by phosphorylation. Biochem Soc Symp. 1985;50:43–58. [PubMed] [Google Scholar]
  26. Malewicz B., Kumar V. V., Johnson R. G., Baumann W. J. Lipids in gap junction assembly and function. Lipids. 1990 Aug;25(8):419–427. doi: 10.1007/BF02538083. [DOI] [PubMed] [Google Scholar]
  27. Manjunath C. K., Goings G. E., Page E. Cytoplasmic surface and intramembrane components of rat heart gap junctional proteins. Am J Physiol. 1984 Jun;246(6 Pt 2):H865–H875. doi: 10.1152/ajpheart.1984.246.6.H865. [DOI] [PubMed] [Google Scholar]
  28. Manjunath C. K., Page E. Rat heart gap junctions as disulfide-bonded connexon multimers: their depolymerization and solubilization in deoxycholate. J Membr Biol. 1986;90(1):43–57. doi: 10.1007/BF01869685. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Matter K., Brauchbar M., Bucher K., Hauri H. P. Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2). Cell. 1990 Feb 9;60(3):429–437. doi: 10.1016/0092-8674(90)90594-5. [DOI] [PubMed] [Google Scholar]
  31. Meda P., Bruzzone R., Knodel S., Orci L. Blockage of cell-to-cell communication within pancreatic acini is associated with increased basal release of amylase. J Cell Biol. 1986 Aug;103(2):475–483. doi: 10.1083/jcb.103.2.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mege R. M., Matsuzaki F., Gallin W. J., Goldberg J. I., Cunningham B. A., Edelman G. M. Construction of epithelioid sheets by transfection of mouse sarcoma cells with cDNAs for chicken cell adhesion molecules. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7274–7278. doi: 10.1073/pnas.85.19.7274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mehta P. P., Bertram J. S., Loewenstein W. R. Growth inhibition of transformed cells correlates with their junctional communication with normal cells. Cell. 1986 Jan 17;44(1):187–196. doi: 10.1016/0092-8674(86)90497-6. [DOI] [PubMed] [Google Scholar]
  34. Miledi R., Molinoff P., Potter L. T. Isolation of the cholinergic receptor protein of Torpedo electric tissue. Nature. 1971 Feb 19;229(5286):554–557. doi: 10.1038/229554a0. [DOI] [PubMed] [Google Scholar]
  35. Musil L. S., Beyer E. C., Goodenough D. A. Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J Membr Biol. 1990 Jun;116(2):163–175. doi: 10.1007/BF01868674. [DOI] [PubMed] [Google Scholar]
  36. Musil L. S., Cunningham B. A., Edelman G. M., Goodenough D. A. Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J Cell Biol. 1990 Nov;111(5 Pt 1):2077–2088. doi: 10.1083/jcb.111.5.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Musil L. S., Goodenough D. A. Gap junctional intercellular communication and the regulation of connexin expression and function. Curr Opin Cell Biol. 1990 Oct;2(5):875–880. doi: 10.1016/0955-0674(90)90086-t. [DOI] [PubMed] [Google Scholar]
  38. Nagafuchi A., Shirayoshi Y., Okazaki K., Yasuda K., Takeichi M. Transformation of cell adhesion properties by exogenously introduced E-cadherin cDNA. Nature. 1987 Sep 24;329(6137):341–343. doi: 10.1038/329341a0. [DOI] [PubMed] [Google Scholar]
  39. Nelson W. J. Topogenesis of plasma membrane domains in polarized epithelial cells. Curr Opin Cell Biol. 1989 Aug;1(4):660–668. doi: 10.1016/0955-0674(89)90031-8. [DOI] [PubMed] [Google Scholar]
  40. Peracchia C., Peracchia L. L. Gap junction dynamics: reversible effects of divalent cations. J Cell Biol. 1980 Dec;87(3 Pt 1):708–718. doi: 10.1083/jcb.87.3.708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Peters B. P., Brooks M., Hartle R. J., Krzesicki R. F., Perini F., Ruddon R. W. The use of drugs to dissect the pathway for secretion of the glycoprotein hormone chorionic gonadotropin by cultured human trophoblastic cells. J Biol Chem. 1983 Dec 10;258(23):14505–14515. [PubMed] [Google Scholar]
  42. Rook M. B., de Jonge B., Jongsma H. J., Masson-Pévet M. A. Gap junction formation and functional interaction between neonatal rat cardiocytes in culture: a correlative physiological and ultrastructural study. J Membr Biol. 1990 Nov;118(2):179–192. doi: 10.1007/BF01868475. [DOI] [PubMed] [Google Scholar]
  43. Saez J. C., Spray D. C., Nairn A. C., Hertzberg E., Greengard P., Bennett M. V. cAMP increases junctional conductance and stimulates phosphorylation of the 27-kDa principal gap junction polypeptide. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2473–2477. doi: 10.1073/pnas.83.8.2473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sargiacomo M., Lisanti M., Graeve L., Le Bivic A., Rodriguez-Boulan E. Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells. J Membr Biol. 1989 Mar;107(3):277–286. doi: 10.1007/BF01871942. [DOI] [PubMed] [Google Scholar]
  45. Schuetze S. M., Goodenough D. A. Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO2 treatment with development. J Cell Biol. 1982 Mar;92(3):694–705. doi: 10.1083/jcb.92.3.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Spray D. C., Burt J. M. Structure-activity relations of the cardiac gap junction channel. Am J Physiol. 1990 Feb;258(2 Pt 1):C195–C205. doi: 10.1152/ajpcell.1990.258.2.C195. [DOI] [PubMed] [Google Scholar]
  47. Stagg R. B., Fletcher W. H. The hormone-induced regulation of contact-dependent cell-cell communication by phosphorylation. Endocr Rev. 1990 May;11(2):302–325. doi: 10.1210/edrv-11-2-302. [DOI] [PubMed] [Google Scholar]
  48. Stevenson B. R., Paul D. L. The molecular constituents of intercellular junctions. Curr Opin Cell Biol. 1989 Oct;1(5):884–891. doi: 10.1016/0955-0674(89)90054-9. [DOI] [PubMed] [Google Scholar]
  49. Swenson K. I., Piwnica-Worms H., McNamee H., Paul D. L. Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication. Cell Regul. 1990 Dec;1(13):989–1002. doi: 10.1091/mbc.1.13.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Traub O., Look J., Paul D., Willecke K. Cyclic adenosine monophosphate stimulates biosynthesis and phosphorylation of the 26 kDa gap junction protein in cultured mouse hepatocytes. Eur J Cell Biol. 1987 Feb;43(1):48–54. [PubMed] [Google Scholar]
  51. Yamasaki H. Gap junctional intercellular communication and carcinogenesis. Carcinogenesis. 1990 Jul;11(7):1051–1058. doi: 10.1093/carcin/11.7.1051. [DOI] [PubMed] [Google Scholar]
  52. Yu J., Fischman D. A., Steck T. L. Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J Supramol Struct. 1973;1(3):233–248. doi: 10.1002/jss.400010308. [DOI] [PubMed] [Google Scholar]
  53. Zampighi G., Robertson J. D. Fine structure of the synaptic discs separated from the goldfish medulla oblongata. J Cell Biol. 1973 Jan;56(1):92–105. doi: 10.1083/jcb.56.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. el-Fouly M. H., Trosko J. E., Chang C. C. Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp Cell Res. 1987 Feb;168(2):422–430. doi: 10.1016/0014-4827(87)90014-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES