Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Dec 2;115(6):1585–1594. doi: 10.1083/jcb.115.6.1585

Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits

PMCID: PMC2289209  PMID: 1661731

Abstract

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild- type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol- acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Alvarez E., Gironès N., Davis R. J. A point mutation in the cytoplasmic domain of the transferrin receptor inhibits endocytosis. Biochem J. 1990 Apr 1;267(1):31–35. doi: 10.1042/bj2670031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. G., Brown M. S., Goldstein J. L. Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell. 1977 Mar;10(3):351–364. doi: 10.1016/0092-8674(77)90022-8. [DOI] [PubMed] [Google Scholar]
  3. Axelrod D., Koppel D. E., Schlessinger J., Elson E., Webb W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 1976 Sep;16(9):1055–1069. doi: 10.1016/S0006-3495(76)85755-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barak L. S., Webb W. W. Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts. J Cell Biol. 1981 Sep;90(3):595–604. doi: 10.1083/jcb.90.3.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brandtzaeg P. Conjugates of immunoglobulin G with different fluorochromes. I. Characterization by anionic-exchange chromatography. Scand J Immunol. 1973;2(3):273–290. doi: 10.1111/j.1365-3083.1973.tb02037.x. [DOI] [PubMed] [Google Scholar]
  6. Bretscher M. S., Pearse B. M. Coated pits in action. Cell. 1984 Aug;38(1):3–4. doi: 10.1016/0092-8674(84)90519-1. [DOI] [PubMed] [Google Scholar]
  7. Bretscher M. S., Thomson J. N., Pearse B. M. Coated pits act as molecular filters. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4156–4159. doi: 10.1073/pnas.77.7.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen W. J., Goldstein J. L., Brown M. S. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J Biol Chem. 1990 Feb 25;265(6):3116–3123. [PubMed] [Google Scholar]
  9. Chen W. S., Lazar C. S., Lund K. A., Welsh J. B., Chang C. P., Walton G. M., Der C. J., Wiley H. S., Gill G. N., Rosenfeld M. G. Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation. Cell. 1989 Oct 6;59(1):33–43. doi: 10.1016/0092-8674(89)90867-2. [DOI] [PubMed] [Google Scholar]
  10. Collawn J. F., Stangel M., Kuhn L. A., Esekogwu V., Jing S. Q., Trowbridge I. S., Tainer J. A. Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell. 1990 Nov 30;63(5):1061–1072. doi: 10.1016/0092-8674(90)90509-d. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Davoust J., Gruenberg J., Howell K. E. Two threshold values of low pH block endocytosis at different stages. EMBO J. 1987 Dec 1;6(12):3601–3609. doi: 10.1002/j.1460-2075.1987.tb02691.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Doyle C., Roth M. G., Sambrook J., Gething M. J. Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport. J Cell Biol. 1985 Mar;100(3):704–714. doi: 10.1083/jcb.100.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Duband J. L., Nuckolls G. H., Ishihara A., Hasegawa T., Yamada K. M., Thiery J. P., Jacobson K. Fibronectin receptor exhibits high lateral mobility in embryonic locomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells. J Cell Biol. 1988 Oct;107(4):1385–1396. doi: 10.1083/jcb.107.4.1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dunn W. A., Hubbard A. L. Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics. J Cell Biol. 1984 Jun;98(6):2148–2159. doi: 10.1083/jcb.98.6.2148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ellens H., Bentz J., Mason D., Zhang F., White J. M. Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density. Biochemistry. 1990 Oct 16;29(41):9697–9707. doi: 10.1021/bi00493a027. [DOI] [PubMed] [Google Scholar]
  17. Elson E. L., Reidler J. A. Analysis of cell surface interactions by measurements of lateral mobility. J Supramol Struct. 1979;12(4):481–489. doi: 10.1002/jss.400120408. [DOI] [PubMed] [Google Scholar]
  18. Glenney J. R., Jr, Chen W. S., Lazar C. S., Walton G. M., Zokas L. M., Rosenfeld M. G., Gill G. N. Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies. Cell. 1988 Mar 11;52(5):675–684. doi: 10.1016/0092-8674(88)90405-9. [DOI] [PubMed] [Google Scholar]
  19. Goldstein B., Wofsy C., Bell G. Interactions of low density lipoprotein receptors with coated pits on human fibroblasts: estimate of the forward rate constant and comparison with the diffusion limit. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5695–5698. doi: 10.1073/pnas.78.9.5695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldstein J. L., Brown M. S., Anderson R. G., Russell D. W., Schneider W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. doi: 10.1146/annurev.cb.01.110185.000245. [DOI] [PubMed] [Google Scholar]
  21. Henis Y. I., Gutman O. Lateral diffusion and patch formation of H-2Kk antigens on mouse spleen lymphocytes. Biochim Biophys Acta. 1983 Apr 5;762(2):281–288. doi: 10.1016/0167-4889(83)90082-4. [DOI] [PubMed] [Google Scholar]
  22. Henis Y. I., Gutman O., Loyter A. Sendai virus envelope glycoproteins become laterally mobile on the surface of human erythrocytes following fusion. Exp Cell Res. 1985 Oct;160(2):514–526. doi: 10.1016/0014-4827(85)90198-3. [DOI] [PubMed] [Google Scholar]
  23. Henis Y. I., Katzir Z., Shia M. A., Lodish H. F. Oligomeric structure of the human asialoglycoprotein receptor: nature and stoichiometry of mutual complexes containing H1 and H2 polypeptides assessed by fluorescence photobleaching recovery. J Cell Biol. 1990 Oct;111(4):1409–1418. doi: 10.1083/jcb.111.4.1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Henis Y. I. Lateral mobility measurement of cell surface components: applications for molecular pharmacology. Trends Pharmacol Sci. 1989 Mar;10(3):95–98. doi: 10.1016/0165-6147(89)90201-0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Heuser J. Effects of cytoplasmic acidification on clathrin lattice morphology. J Cell Biol. 1989 Feb;108(2):401–411. doi: 10.1083/jcb.108.2.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hopkins C. R., Trowbridge I. S. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells. J Cell Biol. 1983 Aug;97(2):508–521. doi: 10.1083/jcb.97.2.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hubbard A. L. Endocytosis. Curr Opin Cell Biol. 1989 Aug;1(4):675–683. doi: 10.1016/0955-0674(89)90033-1. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Jacobson K., Ishihara A., Inman R. Lateral diffusion of proteins in membranes. Annu Rev Physiol. 1987;49:163–175. doi: 10.1146/annurev.ph.49.030187.001115. [DOI] [PubMed] [Google Scholar]
  31. Jing S. Q., Spencer T., Miller K., Hopkins C., Trowbridge I. S. Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization. J Cell Biol. 1990 Feb;110(2):283–294. doi: 10.1083/jcb.110.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Johnson K. F., Chan W., Kornfeld S. Cation-dependent mannose 6-phosphate receptor contains two internalization signals in its cytoplasmic domain. Proc Natl Acad Sci U S A. 1990 Dec;87(24):10010–10014. doi: 10.1073/pnas.87.24.10010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Koppel D. E., Axelrod D., Schlessinger J., Elson E. L., Webb W. W. Dynamics of fluorescence marker concentration as a probe of mobility. Biophys J. 1976 Nov;16(11):1315–1329. doi: 10.1016/S0006-3495(76)85776-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Koppel D. E., Sheetz M. P. Fluorescence photobleaching does not alter the lateral mobility of erythrocyte membrane glycoproteins. Nature. 1981 Sep 10;293(5828):159–161. doi: 10.1038/293159a0. [DOI] [PubMed] [Google Scholar]
  35. Ktistakis N. T., Thomas D., Roth M. G. Characteristics of the tyrosine recognition signal for internalization of transmembrane surface glycoproteins. J Cell Biol. 1990 Oct;111(4):1393–1407. doi: 10.1083/jcb.111.4.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lazarovits J., Roth M. A single amino acid change in the cytoplasmic domain allows the influenza virus hemagglutinin to be endocytosed through coated pits. Cell. 1988 Jun 3;53(5):743–752. doi: 10.1016/0092-8674(88)90092-x. [DOI] [PubMed] [Google Scholar]
  37. Lehrman M. A., Goldstein J. L., Brown M. S., Russell D. W., Schneider W. J. Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain. Cell. 1985 Jul;41(3):735–743. doi: 10.1016/s0092-8674(85)80054-4. [DOI] [PubMed] [Google Scholar]
  38. Lemansky P., Fatemi S. H., Gorican B., Meyale S., Rossero R., Tartakoff A. M. Dynamics and longevity of the glycolipid-anchored membrane protein, Thy-1. J Cell Biol. 1990 May;110(5):1525–1531. doi: 10.1083/jcb.110.5.1525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Livneh E., Benveniste M., Prywes R., Felder S., Kam Z., Schlessinger J. Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility. J Cell Biol. 1986 Aug;103(2):327–331. doi: 10.1083/jcb.103.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lobel P., Fujimoto K., Ye R. D., Griffiths G., Kornfeld S. Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis. Cell. 1989 Jun 2;57(5):787–796. doi: 10.1016/0092-8674(89)90793-9. [DOI] [PubMed] [Google Scholar]
  41. Lund K. A., Opresko L. K., Starbuck C., Walsh B. J., Wiley H. S. Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization. J Biol Chem. 1990 Sep 15;265(26):15713–15723. [PubMed] [Google Scholar]
  42. McGraw T. E., Pytowski B., Arzt J., Ferrone C. Mutagenesis of the human transferrin receptor: two cytoplasmic phenylalanines are required for efficient internalization and a second-site mutation is capable of reverting an internalization-defective phenotype. J Cell Biol. 1991 Mar;112(5):853–861. doi: 10.1083/jcb.112.5.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Miettinen H. M., Rose J. K., Mellman I. Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity. Cell. 1989 Jul 28;58(2):317–327. doi: 10.1016/0092-8674(89)90846-5. [DOI] [PubMed] [Google Scholar]
  44. Miller K., Shipman M., Trowbridge I. S., Hopkins C. R. Transferrin receptors promote the formation of clathrin lattices. Cell. 1991 May 17;65(4):621–632. doi: 10.1016/0092-8674(91)90094-f. [DOI] [PubMed] [Google Scholar]
  45. Mostov K. E., de Bruyn Kops A., Deitcher D. L. Deletion of the cytoplasmic domain of the polymeric immunoglobulin receptor prevents basolateral localization and endocytosis. Cell. 1986 Nov 7;47(3):359–364. doi: 10.1016/0092-8674(86)90592-1. [DOI] [PubMed] [Google Scholar]
  46. Pearse B. M., Crowther R. A. Structure and assembly of coated vesicles. Annu Rev Biophys Biophys Chem. 1987;16:49–68. doi: 10.1146/annurev.bb.16.060187.000405. [DOI] [PubMed] [Google Scholar]
  47. Pipas J. M., Adler S. P., Peden K. W., Nathans D. Deletion mutants of SV40 that affect the structure of viral tumor antigens. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):285–291. doi: 10.1101/sqb.1980.044.01.032. [DOI] [PubMed] [Google Scholar]
  48. Roth M. G., Doyle C., Sambrook J., Gething M. J. Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway. J Cell Biol. 1986 Apr;102(4):1271–1283. doi: 10.1083/jcb.102.4.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rothenberger S., Iacopetta B. J., Kühn L. C. Endocytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site. Cell. 1987 May 8;49(3):423–431. doi: 10.1016/0092-8674(87)90295-9. [DOI] [PubMed] [Google Scholar]
  50. Saffman P. G., Delbrück M. Brownian motion in biological membranes. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3111–3113. doi: 10.1073/pnas.72.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. 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]
  53. Schlessinger J., Schreiber A. B., Levi A., Lax I., Libermann T., Yarden Y. Regulation of cell proliferation by epidermal growth factor. CRC Crit Rev Biochem. 1983;14(2):93–111. doi: 10.3109/10409238309102791. [DOI] [PubMed] [Google Scholar]
  54. Schmid S. L., Carter L. L. ATP is required for receptor-mediated endocytosis in intact cells. J Cell Biol. 1990 Dec;111(6 Pt 1):2307–2318. doi: 10.1083/jcb.111.6.2307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sheetz M. P., Turney S., Qian H., Elson E. L. Nanometre-level analysis demonstrates that lipid flow does not drive membrane glycoprotein movements. Nature. 1989 Jul 27;340(6231):284–288. doi: 10.1038/340284a0. [DOI] [PubMed] [Google Scholar]
  56. Wolf D. E., Edidin M., Dragsten P. R. Effect of bleaching light on measurements of lateral diffusion in cell membranes by the fluorescence photobleaching recovery method. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2043–2045. doi: 10.1073/pnas.77.4.2043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yamashiro D. J., Tycko B., Fluss S. R., Maxfield F. R. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell. 1984 Jul;37(3):789–800. doi: 10.1016/0092-8674(84)90414-8. [DOI] [PubMed] [Google Scholar]

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

RESOURCES