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. 1981 Feb 1;88(2):430–440. doi: 10.1083/jcb.88.2.430

Spectrin phosphorylation and shape change of human erythrocyte ghosts

PMCID: PMC2111749  PMID: 7204501

Abstract

Human erthrocyte membranes in isotonic medium change shape from crenated spheres to biconcave disks and cup-forms when incubated at 37 degrees C in the presence of MgATP (M. P. Sheetz and S. J. Singer, 1977, J. Cell Biol. 73:638-646). The postulated relationship between spectrin phosphorylation and shape change (W. Birchmeier and S. J. Singer, 1977, J. Cell Biol. 73:647-659) is examined in this report. Salt extraction of white ghosts reduced spectrin phosphorylation during shape changes by 85-95%. Salt extraction did not alter crenation, rate of MgATP-dependent shape change, or the fraction (greater than 80%) ultimately converted to disks and cup-forms after 1 h. Spectrin was partially dephosphorylated in intact cells by subjection to metabolic depletion in vitro. Membranes from depleted cells exhibited normal shape-change behavior. Shape-change behavior was influenced by the hemolysis buffer and temperature and by the time required for membrane preparation. Tris and phosphate ghosts lost the capacity to change shape after standing for 1-2 h at 0 degrees C. Hemolysis in HEPES or N- tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid yielded ghosts that were converted rapidly to disks in the absence of ATP and did not undergo further conversion to cup-forms. These effects could not be attributed to differential dephsphorylation of spectrin, because dephosphorylation during ghost preparation and incubation was negligible. These results suggest that spectrin phosphorylation is not required for MgATP-dependent shape change. It is proposed that other biochemical events induce membrane curvature changes and that the role of spectrin is passive.

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Selected References

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  1. Allan D., Thomas P., Michell R. H. Rapid transbilayer diffusion of 1,2-diacylglycerol and its relevance to control of membrane curvature. Nature. 1978 Nov 16;276(5685):289–290. doi: 10.1038/276289a0. [DOI] [PubMed] [Google Scholar]
  2. Allan D., Watts R., Michell R. H. Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187. Biochem J. 1976 May 15;156(2):225–232. doi: 10.1042/bj1560225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson J. M., Tyler J. M. State of spectrin phosphorylation does not affect erythrocyte shape or spectrin binding to erythrocyte membranes. J Biol Chem. 1980 Feb 25;255(4):1259–1265. [PubMed] [Google Scholar]
  4. Avruch J., Fairbanks G., Crapo L. M. Regulation of plasma membrane protein phosphorylation in two mammalian cell types. J Cell Physiol. 1976 Dec;89(4):815–826. doi: 10.1002/jcp.1040890449. [DOI] [PubMed] [Google Scholar]
  5. Avruch J., Fairbanks G. Phosphorylation of endogenous substrates by erythrocyte membrane protein kinases. I. A monovalent cation-stimulated reaction. Biochemistry. 1974 Dec 31;13(27):5507–5514. doi: 10.1021/bi00724a009. [DOI] [PubMed] [Google Scholar]
  6. Bennett V., Branton D. Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin. J Biol Chem. 1977 Apr 25;252(8):2753–2763. [PubMed] [Google Scholar]
  7. Birchmeier W., Singer S. J. Muscle G-actin is an inhibitor of ATP-induced erythrocyte ghost shape changes and endocytosis. Biochem Biophys Res Commun. 1977 Aug 22;77(4):1354–1360. doi: 10.1016/s0006-291x(77)80128-9. [DOI] [PubMed] [Google Scholar]
  8. Birchmeier W., Singer S. J. On the mechanism of ATP-induced shape changes in human erythrocyte membranes. II. The role of ATP. J Cell Biol. 1977 Jun;73(3):647–659. doi: 10.1083/jcb.73.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bodemann H., Passow H. Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis. J Membr Biol. 1972;8(1):1–26. doi: 10.1007/BF01868092. [DOI] [PubMed] [Google Scholar]
  10. Brailsford J. D., Korpman R. A., Bull B. S. The red cell shape form discocyte to hypotonic spherocyte--a mathematical delineation based on a uniform shell hypothesis. J Theor Biol. 1976 Jul 21;60(01):131–145. doi: 10.1016/0022-5193(76)90159-4. [DOI] [PubMed] [Google Scholar]
  11. Brenner S. L., Korn E. D. Spectrin-actin interaction. Phosphorylated and dephosphorylated spectrin tetramer cross-link F-actin. J Biol Chem. 1979 Sep 10;254(17):8620–8627. [PubMed] [Google Scholar]
  12. Bull B. S., Brailsford J. D. The biconcavity of the red cell: an analysis of several hypotheses. Blood. 1973 Jun;41(6):833–844. [PubMed] [Google Scholar]
  13. Cashel M., Lazzarini R. A., Kalbacher B. An improved method for thin-layer chromatography of nucleotide mixtures containing 32P-labelled orthophosphate. J Chromatogr. 1969 Mar 11;40(1):103–109. doi: 10.1016/s0021-9673(01)96624-5. [DOI] [PubMed] [Google Scholar]
  14. Cohen C. M., Branton D. The role of spectrin in erythrocyte membrane-stimulated actin polymerisation. Nature. 1979 May 10;279(5709):163–165. doi: 10.1038/279163a0. [DOI] [PubMed] [Google Scholar]
  15. Deuling H. J., Helfrich W. Red blood cell shapes as explained on the basis of curvature elasticity. Biophys J. 1976 Aug;16(8):861–868. doi: 10.1016/S0006-3495(76)85736-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dunbar J. C., Ralston G. B. The incorporation of 32 P into spectrin aggregates following incubation of erythrocytes in 32 P-labelled inorganic phosphate. Biochim Biophys Acta. 1978 Jul 4;510(2):283–291. doi: 10.1016/0005-2736(78)90028-7. [DOI] [PubMed] [Google Scholar]
  17. Elgsaeter A., Shotton D. M., Branton D. Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation. Biochim Biophys Acta. 1976 Feb 19;426(1):101–122. doi: 10.1016/0005-2736(76)90433-8. [DOI] [PubMed] [Google Scholar]
  18. England P. J., Walsh D. A. A rapid method for the measurement of [gamma-32P]ATP specific radioactivity in tissue extracts and its application to the study of 32Pi uptake in perfused rat heart. Anal Biochem. 1976 Oct;75(2):429–435. doi: 10.1016/0003-2697(76)90096-8. [DOI] [PubMed] [Google Scholar]
  19. Evans E. A. Bending resistance and chemically induced moments in membrane bilayers. Biophys J. 1974 Dec;14(12):923–931. doi: 10.1016/S0006-3495(74)85959-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fairbanks G., Avruch J., Dino J. E., Patel V. P. Phosphorylation and dephosphorylation of spectrin. J Supramol Struct. 1978;9(1):97–112. doi: 10.1002/jss.400090110. [DOI] [PubMed] [Google Scholar]
  21. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  22. Fenner C., Traut R. R., Mason D. T., Wikman-Coffelt J. Quantification of Coomassie Blue stained proteins in polyacrylamide gels based on analyses of eluted dye. Anal Biochem. 1975 Feb;63(2):595–602. doi: 10.1016/0003-2697(75)90386-3. [DOI] [PubMed] [Google Scholar]
  23. Féo C. J., Leblond P. F. The discocyte-echinocyte transformation: comparison of normal and ATP-enriched human erythrocytes. Blood. 1974 Nov;44(5):639–647. [PubMed] [Google Scholar]
  24. Garrett N. E., Garrett R. J., Talwalkar R. T., Lester R. L. Rapid breakdown of diphosphoinositide and triphosphoinositide in erythrocyte membranes. J Cell Physiol. 1976 Jan;87(1):63–69. doi: 10.1002/jcp.1040870109. [DOI] [PubMed] [Google Scholar]
  25. Graham C., Avruch J., Fairbanks G. Phosphoprotein phosphatase of the human erythrocyte. Biochem Biophys Res Commun. 1976 Sep 20;72(2):701–708. doi: 10.1016/s0006-291x(76)80096-4. [DOI] [PubMed] [Google Scholar]
  26. Gratzer W. B., Beaven G. H. Properties of the high-molecular-weight protein (spectrin) from human-erythrocyte membranes. Eur J Biochem. 1975 Oct 15;58(2):403–409. doi: 10.1111/j.1432-1033.1975.tb02387.x. [DOI] [PubMed] [Google Scholar]
  27. Guthrow C. E., Jr, Allen J. E., Rasmussen H. Phosphorylation of an endogenous membrane protein by an endogenous, membrane-associated cyclic adenosine 3',5'-monophosphate-dependent protein kinase in human erythrocyte ghosts. J Biol Chem. 1972 Dec 25;247(24):8145–8153. [PubMed] [Google Scholar]
  28. HOKIN L. E., HOKIN M. R. THE INCORPORATION OF 32P FROM TRIPHOSPHATE INTO POLYPHOSPHOINOSITIDES (GAMMA-32P)ADENOSINE AND PHOSPHATIDIC ACID IN ERYTHROCYTE MEMBRANES. Biochim Biophys Acta. 1964 Oct 2;84:563–575. doi: 10.1016/0926-6542(64)90126-x. [DOI] [PubMed] [Google Scholar]
  29. Hayashi H., Jarrett H. W., Penniston J. T. Peripheral proteins and smooth membrane from erythrocyte ghosts. Segregation of ATP-utilizing enzymes into smooth membrane. J Cell Biol. 1978 Jan;76(1):105–115. doi: 10.1083/jcb.76.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kirkpatrick F. Spectrin: current understanding of its physical, biochemical, and functional properties. Life Sci. 1976 Jul 1;19(1):1–17. doi: 10.1016/0024-3205(76)90368-4. [DOI] [PubMed] [Google Scholar]
  31. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  32. Lux S. E., John K. M., Karnovsky M. J. Irreversible deformation of the spectrin-actin lattice in irreversibly sickled cells. J Clin Invest. 1976 Oct;58(4):955–963. doi: 10.1172/JCI108549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lux S. E. Spectrin-actin membrane skeleton of normal and abnormal red blood cells. Semin Hematol. 1979 Jan;16(1):21–51. [PubMed] [Google Scholar]
  34. Meiselman H. J., Evans E. A., Hochmuth R. M. Membrane mechanical properties of ATP-depleted human erythrocytes. Blood. 1978 Sep;52(3):499–504. [PubMed] [Google Scholar]
  35. NAKAO M., NAKAO T., YAMAZOE S. Adenosine triphosphate and maintenance of shape of the human red cells. Nature. 1960 Sep 10;187:945–946. doi: 10.1038/187945a0. [DOI] [PubMed] [Google Scholar]
  36. Palmer F. B., Verpoorte J. A. The phosphorus components of solubilized erythrocyte membrane protein. Can J Biochem. 1971 Mar;49(3):337–347. doi: 10.1139/o71-050. [DOI] [PubMed] [Google Scholar]
  37. Roses A. D., Appel S. H. Erythrocyte protein phosphorylation. J Biol Chem. 1973 Feb 25;248(4):1408–1411. [PubMed] [Google Scholar]
  38. Rubin C. S., Rosen O. M. The role of cyclic AMP in the phosphorylation of proteins in human erythrocyte membranes. Biochem Biophys Res Commun. 1973 Jan 23;50(2):421–429. doi: 10.1016/0006-291x(73)90857-7. [DOI] [PubMed] [Google Scholar]
  39. Sheetz M. P. Cation effects on cell shape. Prog Clin Biol Res. 1977;17:559–567. [PubMed] [Google Scholar]
  40. Sheetz M. P., Koppel D. E. Membrane damage caused by irradiation of fluorescent concanavalin A. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3314–3317. doi: 10.1073/pnas.76.7.3314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sheetz M. P., Painter R. G., Singer S. J. Relationships of the spectrin complex of human erythrocyte membranes to the actomyosins of muscle cells. Biochemistry. 1976 Oct 5;15(20):4486–4492. doi: 10.1021/bi00665a024. [DOI] [PubMed] [Google Scholar]
  42. Sheetz M. P., Sawyer D., Jackowski S. The ATP-dependent red cell membrane shape change: a molecular explanation. Prog Clin Biol Res. 1978;21:431–452. [PubMed] [Google Scholar]
  43. Sheetz M. P., Singer S. J. Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4457–4461. doi: 10.1073/pnas.71.11.4457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sheetz M. P., Singer S. J. On the mechanism of ATP-induced shape changes in human erythrocyte membranes. I. The role of the spectrin complex. J Cell Biol. 1977 Jun;73(3):638–646. doi: 10.1083/jcb.73.3.638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Steck T. L., Kant J. A. Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes. Methods Enzymol. 1974;31:172–180. doi: 10.1016/0076-6879(74)31019-1. [DOI] [PubMed] [Google Scholar]
  46. Steck T. L. The organization of proteins in the human red blood cell membrane. A review. J Cell Biol. 1974 Jul;62(1):1–19. doi: 10.1083/jcb.62.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tokuyasu K. T., Schekman R., Singer S. J. Domains of receptor mobility and endocytosis in the membranes of neonatal human erythrocytes and reticulocytes are deficient in spectrin. J Cell Biol. 1979 Feb;80(2):481–486. doi: 10.1083/jcb.80.2.481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ungewickell E., Gratzer W. Self-association of human spectrin. A thermodynamic and kinetic study. Eur J Biochem. 1978 Aug 1;88(2):379–385. doi: 10.1111/j.1432-1033.1978.tb12459.x. [DOI] [PubMed] [Google Scholar]
  49. Vickers J. D., Brierley J., Rathbone M. P. Phosphorylation of casein by human erythrocyte membrane-bound protein kinase: competition of casein with endogenous substrates. J Membr Biol. 1979 Aug;49(2):123–138. doi: 10.1007/BF01868721. [DOI] [PubMed] [Google Scholar]
  50. Weed R. I., LaCelle P. L., Merrill E. W. Metabolic dependence of red cell deformability. J Clin Invest. 1969 May;48(5):795–809. doi: 10.1172/JCI106038. [DOI] [PMC free article] [PubMed] [Google Scholar]

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