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. 1981 Dec 1;91(3):637–646. doi: 10.1083/jcb.91.3.637

Erythrocyte membrane protein band 3: its biosynthesis and incorporation into membranes

PMCID: PMC2112817  PMID: 7328113

Abstract

Band 3, a transmembrane protein that provides the anion channel of the erythrocyte plasma membrane, crosses the membrane more than once and has a large amino terminal segment exposes on the cytoplasmic side of the membrane. The biosynthesis of band 3 and the process of its incorporation into membranes were studied in vivo in erythroid spleen cells of anemic mice and in vitro in protein synthesizing cell-free systems programmed with polysomes and messenger RNA (mRNA). In intact cells newly synthesized band 3 is rapidly incorporated into intracellular membranes where it is glycosylated and it is subsequently transferred to the plasma membrane where it becomes sensitive to digestion by exogenous chymotrypsin. The appearance of band 3 in the cell surface is not contingent upon its glycosylation because it proceeds efficiently in cells treated with tunicamycin. The site of synthesis of band 3 in bound polysomes was established directly by in vitro translation experiments with purified polysomes or with mRNA extracted from them. The band-3 polypeptide synthesized in an mRNA- dependent system had the same electrophoretic mobility as that synthesized in cells treated with tunicamycin. When microsomal membranes were present during translation, the in vitro synthesized band-3 polypeptide was cotranslationally glycosylated and inserted into the membranes. This was inferred from the facts that when synthesis was carried out in the presence of membranes the product had a lower electrophoretic mobility and showed partial resistance to protease digestion. Our observations indicate that the primary translation product of band-3 mRNA is not proteolytically processed either co- or posttranslationally. It is, therefore, proposed that the incorporation of band 3 into the endoplasmic reticulum (ER) membrane is initiated by a permanent insertion signal. To account for the cytoplasmic exposure of the amino terminus of the polypeptide we suggest that this signal is located within the interior of the polypeptide. a mechanism that explains the final transmembrane disposition of band 3 in the plasma membrane as resulting from the mode of its incorporation into the ER is presented.

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Selected References

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  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boime I., Szczesna E., Smith D. Membrane-dependent cleavage of the human placental lactogen precursor to its native form in ascites cell-free extracts. Eur J Biochem. 1977 Mar 1;73(2):515–520. doi: 10.1111/j.1432-1033.1977.tb11345.x. [DOI] [PubMed] [Google Scholar]
  4. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. Bretscher M. S. A major protein which spans the human erythrocyte membrane. J Mol Biol. 1971 Jul 28;59(2):351–357. doi: 10.1016/0022-2836(71)90055-6. [DOI] [PubMed] [Google Scholar]
  6. Cabantchik Z. I., Rothstein A. Membrane proteins related to anion permeability of human red blood cells. I. Localization of disulfonic stilbene binding sites in proteins involved in permeation. J Membr Biol. 1974;15(3):207–226. doi: 10.1007/BF01870088. [DOI] [PubMed] [Google Scholar]
  7. Chang H., Langer P. J., Lodish H. F. Asynchronous synthesis of erythrocyte membrane proteins. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3206–3210. doi: 10.1073/pnas.73.9.3206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng T. C., Kazazian H. H., Jr Unequal accumulation of alpha- and beta-globin mRNA in erythropoietic mouse spleen. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1811–1815. doi: 10.1073/pnas.73.6.1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng T., Polmar S. K., Kazazian H. H., Jr Isolation and characterization of modified globin messenger ribonucleic acid from erythropoietic mouse spleen. J Biol Chem. 1974 Mar 25;249(6):1781–1786. [PubMed] [Google Scholar]
  10. Conkie D., Kleiman L., Harrison P. R., Paul J. Increase in the accumulation of globin mRNA in immature erythroblasts in response to erythropoietin in vivo or in vitro. Exp Cell Res. 1975 Jul;93(2):315–324. doi: 10.1016/0014-4827(75)90456-5. [DOI] [PubMed] [Google Scholar]
  11. Cotmore S. F., Furthmayr H., Marchesi V. T. Immunochemical evidence for the transmembrane orientation of glycophorin A. Localization of ferritin-antibody conjugates in intact cells. J Mol Biol. 1977 Jul 5;113(3):539–553. doi: 10.1016/0022-2836(77)90237-6. [DOI] [PubMed] [Google Scholar]
  12. Dickerman H. W., Cheng T. C., Kazazian H. H., Jr, Spivak J. L. The erythropoietic mouse spleen-a model system of development. Arch Biochem Biophys. 1976 Nov;177(1):1–9. doi: 10.1016/0003-9861(76)90408-2. [DOI] [PubMed] [Google Scholar]
  13. Drickamer L. K. Fragmentation of the band 3 polypeptide from human erythrocyte membranes. Identification of regions likely to interact with the lipid bilayer. J Biol Chem. 1977 Oct 10;252(19):6909–6917. [PubMed] [Google Scholar]
  14. Drickamer L. K. Orientation of the band 3 polypeptide from human erythrocyte membranes. Identification of NH2-terminal sequence and site of carbohydrate attachment. J Biol Chem. 1978 Oct 25;253(20):7242–7248. [PubMed] [Google Scholar]
  15. Duksin D., Bornstein P. Impaired conversion of procollagen to collagen by fibroblasts and bone treated with tunicamycin, an inhibitor of protein glycosylation. J Biol Chem. 1977 Feb 10;252(3):955–962. [PubMed] [Google Scholar]
  16. Eisen H., Bach R., Emery R. Induction of spectrin in erythroleukemic cells transformed by Friend virus. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3898–3902. doi: 10.1073/pnas.74.9.3898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Fukuda M., Eshdat Y., Tarone G., Marchesi V. T. Isolation and characterization of peptides derived from the cytoplasmic segment of band 3, the predominant intrinsic membrane protein of the human erythrocyte. J Biol Chem. 1978 Apr 10;253(7):2419–2428. [PubMed] [Google Scholar]
  19. Gahmberg C. G., Jokinen M., Andersson L. C. Expression of the major red cell sialoglycoprotein, glycophorin A, in the human leukemic cell line K562. J Biol Chem. 1979 Aug 10;254(15):7442–7448. [PubMed] [Google Scholar]
  20. Gahmberg C. G., Jokinen M., Karhi K. K., Andersson L. C. Effect of tunicamycin on the biosynthesis of the major human red cell sialoglycoprotein, glycophorin A, in the leukemia cell line K562. J Biol Chem. 1980 Mar 10;255(5):2169–2175. [PubMed] [Google Scholar]
  21. Gibson R., Leavitt R., Kornfeld S., Schlesinger S. Synthesis and infectivity of vesicular stomatitis virus containing nonglycosylated G protein. Cell. 1978 Apr;13(4):671–679. doi: 10.1016/0092-8674(78)90217-9. [DOI] [PubMed] [Google Scholar]
  22. Gibson R., Schlesinger S., Kornfeld S. The nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures. J Biol Chem. 1979 May 10;254(9):3600–3607. [PubMed] [Google Scholar]
  23. Goldman B. M., Blobel G. Biogenesis of peroxisomes: intracellular site of synthesis of catalase and uricase. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5066–5070. doi: 10.1073/pnas.75.10.5066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Green R. F., Meiss H. K., Rodriguez-Boulan E. Glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells. J Cell Biol. 1981 May;89(2):230–239. doi: 10.1083/jcb.89.2.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. HAM R. G. CLONAL GROWTH OF MAMMALIAN CELLS IN A CHEMICALLY DEFINED, SYNTHETIC MEDIUM. Proc Natl Acad Sci U S A. 1965 Feb;53:288–293. doi: 10.1073/pnas.53.2.288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hickman S., Kulczycki A., Jr, Lynch R. G., Kornfeld S. Studies of the mechanism of tunicamycin in hibition of IgA and IgE secretion by plasma cells. J Biol Chem. 1977 Jun 25;252(12):4402–4408. [PubMed] [Google Scholar]
  27. Inouye M., Halegoua S. Secretion and membrane localization of proteins in Escherichia coli. CRC Crit Rev Biochem. 1980;7(4):339–371. doi: 10.3109/10409238009105465. [DOI] [PubMed] [Google Scholar]
  28. Jacobson B. S., Branton D. Plasma membrane: rapid isolation and exposure of the cytoplasmic surface by use of positively charged beads. Science. 1977 Jan 21;195(4275):302–304. doi: 10.1126/science.831278. [DOI] [PubMed] [Google Scholar]
  29. Jenkins R. E., Tanner J. A. The major human erythrocyte membrane protein. Evidence for an S-shaped structure which traverses the membrane twice and contains a duplicated set of sites. Biochem J. 1975 Jun;147(3):393–399. doi: 10.1042/bj1470393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Katz F. N., Rothman J. E., Knipe D. M., Lodish H. F. Membrane assembly: synthesis and intracellular processing of the vesicular stomatitis viral glycoprotein. J Supramol Struct. 1977;7(3-4):353–370. doi: 10.1002/jss.400070308. [DOI] [PubMed] [Google Scholar]
  31. Kazazian H. H., Jr, cheng T., Polmar S. K., Ginder G. D. Globin mRNA of mouse spleen erythroblasts. Ann N Y Acad Sci. 1974 Nov 29;241(0):170–182. doi: 10.1111/j.1749-6632.1974.tb21876.x. [DOI] [PubMed] [Google Scholar]
  32. Kehry M., Ewald S., Douglas R., Sibley C., Raschke W., Fambrough D., Hood L. The immunoglobulin mu chains of membrane-bound and secreted IgM molecules differ in their C-terminal segments. Cell. 1980 Sep;21(2):393–406. doi: 10.1016/0092-8674(80)90476-6. [DOI] [PubMed] [Google Scholar]
  33. Kiehm D. J., Ji T. H. Photochemical cross-linking of cell membranes. A test for natural and random collisional cross-links by millisecond cross-linking. J Biol Chem. 1977 Dec 10;252(23):8524–8531. [PubMed] [Google Scholar]
  34. Kreibich G., Czakó-Graham M., Grebenau R. C., Sabatini D. D. Functional and structural characteristics of endoplasmic reticulum proteins associated with ribosome binding sites. Ann N Y Acad Sci. 1980;343:17–33. doi: 10.1111/j.1749-6632.1980.tb47239.x. [DOI] [PubMed] [Google Scholar]
  35. Kreibich G., Sabatini D. D. Selective release of content from microsomal vesicles without membrane disassembly. II. Electrophoretic and immunological characterization of microsomal subfractions. J Cell Biol. 1974 Jun;61(3):789–807. doi: 10.1083/jcb.61.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lingappa V. R., Katz F. N., Lodish H. F., Blobel G. A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function. J Biol Chem. 1978 Dec 25;253(24):8667–8670. [PubMed] [Google Scholar]
  37. Marchesi V. T., Furthmayr H., Tomita M. The red cell membrane. Annu Rev Biochem. 1976;45:667–698. doi: 10.1146/annurev.bi.45.070176.003315. [DOI] [PubMed] [Google Scholar]
  38. Merkel C. G., Kwan S., Lingrel J. B. Size of the polyadenylic acid region of newly synthesized globin messenger ribonucleic acid. J Biol Chem. 1975 May 25;250(10):3725–3728. [PubMed] [Google Scholar]
  39. Palmiter R. D., Gagnon J., Ericsson L. H., Walsh K. A. Precursor of egg white lysozyme. Amino acid sequence of an NH2-terminal extension. J Biol Chem. 1977 Sep 25;252(18):6386–6393. [PubMed] [Google Scholar]
  40. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  41. Peterson P. A., Rask L., Lindblom J. B. Highly purified papain-solubilized HL-A antigens contain beta2-microglobulin. Proc Natl Acad Sci U S A. 1974 Jan;71(1):35–39. doi: 10.1073/pnas.71.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ramsey J. C., Steele W. J. A method for visualizing and quantifying the total complement of free and membrane-bound ribosomes in rat liver. Anal Biochem. 1979 Jan 15;92(2):305–313. doi: 10.1016/0003-2697(79)90663-8. [DOI] [PubMed] [Google Scholar]
  43. Ramsey J. C., Steele W. J. A procedure for the quantitative recovery of homogeneous populations of undegraded free and bound polysomes from rat liver. Biochemistry. 1976 Apr 20;15(8):1704–1712. doi: 10.1021/bi00653a018. [DOI] [PubMed] [Google Scholar]
  44. Ramsey J. C., Steele W. J. Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes. Biochem J. 1977 Oct 15;168(1):1–8. doi: 10.1042/bj1680001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Roman R., Brooker J. D., Seal S. N., Marcus A. Inhibition of the transition of a 40 S ribosome-Met-tRNA-i-Met complex to an 80 S ribosome-Met-tRNA-i-Met- complex by 7-Methylguanosine-5'-phosphate. Nature. 1976 Mar 25;260(5549):359–360. doi: 10.1038/260359a0. [DOI] [PubMed] [Google Scholar]
  46. Rose J. K., Welch W. J., Sefton B. M., Esch F. S., Ling N. C. Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3884–3888. doi: 10.1073/pnas.77.7.3884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Roth M. G., Fitzpatrick J. P., Compans R. W. Polarity of influenza and vesicular stomatitis virus maturation in MDCK cells: lack of a requirement for glycosylation of viral glycoproteins. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6430–6434. doi: 10.1073/pnas.76.12.6430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rothman J. E., Lodish H. F. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature. 1977 Oct 27;269(5631):775–780. doi: 10.1038/269775a0. [DOI] [PubMed] [Google Scholar]
  49. Sabban E. L., Sabatini D. D., Marchesi V. T., Adesnik M. Biosynthesis of erythrocyte membrane protein band 3 in DMSO-induced Friend erythroleukemia cells. J Cell Physiol. 1980 Aug;104(2):261–268. doi: 10.1002/jcp.1041040217. [DOI] [PubMed] [Google Scholar]
  50. Shields D., Blobel G. Efficient cleavage and segregation of nascent presecretory proteins in a reticulocyte lysate supplemented with microsomal membranes. J Biol Chem. 1978 Jun 10;253(11):3753–3756. [PubMed] [Google Scholar]
  51. Spivak J. L., Marmor J., Dickerman H. W. Studies on splenic erythropoiesis in the mouse. I. Ribosomal ribonucleic acid metabolism. J Lab Clin Med. 1972 Apr;79(4):526–540. [PubMed] [Google Scholar]
  52. Steck T. L. Cross-linking the major proteins of the isolated erythrocyte membrane. J Mol Biol. 1972 May 14;66(2):295–305. doi: 10.1016/0022-2836(72)90481-0. [DOI] [PubMed] [Google Scholar]
  53. Steck T. L., Ramos B., Strapazon E. Proteolytic dissection of band 3, the predominant transmembrane polypeptide of the human erythrocyte membrane. Biochemistry. 1976 Mar 9;15(5):1153–1161. doi: 10.1021/bi00650a030. [DOI] [PubMed] [Google Scholar]
  54. Steck T. L. The band 3 protein of the human red cell membrane: a review. J Supramol Struct. 1978;8(3):311–324. doi: 10.1002/jss.400080309. [DOI] [PubMed] [Google Scholar]
  55. Steiner D. F., Quinn P. S., Chan S. J., Marsh J., Tager H. S. Processing mechanisms in the biosynthesis of proteins. Ann N Y Acad Sci. 1980;343:1–16. doi: 10.1111/j.1749-6632.1980.tb47238.x. [DOI] [PubMed] [Google Scholar]
  56. Struck D. K., Lennarz W. J. Evidence for the participation of saccharide-lipids in the synthesis of the oligosaccharide chain of ovalbumin. J Biol Chem. 1977 Feb 10;252(3):1007–1013. [PubMed] [Google Scholar]
  57. Tkacz J. S., Lampen O. Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem Biophys Res Commun. 1975 Jul 8;65(1):248–257. doi: 10.1016/s0006-291x(75)80086-6. [DOI] [PubMed] [Google Scholar]
  58. Tomita M., Marchesi V. T. Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2964–2968. doi: 10.1073/pnas.72.8.2964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tsuji T., Irimura T., Osawa T. The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes. Biochem J. 1980 Jun 1;187(3):677–686. doi: 10.1042/bj1870677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wang K., Richards F. M. An approach to nearest neighbor analysis of membrane proteins. Application to the human erythrocyte membrane of a method employing cleavable cross-linkages. J Biol Chem. 1974 Dec 25;249(24):8005–8018. [PubMed] [Google Scholar]
  61. Yamane K., Nathenson S. G. Biochemical similarity of papain-solubilized H-2d alloantigens from tumor cells and from normal cells. Biochemistry. 1970 Nov 24;9(24):4743–4750. doi: 10.1021/bi00826a018. [DOI] [PubMed] [Google Scholar]

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