Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Jul 1;107(1):191–200. doi: 10.1083/jcb.107.1.191

Properties and topography of the major integral plasma membrane protein of a unicellular organism

PMCID: PMC2115185  PMID: 3134363

Abstract

The cellular distribution, membrane orientation, and biochemical properties of the two major NaOH-insoluble (integral) plasma membrane proteins of Euglena are detailed. We present evidence which suggests that these two polypeptides (Mr 68 and 39 kD) are dimer and monomer of the same protein: (a) Antibodies directed against either the 68- or the 39-kD polypeptide bind to both 68- and 39-kD bands in Western blots. (b) Trypsin digests of the 68- and 39-kD polypeptides yield similar peptide fragments. (c) The 68- and 39-kD polypeptides interconvert during successive electrophoresis runs in the presence of SDS and beta- mercaptoethanol. (d) The 39-kD band is the only major integral membrane protein evident after isoelectric focusing in acrylamide gels. The apparent shift from 68 to 39 kD in focusing gels has been duplicated in denaturing SDS gels by adding ampholyte solutions directly to the protein samples. The membrane orientation of the 39-kD protein and its 68-kD dimer has been assessed by radioiodination in situ using intact cells or purified plasma membranes. Putative monomers and dimers are labeled only when the cytoplasmic side of the membrane is exposed. These results together with trypsin digestion data suggest that the 39- kD protein and its dimer have an asymmetric membrane orientation with a substantial cytoplasmic domain but with no detectable extracellular region. Immunolabeling of sectioned cells indicates that the plasma membrane is the only cellular membrane with significant amounts of 39- kD protein. No major 68- or 39-kD polypeptide bands are evident in SDS acrylamide gels or immunoblots of electrophoresed whole flagella or preparations enriched in flagellar membrane vesicles, nor is there a detectable shift in any flagellar polypeptide in the presence of ampholyte solutions. These findings are considered with respect to the well-known internal crystalline organization of the euglenoid plasma membrane and to the potential for these proteins to serve as anchors for membrane skeletal proteins.

Full Text

The Full Text of this article is available as a PDF (3.0 MB).

Selected References

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

  1. Altman L. G., Schneider B. G., Papermaster D. S. Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy. J Histochem Cytochem. 1984 Nov;32(11):1217–1223. doi: 10.1177/32.11.6436366. [DOI] [PubMed] [Google Scholar]
  2. Bennett H., Condeelis J. A gradient in the density of intramembrane particles is formed during capping induced by concanavalin A. J Cell Sci. 1986 Jul;83:61–76. doi: 10.1242/jcs.83.1.61. [DOI] [PubMed] [Google Scholar]
  3. Bennett V. The membrane skeleton of human erythrocytes and its implications for more complex cells. Annu Rev Biochem. 1985;54:273–304. doi: 10.1146/annurev.bi.54.070185.001421. [DOI] [PubMed] [Google Scholar]
  4. Brown S. S., Petzold A. S. Using antibodies against Dictyostelium membranes to identify an actin-binding membrane protein. J Cell Biol. 1987 Mar;104(3):513–518. doi: 10.1083/jcb.104.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen S. J., Bouck G. B. Endogenous glycosyltransferases glucosylate lipids in flagella of Euglena. J Cell Biol. 1984 May;98(5):1825–1835. doi: 10.1083/jcb.98.5.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clarke S. The size and detergent binding of membrane proteins. J Biol Chem. 1975 Jul 25;250(14):5459–5469. [PubMed] [Google Scholar]
  7. Dubreuil R. R., Bouck G. B. The membrane skeleton of a unicellular organism consists of bridged, articulating strips. J Cell Biol. 1985 Nov;101(5 Pt 1):1884–1896. doi: 10.1083/jcb.101.5.1884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elder J. H., Pickett R. A., 2nd, Hampton J., Lerner R. A. Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. J Biol Chem. 1977 Sep 25;252(18):6510–6515. [PubMed] [Google Scholar]
  9. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  10. Furthmayr H., Marchesi V. T. Subunit structure of human erythrocyte glycophorin A. Biochemistry. 1976 Mar 9;15(5):1137–1144. doi: 10.1021/bi00650a028. [DOI] [PubMed] [Google Scholar]
  11. Granger B. L., Lazarides E. Desmin and vimentin coexist at the periphery of the myofibril Z disc. Cell. 1979 Dec;18(4):1053–1063. doi: 10.1016/0092-8674(79)90218-6. [DOI] [PubMed] [Google Scholar]
  12. Granger B. L., Lazarides E. Membrane skeletal protein 4.1 of avian erythrocytes is composed of multiple variants that exhibit tissue-specific expression. Cell. 1984 Jun;37(2):595–607. doi: 10.1016/0092-8674(84)90390-8. [DOI] [PubMed] [Google Scholar]
  13. Helenius A., Simons K. Charge shift electrophoresis: simple method for distinguishing between amphiphilic and hydrophilic proteins in detergent solution. Proc Natl Acad Sci U S A. 1977 Feb;74(2):529–532. doi: 10.1073/pnas.74.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hofmann C., Bouck G. B. Immunological and structural evidence for patterned intussusceptive surface growth in a unicellular organism. A postulated role for submembranous proteins and microtubules. J Cell Biol. 1976 Jun;69(3):693–715. doi: 10.1083/jcb.69.3.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnson G. D., Nogueira Araujo G. M. A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods. 1981;43(3):349–350. doi: 10.1016/0022-1759(81)90183-6. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Lane L. C. A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis. Anal Biochem. 1978 Jun 1;86(2):655–664. doi: 10.1016/0003-2697(78)90792-3. [DOI] [PubMed] [Google Scholar]
  18. Lefort-Tran M., Bre M. H., Ranck J. L., Pouphile M. Euglena plasma membrane during normal and vitamin B12 starvation growth. J Cell Sci. 1980 Feb;41:245–261. doi: 10.1242/jcs.41.1.245. [DOI] [PubMed] [Google Scholar]
  19. Melkonian M., Robenek H., Rassat J. Flagellar membrane specializations and their relationship to mastigonemes and microtubules in Euglena gracilis. J Cell Sci. 1982 Jun;55:115–135. doi: 10.1242/jcs.55.1.115. [DOI] [PubMed] [Google Scholar]
  20. Miller K. R., Miller G. J. Organization of the cell membrane in Euglena. Protoplasma. 1978;95(1-2):11–24. doi: 10.1007/BF01279691. [DOI] [PubMed] [Google Scholar]
  21. Murray J. M. Disassembly and reconstitution of a membrane-microtubule complex. J Cell Biol. 1984 Apr;98(4):1481–1487. doi: 10.1083/jcb.98.4.1481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Murray J. M. Three-dimensional structure of a membrane-microtubule complex. J Cell Biol. 1984 Jan;98(1):283–295. doi: 10.1083/jcb.98.1.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Newby A. C., Chrambach A. Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents. Biochem J. 1979 Feb 1;177(2):623–630. doi: 10.1042/bj1770623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Prat J. P., Lamy J. N., Weill J. D. Coloration des lipoprotéines après électrophorèse en gel de polyacrylamide. Bull Soc Chim Biol (Paris) 1969 Dec 18;51(9):1367–1367. [PubMed] [Google Scholar]
  25. Rogalski A. A., Bouck G. B. Characterization and localization of a flagellar-specific membrane glycoprotein in Euglena. J Cell Biol. 1980 Aug;86(2):424–435. doi: 10.1083/jcb.86.2.424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sleytr U. B., Robards A. W. Plastic deformation during freeze-cleavage: a review. J Microsc. 1977 May;110(1):1–25. doi: 10.1111/j.1365-2818.1977.tb00009.x. [DOI] [PubMed] [Google Scholar]
  27. Steck T. L., Yu J. Selective solubilization of proteins from red blood cell membranes by protein perturbants. J Supramol Struct. 1973;1(3):220–232. doi: 10.1002/jss.400010307. [DOI] [PubMed] [Google Scholar]
  28. Switzer R. C., 3rd, Merril C. R., Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem. 1979 Sep 15;98(1):231–237. doi: 10.1016/0003-2697(79)90732-2. [DOI] [PubMed] [Google Scholar]
  29. Tijssen P., Kurstak E. An efficient two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis method for simultaneous peptide mapping of protein contained in a mixture. Anal Biochem. 1983 Jan;128(1):26–35. doi: 10.1016/0003-2697(83)90339-1. [DOI] [PubMed] [Google Scholar]
  30. Tokuyasu K. T. A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol. 1973 May;57(2):551–565. doi: 10.1083/jcb.57.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]

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

RESOURCES