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. 1985 Feb 1;100(2):574–587. doi: 10.1083/jcb.100.2.574

Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin

PMCID: PMC2113453  PMID: 3155750

Abstract

Isolated Xenopus laevis retinas were incubated with 3H-labeled mannose or leucine in the presence or absence of tunicamycin (TM), a selective inhibitor of dolichyl phosphate-dependent protein glycosylation. At a TM concentration of 20 micrograms/ml, the incorporation of [3H]mannose and [3H]leucine into retinal macromolecules was inhibited by approximately 66 and 12-16%, respectively, relative to controls. Cellular uptake of the radiolabeled substrates was not inhibited at this TM concentration. Polyacrylamide gel electrophoresis revealed that TM had little effect on the incorporation of [3H]leucine into the proteins of whole retinas and that labeling of proteins (especially opsin) in isolated rod outer segment (ROS) membranes was negligible. The incorporation of [3H]mannose into proteins of whole retinas and ROS membranes was nearly abolished in the presence of TM. Autoradiograms of control retinas incubated with either [3H]mannose or [3H]leucine exhibited a discrete concentration of silver grains over ROS basal disc membranes. In TM-treated retinas, the extracellular space between rod inner and outer segments was dilated and filled with numerous heterogeneously size vesicles, which were labeled with [3H]leucine but not with [3H]mannose. ROS disc membranes per se were not labeled in the TM-treated retinas. Quantitative light microscopic autoradiography of retinas pulse-labeled with [3H]leucine showed no differences in labeling of rod cellular compartments in the presence or absence of TM as a function of increasing chase time. These results demonstrate that TM can block retinal protein glycosylation and normal disc membrane assembly under conditions where synthesis and intracellular transport of rod cell proteins (e.g., opsin) are not inhibited.

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Selected References

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  1. Ashwell G., Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531–554. doi: 10.1146/annurev.bi.51.070182.002531. [DOI] [PubMed] [Google Scholar]
  2. Barondes S. H. Soluble lectins: a new class of extracellular proteins. Science. 1984 Mar 23;223(4642):1259–1264. doi: 10.1126/science.6367039. [DOI] [PubMed] [Google Scholar]
  3. Basinger S. F., Hall M. O. Rhodopsin biosynthesis in vitro. Biochemistry. 1973 May 8;12(10):1996–2003. doi: 10.1021/bi00734a025. [DOI] [PubMed] [Google Scholar]
  4. Bernard B. A., Yamada K. M., Olden K. Carbohydrates selectively protect a specific domain of fibronectin against proteases. J Biol Chem. 1982 Jul 25;257(14):8549–8554. [PubMed] [Google Scholar]
  5. Besharse J. C., Hollyfield J. G., Rayborn M. E. Photoreceptor outer segments: accelerated membrane renewal in rods after exposure to light. Science. 1977 Apr 29;196(4289):536–538. doi: 10.1126/science.300504. [DOI] [PubMed] [Google Scholar]
  6. Besharse J. C., Pfenninger K. H. Membrane assembly in retinal photoreceptors I. Freeze-fracture analysis of cytoplasmic vesicles in relationship to disc assembly. J Cell Biol. 1980 Nov;87(2 Pt 1):451–463. doi: 10.1083/jcb.87.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bok D., Basinger S. F., Hall M. O. Proceedings: Autoradiographic and radiobiochemical studies on the incorporation of (6-3H)glucosamine into frog rhodopsin. Exp Eye Res. 1974 Mar;18(3):225–240. doi: 10.1016/0014-4835(74)90151-1. [DOI] [PubMed] [Google Scholar]
  8. Duksin D., Mahoney W. C. Relationship of the structure and biological activity of the natural homologues of tunicamycin. J Biol Chem. 1982 Mar 25;257(6):3105–3109. [PubMed] [Google Scholar]
  9. Fliesler S. J., Tabor G. A., Hollyfield J. G. Glycoprotein synthesis in the human retina: localization of the lipid intermediate pathway. Exp Eye Res. 1984 Aug;39(2):153–173. doi: 10.1016/0014-4835(84)90005-8. [DOI] [PubMed] [Google Scholar]
  10. Fukuda M. N., Papermaster D. S., Hargrave P. A. Rhodopsin carbohydrate. Structure of small oligosaccharides attached at two sites near the NH2 terminus. J Biol Chem. 1979 Sep 10;254(17):8201–8207. [PubMed] [Google Scholar]
  11. Goldman B. M., Blobel G. In vitro biosynthesis, core glycosylation, and membrane integration of opsin. J Cell Biol. 1981 Jul;90(1):236–242. doi: 10.1083/jcb.90.1.236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hall M. O., Bok D., Bacharach A. D. Biosynthesis and assembly of the rod outer segment membrane system. Formation and fate of visual pigment in the frog retina. J Mol Biol. 1969 Oct 28;45(2):397–406. doi: 10.1016/0022-2836(69)90114-4. [DOI] [PubMed] [Google Scholar]
  13. Heath A. R., Basinger S. F. Simple sugars inhibit rod outer segment disc shedding by the frog retina. Vision Res. 1983;23(12):1371–1377. doi: 10.1016/0042-6989(83)90148-7. [DOI] [PubMed] [Google Scholar]
  14. Heller J., Lawrence M. A. Structure of the glycopeptide from bovine visual pigment 500. Biochemistry. 1970 Feb 17;9(4):864–869. doi: 10.1021/bi00806a021. [DOI] [PubMed] [Google Scholar]
  15. Heller J. Structure of visual pigments. I. Purification, molecular weight, and composition of bovine visual pigment500. Biochemistry. 1968 Aug;7(8):2906–2913. doi: 10.1021/bi00848a030. [DOI] [PubMed] [Google Scholar]
  16. Hollyfield J. G., Besharse J. C., Rayborn M. E. Turnover of rod photoreceptor outer segments. I. Membrane addition and loss in relationship to temperature. J Cell Biol. 1977 Nov;75(2 Pt 1):490–506. doi: 10.1083/jcb.75.2.490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hollyfield J. G., Rayborn M. E., Sarthy P. V., Lam D. M. The emergence, localization and maturation of neurotransmitter systems during development of the retina in Xenopus laevis. I. Gamma aminobutyric acid. J Comp Neurol. 1979 Dec 15;188(4):587–598. doi: 10.1002/cne.901880406. [DOI] [PubMed] [Google Scholar]
  18. Hollyfield J. G., Rayborn M. E., Verner G. E., Maude M. B., Anderson R. E. Membrane addition to rod photoreceptor outer segments: light stimulates membrane assembly in the absence of increased membrane biosynthesis. Invest Ophthalmol Vis Sci. 1982 Apr;22(4):417–427. [PubMed] [Google Scholar]
  19. Kinney M. S., Fisher S. K. The photoreceptors and pigment epithelium of the larval Xenopus retina: morphogenesis and outer segment renewal. Proc R Soc Lond B Biol Sci. 1978 May 5;201(1143):149–167. doi: 10.1098/rspb.1978.0037. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Lehle L., Tanner W. The specific site of tunicamycin inhibition in the formation of dolichol-bound N-acetylglucosamine derivatives. FEBS Lett. 1976 Nov 15;72(1):167–170. doi: 10.1016/0014-5793(76)80922-2. [DOI] [PubMed] [Google Scholar]
  22. Liang C. J., Yamashita K., Muellenberg C. G., Shichi H., Kobata A. Structure of the carbohydrate moieties of bovine rhodopsin. J Biol Chem. 1979 Jul 25;254(14):6414–6418. [PubMed] [Google Scholar]
  23. Marshall R. D. Some observations on why many proteins are glycosylated. Biochem Soc Trans. 1979 Aug;7(4):800–805. doi: 10.1042/bst0070800. [DOI] [PubMed] [Google Scholar]
  24. Matheke M. L., Fliesler S. J., Basinger S. F., Holtzman E. The effects of monensin on transport of membrane components in the frog retinal photoreceptor. I. Light microscopic autoradiography and biochemical analysis. J Neurosci. 1984 Apr;4(4):1086–1092. doi: 10.1523/JNEUROSCI.04-04-01086.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Montreuil J. Primary structure of glycoprotein glycans: basis for the molecular biology of glycoproteins. Adv Carbohydr Chem Biochem. 1980;37:157–223. doi: 10.1016/s0065-2318(08)60021-9. [DOI] [PubMed] [Google Scholar]
  26. O'Brien P. J. Differential effects of puromycin on the incorporation of precursors of rhodopsin in bovine retina. Biochemistry. 1977 Mar 8;16(5):953–958. doi: 10.1021/bi00624a022. [DOI] [PubMed] [Google Scholar]
  27. O'Brien P. J. Incorporation of mannose into rhodopsin in isolated bovine retina. Exp Eye Res. 1977 May;24(5):449–458. doi: 10.1016/0014-4835(77)90266-4. [DOI] [PubMed] [Google Scholar]
  28. O'Brien P. J., Muellenberg C. G. Proceedings: The biosynthesis of rhodopsin in vitro. Exp Eye Res. 1974 Mar;18(3):241–252. doi: 10.1016/0014-4835(74)90152-3. [DOI] [PubMed] [Google Scholar]
  29. O'Brien P. J., Muellenberg C. G. Synthesis of rhodopsin and opsin in vitro. Biochemistry. 1975 Apr 22;14(8):1695–1700. doi: 10.1021/bi00679a023. [DOI] [PubMed] [Google Scholar]
  30. O'Brien P. J. Rhodopsin as a glycoprotein: a possible role for the oligosaccharide in phagocytosis. Exp Eye Res. 1976 Aug;23(2):127–137. doi: 10.1016/0014-4835(76)90196-2. [DOI] [PubMed] [Google Scholar]
  31. Olden K., Bernard B. A., White S. L., Parent J. B. Function of the carbohydrate moieties of glycoproteins. J Cell Biochem. 1982;18(3):313–335. doi: 10.1002/jcb.1982.240180306. [DOI] [PubMed] [Google Scholar]
  32. Olden K., Parent J. B., White S. L. Carbohydrate moieties of glycoproteins. A re-evaluation of their function. Biochim Biophys Acta. 1982 May 12;650(4):209–232. doi: 10.1016/0304-4157(82)90017-x. [DOI] [PubMed] [Google Scholar]
  33. Papermaster D. S., Dreyer W. J. Rhodopsin content in the outer segment membranes of bovine and frog retinal rods. Biochemistry. 1974 May 21;13(11):2438–2444. doi: 10.1021/bi00708a031. [DOI] [PubMed] [Google Scholar]
  34. Papermaster D. S., Schneider B. G., Zorn M. A., Kraehenbuhl J. P. Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas. J Cell Biol. 1978 Apr;77(1):196–210. doi: 10.1083/jcb.77.1.196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Peters K. R., Palade G. E., Schneider B. G., Papermaster D. S. Fine structure of a periciliary ridge complex of frog retinal rod cells revealed by ultrahigh resolution scanning electron microscopy. J Cell Biol. 1983 Jan;96(1):265–276. doi: 10.1083/jcb.96.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Plantner J. J., Kean E. L. Carbohydrate composition of bovine rhodopsin. J Biol Chem. 1976 Mar 25;251(6):1548–1552. [PubMed] [Google Scholar]
  37. Plantner J. J., Poncz L., Kean E. L. Effect of tunicamycin on the glycosylation of rhodopsin. Arch Biochem Biophys. 1980 May;201(2):527–532. doi: 10.1016/0003-9861(80)90541-x. [DOI] [PubMed] [Google Scholar]
  38. Pratt R. M., Yamada K. M., Olden K., Ohanian S. H., Hascall V. C. Tunicamycin-induced alterations in the synthesis of sulfated proteoglycans and cell surface morphology in the chick embryo fibroblast. Exp Cell Res. 1979 Feb;118(2):245–252. doi: 10.1016/0014-4827(79)90149-6. [DOI] [PubMed] [Google Scholar]
  39. Schwarz R. T., Datema R. The lipid pathway of protein glycosylation and its inhibitors: the biological significance of protein-bound carbohydrates. Adv Carbohydr Chem Biochem. 1982;40:287–379. doi: 10.1016/s0065-2318(08)60111-0. [DOI] [PubMed] [Google Scholar]
  40. Steinberg R. H., Fisher S. K., Anderson D. H. Disc morphogenesis in vertebrate photoreceptors. J Comp Neurol. 1980 Apr 1;190(3):501–508. doi: 10.1002/cne.901900307. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Udenfriend S., Stein S., Böhlen P., Dairman W., Leimgruber W., Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972 Nov 24;178(4063):871–872. doi: 10.1126/science.178.4063.871. [DOI] [PubMed] [Google Scholar]
  44. Young R. W. The renewal of photoreceptor cell outer segments. J Cell Biol. 1967 Apr;33(1):61–72. doi: 10.1083/jcb.33.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Young R. W. Visual cells and the concept of renewal. Invest Ophthalmol Vis Sci. 1976 Sep;15(9):700–725. [PubMed] [Google Scholar]

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