Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 Jul 1;90(1):236–242. doi: 10.1083/jcb.90.1.236

In vitro biosynthesis, core glycosylation and membrane integration of opsin

BM Goldman, G Blobel
PMCID: PMC2111824  PMID: 6454695

Abstract

A membrane-integrated , core-glycosylated form of bovine opsin was synthesized in vitro when bovine retina mRNA was translated in a wheat germ cell-free system supplemented with dog pancreas microsomal vesicles; glycosylation and integration of opsin into membranes were coupled to translation. Proteolysis with themolysin was used to probe the orientation of opsin within the dog pancreas microsomal membrane, and to compare it with that of opsin in rod cell disk membranes isolated from bovine retina. Intact microsomal or disk vesicles were required for production of discrete, membrane-associated thermolysin fragments of opsin; no discrete opsin fragments were detected when membranes were incubated with thermolysin in the presence of the nonionic detergent, Triton X-100. The major opsin fragments produced by themosylin treatment of intact microsomal vesicles resembled those from disk vesicles in their size, oligosaccharide content, and order of appearance. In each case, the first cleavage of opsin took place at the COOH-terminus, generating a glycosylated fragment, O’, which was only slightly smaller than intact opsin. Both the microsomal and disk membrane forms of O’ were next cleaved internally; glycosylated fragments of similar sizes in both cases were detected which were derived from the NH(2)-terminal portion of O’. Several smaller NH(2)-terminal fragments of opsin were detected only in thermolysin-treated microsomal membranes, and not in disk membranes. The data suggest that the topology of opsin integrated into dog pancreas microsomal vesicles is similar to that in rod cell disk vesicles, although not identical. In each case, the glycosylated NH(2)-terminal region of opsin is located within the lumen of the vesicle, while discrete COOH-terminal and internal segments of opsin apparently emerge at the outer, cytoplasmic face of the membrane. Thus, opsin in the heterologous microsomal membrane, like its counterpart in the native disk membrane, may cross the bilayer at least three times. The internal domain of the polypeptide that emerges at the outer membrane surface is apparently more highly exposed in the case of opsin in microsomal membranes, evidenced by the additional internal thermolysin cleavage sites detected.

Full Text

The Full Text of this article is available as a PDF (789.4 KB).

Selected References

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

  1. 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]
  2. Basinger S., Bok D., Hall M. Rhodopsin in the rod outer segment plasma membrane. J Cell Biol. 1976 Apr;69(1):29–42. doi: 10.1083/jcb.69.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blackburn P., Wilson G., Moore S. Ribonuclease inhibitor from human placenta. Purification and properties. J Biol Chem. 1977 Aug 25;252(16):5904–5910. [PubMed] [Google Scholar]
  4. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blobel G., Walter P., Chang C. N., Goldman B. M., Erickson A. H., Lingappa V. R. Translocation of proteins across membranes: the signal hypothesis and beyond. Symp Soc Exp Biol. 1979;33:9–36. [PubMed] [Google Scholar]
  6. 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]
  7. Chen Y. S., Hubbell W. L. Temperature- and light-dependent structural changes in rhodopsin-lipid membranes. Exp Eye Res. 1973 Dec 24;17(6):517–532. doi: 10.1016/0014-4835(73)90082-1. [DOI] [PubMed] [Google Scholar]
  8. Erickson A. H., Blobel G. Early events in the biosynthesis of the lysosomal enzyme cathepsin D. J Biol Chem. 1979 Dec 10;254(23):11771–11774. [PubMed] [Google Scholar]
  9. 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]
  10. Fung B. K., Hubbell W. L. Organization of rhodopsin in photoreceptor membranes. 1. Proteolysis of bovine rhodopsin in native membranes and the distribution of sulfhydryl groups in the fragments. Biochemistry. 1978 Oct 17;17(21):4396–4402. doi: 10.1021/bi00614a007. [DOI] [PubMed] [Google Scholar]
  11. Fung B. K., Hubbell W. L. Organization of rhodopsin in photoreceptor membranes. 2. Transmembrane organization of bovine rhodopsin: evidence from proteolysis and lactoperoxidase-catalyzed iodination of native and reconstituted membranes. Biochemistry. 1978 Oct 17;17(21):4403–4410. doi: 10.1021/bi00614a008. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Hanover J. A., Lennarz W. J. N-Linked glycoprotein assembly. Evidence that oligosaccharide attachment occurs within the lumen of the endoplasmic reticulum. J Biol Chem. 1980 Apr 25;255(8):3600–3604. [PubMed] [Google Scholar]
  14. Hargrave P. A., Fong S. L. The amino- and carboxyl-terminal sequence of bovine rhodopsin. J Supramol Struct. 1977;6(4):559–570. doi: 10.1002/jss.400060409. [DOI] [PubMed] [Google Scholar]
  15. Hargrave P. A. The amino-terminal tryptic peptide of bovine rhodopsin. A glycopeptide containing two sites of oligosaccharide attachment. Biochim Biophys Acta. 1977 May 27;492(1):83–94. doi: 10.1016/0005-2795(77)90216-1. [DOI] [PubMed] [Google Scholar]
  16. Katz F. N., Rothman J. E., Lingappa V. R., Blobel G., Lodish H. F. Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3278–3282. doi: 10.1073/pnas.74.8.3278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lingappa V. R., Lingappa J. R., Blobel G. Chicken ovalbumin contains an internal signal sequence. Nature. 1979 Sep 13;281(5727):117–121. doi: 10.1038/281117a0. [DOI] [PubMed] [Google Scholar]
  18. Lingappa V. R., Lingappa J. R., Prasad R., Ebner K. E., Blobel G. Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein. Proc Natl Acad Sci U S A. 1978 May;75(5):2338–2342. doi: 10.1073/pnas.75.5.2338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McMullen M. D., Shaw P. H., Martin T. E. Characterization of poly(A+)RNA in free messenger ribonucleoprotein and polysomes of mouse Taper ascites cells. J Mol Biol. 1979 Aug 25;132(4):679–694. doi: 10.1016/0022-2836(79)90382-6. [DOI] [PubMed] [Google Scholar]
  20. NILSSON S. E. RECEPTOR CELL OUTER SEGMENT DEVELOPMENT AND ULTRASTRUCTURE OF THE DISK MEMBRANES IN THE RETINA OF THE TADPOLE (RANA PIPIENS). J Ultrastruct Res. 1964 Dec;11:581–602. doi: 10.1016/s0022-5320(64)80084-8. [DOI] [PubMed] [Google Scholar]
  21. O'Brien P. J., Muellenberg C. G., Bungenberg de Jong J. J. Incorporation of leucine into rhodopsin in isolated bovine retina. Biochemistry. 1972 Jan 4;11(1):64–70. doi: 10.1021/bi00751a012. [DOI] [PubMed] [Google Scholar]
  22. Papermaster D. S., Converse C. A., Siuss J. Membrane biosynthesis in the frog retina: opsin transport in the photoreceptor cell. Biochemistry. 1975 Apr 8;14(7):1343–1352. doi: 10.1021/bi00678a001. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Pober J. S., Iwanij V., Reich E., Stryer L. Transglutaminase-catalyzed insertion of a fluorescent probe into the protease-sensitive region of rhodopsin. Biochemistry. 1978 May 30;17(11):2163–2168. doi: 10.1021/bi00604a021. [DOI] [PubMed] [Google Scholar]
  26. Pober J. S., Stryer L. Letter to the editor: Light dissociates enzymatically-cleaved rhodopsin into two different fragments. J Mol Biol. 1975 Jul 5;95(3):477–481. doi: 10.1016/0022-2836(75)90204-1. [DOI] [PubMed] [Google Scholar]
  27. Röhlich P. Photoreceptor membrane carbohydrate on the intradiscal surface of retinal rod disks. Nature. 1976 Oct 28;263(5580):789–791. doi: 10.1038/263789a0. [DOI] [PubMed] [Google Scholar]
  28. Saari J. C. The accessibility of bovine rhodopsin in photoreceptor membranes. J Cell Biol. 1974 Nov;63(2 Pt 1):480–491. doi: 10.1083/jcb.63.2.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schechter I., Burstein Y., Zemell R., Ziv E., Kantor F., Papermaster D. S. Messenger RNA of opsin from bovine retina: isolation and partial sequence of the in vitro translation product. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2654–2658. doi: 10.1073/pnas.76.6.2654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Young R. W., Droz B. The renewal of protein in retinal rods and cones. J Cell Biol. 1968 Oct;39(1):169–184. doi: 10.1083/jcb.39.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]

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

RESOURCES