Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Nov 1;105(5):2021–2029. doi: 10.1083/jcb.105.5.2021

Antibodies to rat pancreas Golgi subfractions: identification of a 58- kD cis-Golgi protein

PMCID: PMC2114852  PMID: 3316245

Abstract

A 58-kD cis-Golgi protein has been identified by generating polyclonal antibodies against heavy (cis) Golgi subfractions. Total microsomes isolated from rat pancreatic homogenates were subfractionated to yield a rough microsomal fraction (B1) and three smooth membrane subfractions (B2-B4) enriched in cis-, middle, and trans-Golgi elements, respectively. The heavy (cis) subfraction, B2 (d = 1.17 g/ml), was fractionated by Triton X-114 phase separation, and the proteins recovered in the detergent phase were used to immunize rabbits. One of the anti-B2 antibodies obtained gave a "Golgi"-staining pattern when screened by immunofluorescence on normal rat kidney cells and mouse RPC 5.4 myeloma cells. In rat pancreatic exocrine cells the antibody reacted with the plasmalemma as well as elements in the Golgi region. By immunoelectron microscopy, the antigen recognized by anti-B2 IgG was found to be restricted to cis-Golgi elements in myeloma cells where it was concentrated in the fenestrated cis-most cisterna and in some of the tubules and vesicles located along the cis face of the Golgi complex. By immunoprecipitation and immunoblotting, the anti-B2 IgG exclusively recognized a 58-kD protein in myeloma cells. The anti-B2 IgG reacted with several proteins in solubilized pancreatic B2 membranes, including a 58-kD protein, but affinity-purified anti-58-kD IgG reacted exclusively with the 58-kD protein. These results suggest that the 58-kD protein is a specific component of cis-Golgi membranes.

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

Selected References

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

  1. Balch W. E., Keller D. S. ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments. J Biol Chem. 1986 Nov 5;261(31):14690–14696. [PubMed] [Google Scholar]
  2. Balch W. E., Rothman J. E. Characterization of protein transport between successive compartments of the Golgi apparatus: asymmetric properties of donor and acceptor activities in a cell-free system. Arch Biochem Biophys. 1985 Jul;240(1):413–425. doi: 10.1016/0003-9861(85)90046-3. [DOI] [PubMed] [Google Scholar]
  3. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  4. Brown W. J., Constantinescu E., Farquhar M. G. Redistribution of mannose-6-phosphate receptors induced by tunicamycin and chloroquine. J Cell Biol. 1984 Jul;99(1 Pt 1):320–326. doi: 10.1083/jcb.99.1.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chicheportiche Y., Vassalli P., Tartakoff A. M. Characterization of cytoplasmically oriented Golgi proteins with a monoclonal antibody. J Cell Biol. 1984 Dec;99(6):2200–2210. doi: 10.1083/jcb.99.6.2200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coudrier E., Reggio H., Louvard D. Characterization of an integral membrane glycoprotein associated with the microfilaments of pig intestinal microvilli. EMBO J. 1983;2(3):469–475. doi: 10.1002/j.1460-2075.1983.tb01446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dunphy W. G., Brands R., Rothman J. E. Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack. Cell. 1985 Feb;40(2):463–472. doi: 10.1016/0092-8674(85)90161-8. [DOI] [PubMed] [Google Scholar]
  8. Dunphy W. G., Fries E., Urbani L. J., Rothman J. E. Early and late functions associated with the Golgi apparatus reside in distinct compartments. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7453–7457. doi: 10.1073/pnas.78.12.7453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dunphy W. G., Rothman J. E. Compartmental organization of the Golgi stack. Cell. 1985 Aug;42(1):13–21. doi: 10.1016/s0092-8674(85)80097-0. [DOI] [PubMed] [Google Scholar]
  10. Dunphy W. G., Rothman J. E. Compartmentation of asparagine-linked oligosaccharide processing in the Golgi apparatus. J Cell Biol. 1983 Jul;97(1):270–275. doi: 10.1083/jcb.97.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. FRIEND D. S., MURRAY M. J. OSMIUM IMPREGNATION OF THE GOLGI APPARATUS. Am J Anat. 1965 Jul;117:135–149. doi: 10.1002/aja.1001170109. [DOI] [PubMed] [Google Scholar]
  12. Farquhar M. G., Palade G. E. The Golgi apparatus (complex)-(1954-1981)-from artifact to center stage. J Cell Biol. 1981 Dec;91(3 Pt 2):77s–103s. doi: 10.1083/jcb.91.3.77s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Farquhar M. G. Progress in unraveling pathways of Golgi traffic. Annu Rev Cell Biol. 1985;1:447–488. doi: 10.1146/annurev.cb.01.110185.002311. [DOI] [PubMed] [Google Scholar]
  14. Goldberg D. E., Kornfeld S. Evidence for extensive subcellular organization of asparagine-linked oligosaccharide processing and lysosomal enzyme phosphorylation. J Biol Chem. 1983 Mar 10;258(5):3159–3165. [PubMed] [Google Scholar]
  15. Griffiths G., Quinn P., Warren G. Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus. J Cell Biol. 1983 Mar;96(3):835–850. doi: 10.1083/jcb.96.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  17. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. J Cell Biol. 1967 Aug;34(2):577–596. doi: 10.1083/jcb.34.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  19. Lin J. J., Queally S. A. A monoclonal antibody that recognizes Golgi-associated protein of cultured fibroblast cells. J Cell Biol. 1982 Jan;92(1):108–112. doi: 10.1083/jcb.92.1.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  22. Novikoff P. M., Tulsiani D. R., Touster O., Yam A., Novikoff A. B. Immunocytochemical localization of alpha-D-mannosidase II in the Golgi apparatus of rat liver. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4364–4368. doi: 10.1073/pnas.80.14.4364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ottosen P. D., Courtoy P. J., Farquhar M. G. Pathways followed by membrane recovered from the surface of plasma cells and myeloma cells. J Exp Med. 1980 Jul 1;152(1):1–19. doi: 10.1084/jem.152.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pohlmann R., Waheed A., Hasilik A., von Figura K. Synthesis of phosphorylated recognition marker in lysosomal enzymes is located in the cis part of Golgi apparatus. J Biol Chem. 1982 May 25;257(10):5323–5325. [PubMed] [Google Scholar]
  25. Pryde J. G., Phillips J. H. Fractionation of membrane proteins by temperature-induced phase separation in Triton X-114. Application to subcellular fractions of the adrenal medulla. Biochem J. 1986 Jan 15;233(2):525–533. doi: 10.1042/bj2330525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roth J., Berger E. G. Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae. J Cell Biol. 1982 Apr;93(1):223–229. doi: 10.1083/jcb.93.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Roth J., Taatjes D. J., Lucocq J. M., Weinstein J., Paulson J. C. Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. Cell. 1985 Nov;43(1):287–295. doi: 10.1016/0092-8674(85)90034-0. [DOI] [PubMed] [Google Scholar]
  28. Saraste J., Kuismanen E. Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface. Cell. 1984 Sep;38(2):535–549. doi: 10.1016/0092-8674(84)90508-7. [DOI] [PubMed] [Google Scholar]
  29. Saraste J., Palade G. E., Farquhar M. G. Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6425–6429. doi: 10.1073/pnas.83.17.6425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smith Z. D., D'Eugenio-Gumkowski F., Yanagisawa K., Jamieson J. D. Endogenous and monoclonal antibodies to the rat pancreatic acinar cell Golgi complex. J Cell Biol. 1984 Jun;98(6):2035–2046. doi: 10.1083/jcb.98.6.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Talian J. C., Olmsted J. B., Goldman R. D. A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture. J Cell Biol. 1983 Oct;97(4):1277–1282. doi: 10.1083/jcb.97.4.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tartakoff A. M. The confined function model of the Golgi complex: center for ordered processing of biosynthetic products of the rough endoplasmic reticulum. Int Rev Cytol. 1983;85:221–252. doi: 10.1016/S0074-7696(08)62374-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tokuyasu K. T. Application of cryoultramicrotomy to immunocytochemistry. J Microsc. 1986 Aug;143(Pt 2):139–149. doi: 10.1111/j.1365-2818.1986.tb02772.x. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Woods J. W., Doriaux M., Farquhar M. G. Transferrin receptors recycle to cis and middle as well as trans Golgi cisternae in Ig-secreting myeloma cells. J Cell Biol. 1986 Jul;103(1):277–286. doi: 10.1083/jcb.103.1.277. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES