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. 1993 Apr 1;121(1):49–59. doi: 10.1083/jcb.121.1.49

Beta-COP localizes mainly to the cis-Golgi side in exocrine pancreas

PMCID: PMC2119769  PMID: 8458872

Abstract

We examined the distribution of the non-clathrin-coated vesicle- associated coat protein beta-COP in rat exocrine pancreatic cells by immunogold cytochemistry. Labeling for beta-COP was found in the Golgi region (48%) where it was associated with vesicles and buds of approximately 50 nm, showing a characteristic approximately 10-nm-thick coat. The other half of the label was present in the cytoplasm, not associated with visible coats or membranes, with a minor fraction present on small clusters of tubules and vesicles. Clathrin-coated vesicles were typically located at the trans-side of the Golgi complex, and showed a thicker coat of approximately 18 nm. Of the total beta-COP labeling over the Golgi region, 68% occurred on the cis-side, 6% on the cisternae, 17% on the rims of the cisternae, and only 9% on the trans- side. For clathrin these figures were 16, 2, 4, and 78%, respectively. At the cis-Golgi side beta-COP was present in transitional areas (TA), on so-called peripheral elements (PE), consisting of tubules and vesicles located between the cup-shaped transitional elements (TE) of the RER and the cis-most Golgi cisternae. Label for Sec23p was also present in TA but was located closer to the TE, while beta-COP labeled PE were located near the cis-Golgi cisternae. Upon energy depletion, Golgi associated beta-COP was almost exclusively (86%) in spherical aggregates of 200-500 nm in diameter, whereas the cis-side (6%), the cisternae (1%), the rims (4%) and trans-side (3%) of the Golgi complex, were barely labeled; 50% of the total label remained in the cytoplasm. The aggregates were predominantly located at the cis-side of the Golgi stack, next to, but distinct from the Sec23p positive TA, that were devoid of beta-COP and had only a few recognizable vesicles left. Incubation with aluminum fluoride resulted in fragmentation of the Golgi complex into large clusters of beta-COP positive vesicles, while 50% of the label remained in the cytoplasm, as in control cells. After 10 min of Brefeldin A treatment 91% of beta-COP was cytoplasmic and only 7% associated with membranes of the Golgi complex. The total label for beta-COP over exocrine cells remained unchanged during the incubation with either of the drugs, indicating that the drugs induce reallocation of beta-COP. Our data suggest that beta-COP plays a role in membrane transport at the cis-side of the Golgi complex.

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Selected References

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  1. Burgoyne R. D. Trimeric G proteins in Golgi transport. Trends Biochem Sci. 1992 Mar;17(3):87–88. [PubMed] [Google Scholar]
  2. Donaldson J. G., Kahn R. A., Lippincott-Schwartz J., Klausner R. D. Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein. Science. 1991 Nov 22;254(5035):1197–1199. doi: 10.1126/science.1957170. [DOI] [PubMed] [Google Scholar]
  3. Donaldson J. G., Lippincott-Schwartz J., Bloom G. S., Kreis T. E., Klausner R. D. Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action. J Cell Biol. 1990 Dec;111(6 Pt 1):2295–2306. doi: 10.1083/jcb.111.6.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Duden R., Griffiths G., Frank R., Argos P., Kreis T. E. Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin. Cell. 1991 Feb 8;64(3):649–665. doi: 10.1016/0092-8674(91)90248-w. [DOI] [PubMed] [Google Scholar]
  5. Geuze J. J., Kramer M. F. Function of coated membranes and multivesicular bodies during membrane regulation in stimulated exocrine pancreas cells. Cell Tissue Res. 1974;156(1):1–20. doi: 10.1007/BF00220098. [DOI] [PubMed] [Google Scholar]
  6. Havinga J. R., Strous G. J., Poort C. Biosynthesis of the major glycoprotein associated with zymogen-granule membranes in the pancreas. Eur J Biochem. 1983 Jun 15;133(2):449–454. doi: 10.1111/j.1432-1033.1983.tb07484.x. [DOI] [PubMed] [Google Scholar]
  7. Howell S. L., Whitfield M. Synthesis and secretion of growth hormone in the rat anterior pituitary. I. The intracellular pathway, its time course and energy requirements. J Cell Sci. 1973 Jan;12(1):1–21. doi: 10.1242/jcs.12.1.1. [DOI] [PubMed] [Google Scholar]
  8. Hsu V. W., Yuan L. C., Nuchtern J. G., Lippincott-Schwartz J., Hammerling G. J., Klausner R. D. A recycling pathway between the endoplasmic reticulum and the Golgi apparatus for retention of unassembled MHC class I molecules. Nature. 1991 Aug 1;352(6334):441–444. doi: 10.1038/352441a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. J Cell Biol. 1968 Dec;39(3):589–603. doi: 10.1083/jcb.39.3.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaiser C. A., Schekman R. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell. 1990 May 18;61(4):723–733. doi: 10.1016/0092-8674(90)90483-u. [DOI] [PubMed] [Google Scholar]
  12. Klausner R. D., Donaldson J. G., Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol. 1992 Mar;116(5):1071–1080. doi: 10.1083/jcb.116.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  14. Kreis T. E. Regulation of vesicular and tubular membrane traffic of the Golgi complex by coat proteins. Curr Opin Cell Biol. 1992 Aug;4(4):609–615. doi: 10.1016/0955-0674(92)90079-r. [DOI] [PubMed] [Google Scholar]
  15. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  16. Lotti L. V., Torrisi M. R., Pascale M. C., Bonatti S. Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein from the intermediate compartment to the Golgi complex. J Cell Biol. 1992 Jul;118(1):43–50. doi: 10.1083/jcb.118.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Malhotra V., Serafini T., Orci L., Shepherd J. C., Rothman J. E. Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack. Cell. 1989 Jul 28;58(2):329–336. doi: 10.1016/0092-8674(89)90847-7. [DOI] [PubMed] [Google Scholar]
  18. Merisko E. M., Farquhar M. G., Palade G. E. Redistribution of clathrin heavy and light chains in anoxic pancreatic acinar cells. Pancreas. 1986;1(2):110–123. doi: 10.1097/00006676-198603000-00002. [DOI] [PubMed] [Google Scholar]
  19. Merisko E. M., Fletcher M., Palade G. E. The reorganization of the Golgi complex in anoxic pancreatic acinar cells. Pancreas. 1986;1(2):95–109. doi: 10.1097/00006676-198603000-00001. [DOI] [PubMed] [Google Scholar]
  20. Orci L., Glick B. S., Rothman J. E. A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack. Cell. 1986 Jul 18;46(2):171–184. doi: 10.1016/0092-8674(86)90734-8. [DOI] [PubMed] [Google Scholar]
  21. Orci L., Malhotra V., Amherdt M., Serafini T., Rothman J. E. Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack. Cell. 1989 Feb 10;56(3):357–368. doi: 10.1016/0092-8674(89)90239-0. [DOI] [PubMed] [Google Scholar]
  22. Orci L., Ravazzola M., Meda P., Holcomb C., Moore H. P., Hicke L., Schekman R. Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8611–8615. doi: 10.1073/pnas.88.19.8611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  24. Pearse B. M. Receptors compete for adaptors found in plasma membrane coated pits. EMBO J. 1988 Nov;7(11):3331–3336. doi: 10.1002/j.1460-2075.1988.tb03204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  26. Pelham H. R. Recycling of proteins between the endoplasmic reticulum and Golgi complex. Curr Opin Cell Biol. 1991 Aug;3(4):585–591. doi: 10.1016/0955-0674(91)90027-v. [DOI] [PubMed] [Google Scholar]
  27. Robinson M. S., Kreis T. E. Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators. Cell. 1992 Apr 3;69(1):129–138. doi: 10.1016/0092-8674(92)90124-u. [DOI] [PubMed] [Google Scholar]
  28. Rothman J. E., Orci L. Molecular dissection of the secretory pathway. Nature. 1992 Jan 30;355(6359):409–415. doi: 10.1038/355409a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Saraste J., Palade G. E., Farquhar M. G. Antibodies to rat pancreas Golgi subfractions: identification of a 58-kD cis-Golgi protein. J Cell Biol. 1987 Nov;105(5):2021–2029. doi: 10.1083/jcb.105.5.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Scheele G. Pancreatic lobules in the in vitro study of pancreatic acinar cell function. Methods Enzymol. 1983;98:17–28. doi: 10.1016/0076-6879(83)98135-1. [DOI] [PubMed] [Google Scholar]
  32. Schweizer A., Fransen J. A., Bächi T., Ginsel L., Hauri H. P. Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. J Cell Biol. 1988 Nov;107(5):1643–1653. doi: 10.1083/jcb.107.5.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schweizer A., Fransen J. A., Matter K., Kreis T. E., Ginsel L., Hauri H. P. Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus. Eur J Cell Biol. 1990 Dec;53(2):185–196. [PubMed] [Google Scholar]
  34. Schweizer A., Matter K., Ketcham C. M., Hauri H. P. The isolated ER-Golgi intermediate compartment exhibits properties that are different from ER and cis-Golgi. J Cell Biol. 1991 Apr;113(1):45–54. doi: 10.1083/jcb.113.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Serafini T., Stenbeck G., Brecht A., Lottspeich F., Orci L., Rothman J. E., Wieland F. T. A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein beta-adaptin. Nature. 1991 Jan 17;349(6306):215–220. doi: 10.1038/349215a0. [DOI] [PubMed] [Google Scholar]
  36. Slot J. W., Geuze H. J., Gigengack S., Lienhard G. E., James D. E. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol. 1991 Apr;113(1):123–135. doi: 10.1083/jcb.113.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Slot J. W., Geuze H. J. Immunoelectron microscopic exploration of the Golgi complex. J Histochem Cytochem. 1983 Aug;31(8):1049–1056. doi: 10.1177/31.8.6863900. [DOI] [PubMed] [Google Scholar]
  38. Slot J. W., Geuze J. J. A morphometrical study of the exocrine pancreatic cell in fasted and fed frogs. J Cell Biol. 1979 Mar;80(3):692–707. doi: 10.1083/jcb.80.3.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Slot J. W., Geuze J. J. Synthesis and intracellular transport of proteins in the exocrine pancreas of the frog (Rana esculenta). II. An in vitro study of the transport process and the influence of temperature. Cell Tissue Res. 1976 Mar 16;167(2):147–165. doi: 10.1007/BF00224324. [DOI] [PubMed] [Google Scholar]
  40. Tooze S. A., Tooze J., Warren G. Site of addition of N-acetyl-galactosamine to the E1 glycoprotein of mouse hepatitis virus-A59. J Cell Biol. 1988 May;106(5):1475–1487. doi: 10.1083/jcb.106.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Warren G. Protein transport. Signals and salvage sequences. Nature. 1987 May 7;327(6117):17–18. doi: 10.1038/327017a0. [DOI] [PubMed] [Google Scholar]
  42. Waters M. G., Serafini T., Rothman J. E. 'Coatomer': a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature. 1991 Jan 17;349(6306):248–251. doi: 10.1038/349248a0. [DOI] [PubMed] [Google Scholar]

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