Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Nov 1;98(9):2146–2157. doi: 10.1172/JCI119021

Ethanol-induced retention of nascent proteins in rat hepatocytes is accompanied by altered distribution of the small GTP-binding protein rab2.

J M Larkin 1, B Oswald 1, M A McNiven 1
PMCID: PMC507660  PMID: 8903335

Abstract

Chronic ethanol consumption induces hepatocellular retention of nascent proteins leading to hepatomegaly. While the molecular mechanisms behind this impairment are undefined, it has been predicted that protein retention results from a disruption of vesicle-mediated secretory processes. Small GTP-binding proteins (rab proteins) have recently been implicated in the regulation of vesicular trafficking in eukaryotic cells. Our objectives were to identify intracellular sites of ethanol-induced protein retention and to determine whether the distribution of secretory rab proteins was altered by ethanol. Transport of hepatic proteins along the secretory pathway in livers from control and ethanol-fed rats was analyzed using subcellular fractionation and immunoprecipitation in the context of in vivo pulse-chase experiments. We show that pre-Golgi and Golgi compartments, as well as secretory vesicles, are sites of ethanol-induced retention of nascent soluble and transmembrane secretory proteins. These results are supported by immunofluorescence localization of hepatic proteins on liver sections. Further, immunoblot analyses of hepatic subcellular fractions from ethanol-damaged livers indicate a dramatic reduction in the association of rab2 with a Golgi compartment as compared with controls. In contrast, rab6 and alpha-mannosidase II, Golgi marker proteins, appear unchanged. These studies provide a detailed analysis of the intracellular site of ethanol-induced protein retention in the hepatocyte and lend novel insight into a potential mechanism behind this impairment. The effects of ethanol exposure on rab proteins and Golgi function are discussed.

Full Text

The Full Text of this article is available as a PDF (684.6 KB).

Selected References

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

  1. Anderson K. L., McNiven M. A. Vesicle dynamics during regulated secretion in a novel pancreatic acinar cell in vitro model. Eur J Cell Biol. 1995 Jan;66(1):25–38. [PubMed] [Google Scholar]
  2. Antony C., Cibert C., Géraud G., Santa Maria A., Maro B., Mayau V., Goud B. The small GTP-binding protein rab6p is distributed from medial Golgi to the trans-Golgi network as determined by a confocal microscopic approach. J Cell Sci. 1992 Nov;103(Pt 3):785–796. doi: 10.1242/jcs.103.3.785. [DOI] [PubMed] [Google Scholar]
  3. Baraona E., Leo M. A., Borowsky S. A., Lieber C. S. Pathogenesis of alcohol-induced accumulation of protein in the liver. J Clin Invest. 1977 Sep;60(3):546–554. doi: 10.1172/JCI108806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baraona E., Pikkarainen P., Salaspuro M., Finkelman F., Lieber C. S. Acute effects of ethanol on hepatic protein synthesis and secretion in the rat. Gastroenterology. 1980 Jul;79(1):104–111. [PubMed] [Google Scholar]
  5. Donohue T. M., Jr, Sorrell M. F., Tuma D. J. Hepatic protein synthetic activity in vivo after ethanol administration. Alcohol Clin Exp Res. 1987 Feb;11(1):80–86. doi: 10.1111/j.1530-0277.1987.tb01267.x. [DOI] [PubMed] [Google Scholar]
  6. Ehrenreich J. H., Bergeron J. J., Siekevitz P., Palade G. E. Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J Cell Biol. 1973 Oct;59(1):45–72. doi: 10.1083/jcb.59.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fernandez-Briera A., Louisot P., Morelis R. Effects of chronic ethanol administration on plasma-membrane-bound glycosyltransferase activities. Pharmacol Biochem Behav. 1990 Jan;35(1):75–84. doi: 10.1016/0091-3057(90)90207-x. [DOI] [PubMed] [Google Scholar]
  8. Fischer von Mollard G., Stahl B., Li C., Südhof T. C., Jahn R. Rab proteins in regulated exocytosis. Trends Biochem Sci. 1994 Apr;19(4):164–168. doi: 10.1016/0968-0004(94)90278-x. [DOI] [PubMed] [Google Scholar]
  9. Gores G. J., Nieminen A. L., Wray B. E., Herman B., Lemasters J. J. Intracellular pH during "chemical hypoxia" in cultured rat hepatocytes. Protection by intracellular acidosis against the onset of cell death. J Clin Invest. 1989 Feb;83(2):386–396. doi: 10.1172/JCI113896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guasch R., Renau-Piqueras J., Guerri C. Chronic ethanol consumption induces accumulation of proteins in the liver Golgi apparatus and decreases galactosyltransferase activity. Alcohol Clin Exp Res. 1992 Oct;16(5):942–948. doi: 10.1111/j.1530-0277.1992.tb01897.x. [DOI] [PubMed] [Google Scholar]
  11. Harris R. A., Burnett R., McQuilkin S., McClard A., Simon F. R. Effects of ethanol on membrane order: fluorescence studies. Ann N Y Acad Sci. 1987;492:125–135. doi: 10.1111/j.1749-6632.1987.tb48660.x. [DOI] [PubMed] [Google Scholar]
  12. Howell K. E., Palade G. E. Hepatic Golgi fractions resolved into membrane and content subfractions. J Cell Biol. 1982 Mar;92(3):822–832. doi: 10.1083/jcb.92.3.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huber L. A., Pimplikar S., Parton R. G., Virta H., Zerial M., Simons K. Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane. J Cell Biol. 1993 Oct;123(1):35–45. doi: 10.1083/jcb.123.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jennett R. B., Tuma D. J., Sorrell M. F. Effect of ethanol and its metabolites on microtubule formation. Pharmacology. 1980;21(5):363–368. doi: 10.1159/000137453. [DOI] [PubMed] [Google Scholar]
  15. Larkin J. M., Palade G. E. Transcytotic vesicular carriers for polymeric IgA receptors accumulate in rat hepatocytes after bile duct ligation. J Cell Sci. 1991 Feb;98(Pt 2):205–216. doi: 10.1242/jcs.98.2.205. [DOI] [PubMed] [Google Scholar]
  16. Lippincott-Schwartz J. Membrane cycling between the ER and Golgi apparatus and its role in biosynthetic transport. Subcell Biochem. 1993;21:95–119. doi: 10.1007/978-1-4615-2912-5_5. [DOI] [PubMed] [Google Scholar]
  17. Marchner H., Tottmar O. A comparative study on the effects of disulfiram, cyanamide and 1-aminocyclopropanol on the acetaldehyde metabolism in rats. Acta Pharmacol Toxicol (Copenh) 1978 Sep;43(3):219–232. doi: 10.1111/j.1600-0773.1978.tb02258.x. [DOI] [PubMed] [Google Scholar]
  18. Martinez O., Schmidt A., Salaméro J., Hoflack B., Roa M., Goud B. The small GTP-binding protein rab6 functions in intra-Golgi transport. J Cell Biol. 1994 Dec;127(6 Pt 1):1575–1588. doi: 10.1083/jcb.127.6.1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matsuda Y., Baraona E., Salaspuro M., Lieber C. S. Effects of ethanol on liver microtubules and Golgi apparatus. Possible role in altered hepatic secretion of plasma proteins. Lab Invest. 1979 Nov;41(5):455–463. [PubMed] [Google Scholar]
  20. Matsuda Y., Takada A., Sato H., Yasuhara M., Takase S. Comparison between ballooned hepatocytes occurring in human alcoholic and nonalcoholic liver diseases. Alcohol Clin Exp Res. 1985 Jul-Aug;9(4):366–370. doi: 10.1111/j.1530-0277.1985.tb05561.x. [DOI] [PubMed] [Google Scholar]
  21. Mauch T. J., Donohue T. M., Jr, Zetterman R. K., Sorrell M. F., Tuma D. J. Covalent binding of acetaldehyde selectively inhibits the catalytic activity of lysine-dependent enzymes. Hepatology. 1986 Mar-Apr;6(2):263–269. doi: 10.1002/hep.1840060218. [DOI] [PubMed] [Google Scholar]
  22. Nuoffer C., Davidson H. W., Matteson J., Meinkoth J., Balch W. E. A GDP-bound of rab1 inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments. J Cell Biol. 1994 Apr;125(2):225–237. doi: 10.1083/jcb.125.2.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pfeffer S. R. Rab GTPases: master regulators of membrane trafficking. Curr Opin Cell Biol. 1994 Aug;6(4):522–526. doi: 10.1016/0955-0674(94)90071-x. [DOI] [PubMed] [Google Scholar]
  24. Plutner H., Cox A. D., Pind S., Khosravi-Far R., Bourne J. R., Schwaninger R., Der C. J., Balch W. E. Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments. J Cell Biol. 1991 Oct;115(1):31–43. doi: 10.1083/jcb.115.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Redman C. M., Banerjee D., Howell K., Palade G. E. Colchicine inhibition of plasma protein release from rat hepatocytes. J Cell Biol. 1975 Jul;66(1):42–59. doi: 10.1083/jcb.66.1.42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Saraste J., Kuismanen E. Pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the Golgi complex. Semin Cell Biol. 1992 Oct;3(5):343–355. doi: 10.1016/1043-4682(92)90020-V. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Saucan L., Palade G. E. Membrane and secretory proteins are transported from the Golgi complex to the sinusoidal plasmalemma of hepatocytes by distinct vesicular carriers. J Cell Biol. 1994 May;125(4):733–741. doi: 10.1083/jcb.125.4.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sorrell M. F., Tuma D. J., Schafer E. C., Barak A. J. Role of acetaldehyde in the ethanol-induced impairment of glycoprotein metabolism in rat liver slices. Gastroenterology. 1977 Jul;73(1):137–144. [PubMed] [Google Scholar]
  29. Sorrell M. F., Tuma D. J. Selective impairment of glycoprotein metabolism by ethanol and acetaldehyde in rat liver slices. Gastroenterology. 1978 Aug;75(2):200–205. [PubMed] [Google Scholar]
  30. Sorrell M. F., Tuma D. J. The functional implications of acetaldehyde binding to cell constituents. Ann N Y Acad Sci. 1987;492:50–62. doi: 10.1111/j.1749-6632.1987.tb48652.x. [DOI] [PubMed] [Google Scholar]
  31. Stibler H., Borg S. Glycoprotein glycosyltransferase activities in serum in alcohol-abusing patients and healthy controls. Scand J Clin Lab Invest. 1991 Feb;51(1):43–51. [PubMed] [Google Scholar]
  32. Sztul E., Kaplin A., Saucan L., Palade G. Protein traffic between distinct plasma membrane domains: isolation and characterization of vesicular carriers involved in transcytosis. Cell. 1991 Jan 11;64(1):81–89. doi: 10.1016/0092-8674(91)90210-p. [DOI] [PubMed] [Google Scholar]
  33. Tisdale E. J., Bourne J. R., Khosravi-Far R., Der C. J., Balch W. E. GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex. J Cell Biol. 1992 Nov;119(4):749–761. doi: 10.1083/jcb.119.4.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tuma D. J., Jennett R. B., Sorrell M. F. Effect of ethanol on the synthesis and secretion of hepatic secretory glycoproteins and albumin. Hepatology. 1981 Nov-Dec;1(6):590–598. doi: 10.1002/hep.1840010606. [DOI] [PubMed] [Google Scholar]
  35. Tuma D. J., Mailliard M. E., Casey C. A., Volentine G. D., Sorrell M. F. Ethanol-induced alterations of plasma membrane assembly in the liver. Biochim Biophys Acta. 1986 Apr 25;856(3):571–577. doi: 10.1016/0005-2736(86)90149-5. [DOI] [PubMed] [Google Scholar]
  36. Tuma D. J., Sorrell M. F. Effects of ethanol on the secretion of glycoproteins by rat liver slices. Gastroenterology. 1981 Feb;80(2):273–278. [PubMed] [Google Scholar]
  37. Tuma D. J., Zetterman R. K., Sorrell M. F. Inhibition of glycoprotein secretion by ethanol and acetaldehyde in rat liver slices. Biochem Pharmacol. 1980 Jan 1;29(1):35–38. doi: 10.1016/0006-2952(80)90240-3. [DOI] [PubMed] [Google Scholar]
  38. Volentine G. D., Ogden K. A., Kortje D. K., Tuma D. J., Sorrell M. F. Role of acetaldehyde in the ethanol-induced impairment of hepatic glycoprotein secretion in the rat in vivo. Hepatology. 1987 May-Jun;7(3):490–495. doi: 10.1002/hep.1840070313. [DOI] [PubMed] [Google Scholar]
  39. Volentine G. D., Tuma D. J., Sorrell M. F. Acute effects of ethanol on hepatic glycoprotein secretion in the rat in vivo. Gastroenterology. 1984 Feb;86(2):225–229. [PubMed] [Google Scholar]
  40. Volentine G. D., Tuma D. J., Sorrell M. F. Subcellular location of secretory proteins retained in the liver during the ethanol-induced inhibition of hepatic protein secretion in the rat. Gastroenterology. 1986 Jan;90(1):158–165. doi: 10.1016/0016-5085(86)90088-0. [DOI] [PubMed] [Google Scholar]
  41. Wilson B. S., Nuoffer C., Meinkoth J. L., McCaffery M., Feramisco J. R., Balch W. E., Farquhar M. G. A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus. J Cell Biol. 1994 May;125(3):557–571. doi: 10.1083/jcb.125.3.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wood C. K., Lamb R. G. The effect of ethanol on glycerolipid biosynthesis by primary monolayer cultures of adult rat hepatocytes. Biochim Biophys Acta. 1979 Jan 29;572(1):121–131. doi: 10.1016/0005-2760(79)90206-6. [DOI] [PubMed] [Google Scholar]
  43. Yamada S., Lieber C. S. Decrease in microviscosity and cholesterol content of rat liver plasma membranes after chronic ethanol feeding. J Clin Invest. 1984 Dec;74(6):2285–2289. doi: 10.1172/JCI111656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zerial M., Stenmark H. Rab GTPases in vesicular transport. Curr Opin Cell Biol. 1993 Aug;5(4):613–620. doi: 10.1016/0955-0674(93)90130-i. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES