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. 1992 Jun 2;117(6):1161–1169. doi: 10.1083/jcb.117.6.1161

Biosynthesis of lipoprotein: location of nascent apoAI and apoB in the rough endoplasmic reticulum of chicken hepatocytes

PMCID: PMC2289498  PMID: 1607380

Abstract

Our previous studies showed that in hepatic RER of young chickens, nascent apoAI is not associated with lipoprotein particles and only becomes part of these lipoprotein structures in the Golgi. In this study, we have used three different methodologies to determine the locations of apoAI and apoB in the RER and compared them to that of albumin. Immunoelectron microscopic examination of the RER cell fractions showed that both apoAI and apoB were associated only with the RER membrane whereas albumin was located both within the lumen and on the limiting membrane of the vesicles. To examine the possibility of membrane integration of nascent apoAI and apoB in the RER, we administered L-[3H]leucine to young chickens for 10 min, isolated RER, treated this cell fraction with buffers of varying pH, and measured the release of radioactive albumin, apoAI, and apoB. The majority of nascent apoAI (64%), nascent apoB (100%), and nascent albumin (97%) was released from RER vesicles at pH 11.2, suggesting that, like albumin, apolipoproteins are not integrated within the membrane. To determine if nascent apoproteins are exposed to the cytoplasmic surface, we administered L-[3H]leucine to young chickens and at various times isolated RER and Golgi cell fractions. Radioactive RER and Golgi cell fractions were treated with exogenous protease and the percent of nascent apoAI and apoB accessible to proteolysis was determined and compared to that of albumin. At 5, 10, and 20 min of labeling, 35-56% of nascent apoAI and 60-75% of apoB in RER were degraded, while albumin was refractive to this treatment. At all times both apolipoproteins and albumin present in Golgi cell fractions were protected from proteolysis. These biochemical and morphological findings indicate that apoAI and apoB are associated with the rough microsomal membrane and are partially exposed to the cytoplasmic surface at early stages of secretion. They may later enter the luminal side of the ER and, on entering the Golgi, form lipoprotein particles.

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Selected References

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  1. Altman L. G., Schneider B. G., Papermaster D. S. Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy. J Histochem Cytochem. 1984 Nov;32(11):1217–1223. doi: 10.1177/32.11.6436366. [DOI] [PubMed] [Google Scholar]
  2. Aris J. P., Blobel G. Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J Cell Biol. 1988 Jul;107(1):17–31. doi: 10.1083/jcb.107.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bamberger M. J., Lane M. D. Assembly of very low density lipoprotein in the hepatocyte. Differential transport of apoproteins through the secretory pathway. J Biol Chem. 1988 Aug 25;263(24):11868–11878. [PubMed] [Google Scholar]
  4. Banerjee D., Mukherjee T. K., Redman C. M. Biosynthesis of high density lipoprotein by chicken liver: intracellular transport and proteolytic processing of nascent apolipoprotein A-1. J Cell Biol. 1985 Oct;101(4):1219–1226. doi: 10.1083/jcb.101.4.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Banerjee D., Redman C. M. Biosynthesis of high density lipoprotein by chicken liver: conjugation of nascent lipids with apoprotein A1. J Cell Biol. 1984 Dec;99(6):1917–1926. doi: 10.1083/jcb.99.6.1917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Banerjee D., Redman C. M. Biosynthesis of high density lipoprotein by chicken liver: nature of nascent intracellular high density lipoprotein. J Cell Biol. 1983 Mar;96(3):651–660. doi: 10.1083/jcb.96.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blue M. L., Ostapchuk P., Gordon J. S., Williams D. L. Synthesis of apolipoprotein AI by peripheral tissues of the rooster. A possible mechanism of cellular cholesterol efflux. J Biol Chem. 1982 Sep 25;257(18):11151–11159. [PubMed] [Google Scholar]
  8. Borchardt R. A., Davis R. A. Intrahepatic assembly of very low density lipoproteins. Rate of transport out of the endoplasmic reticulum determines rate of secretion. J Biol Chem. 1987 Dec 5;262(34):16394–16402. [PubMed] [Google Scholar]
  9. Borén J., Wettesten M., Sjöberg A., Thorlin T., Bondjers G., Wiklund O., Olofsson S. O. The assembly and secretion of apoB 100 containing lipoproteins in Hep G2 cells. Evidence for different sites for protein synthesis and lipoprotein assembly. J Biol Chem. 1990 Jun 25;265(18):10556–10564. [PubMed] [Google Scholar]
  10. Boström K., Borén J., Wettesten M., Sjöberg A., Bondjers G., Wiklund O., Carlsson P., Olofsson S. O. Studies on the assembly of apo B-100-containing lipoproteins in HepG2 cells. J Biol Chem. 1988 Mar 25;263(9):4434–4442. [PubMed] [Google Scholar]
  11. Boström K., Wettesten M., Borén J., Bondjers G., Wiklund O., Olofsson S. O. Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells. J Biol Chem. 1986 Oct 15;261(29):13800–13806. [PubMed] [Google Scholar]
  12. Chapman M. J. Animal lipoproteins: chemistry, structure, and comparative aspects. J Lipid Res. 1980 Sep;21(7):789–853. [PubMed] [Google Scholar]
  13. Chuck S. L., Yao Z., Blackhart B. D., McCarthy B. J., Lingappa V. R. New variation on the translocation of proteins during early biogenesis of apolipoprotein B. Nature. 1990 Jul 26;346(6282):382–385. doi: 10.1038/346382a0. [DOI] [PubMed] [Google Scholar]
  14. Davis R. A., Thrift R. N., Wu C. C., Howell K. E. Apolipoprotein B is both integrated into and translocated across the endoplasmic reticulum membrane. Evidence for two functionally distinct pools. J Biol Chem. 1990 Jun 15;265(17):10005–10011. [PubMed] [Google Scholar]
  15. Dixon J. L., Battini R., Ferrari S., Redman C. M., Banerjee D. Expression and secretion of chicken apolipoprotein AI in transfected COS cells. Biochim Biophys Acta. 1989 Sep 21;1009(1):47–53. doi: 10.1016/0167-4781(89)90077-8. [DOI] [PubMed] [Google Scholar]
  16. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gilmore R., Blobel G. Translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants. Cell. 1985 Sep;42(2):497–505. doi: 10.1016/0092-8674(85)90107-2. [DOI] [PubMed] [Google Scholar]
  18. Gotto A. M., Jr, Pownall H. J., Havel R. J. Introduction to the plasma lipoproteins. Methods Enzymol. 1986;128:3–41. doi: 10.1016/0076-6879(86)28061-1. [DOI] [PubMed] [Google Scholar]
  19. Green P. H., Glickman R. M. Intestinal lipoprotein metabolism. J Lipid Res. 1981 Nov;22(8):1153–1173. [PubMed] [Google Scholar]
  20. Hashimoto S., Fogelman A. M. Smooth microsomes. a trap for cholesteryl ester formed in hepatic microsomes. J Biol Chem. 1980 Sep 25;255(18):8678–8684. [PubMed] [Google Scholar]
  21. Higgins J. A., Fieldsend J. K. Phosphatidylcholine synthesis for incorporation into membranes or for secretion as plasma lipoproteins by Golgi membranes of rat liver. J Lipid Res. 1987 Mar;28(3):268–278. [PubMed] [Google Scholar]
  22. Howell K. E., Palade G. E. Heterogeneity of lipoprotein particles in hepatic Golgi fractions. J Cell Biol. 1982 Mar;92(3):833–845. doi: 10.1083/jcb.92.3.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Janero D. R., Lane M. D. Sequential assembly of very low density lipoprotein apolipoproteins, triacylglycerol, and phosphoglycerides by the intact liver cell. J Biol Chem. 1983 Dec 10;258(23):14496–14504. [PubMed] [Google Scholar]
  24. Norum K. R., Berg T., Helgerud P., Drevon C. A. Transport of cholesterol. Physiol Rev. 1983 Oct;63(4):1343–1419. doi: 10.1152/physrev.1983.63.4.1343. [DOI] [PubMed] [Google Scholar]
  25. Paver J. L., Hawkins H. C., Freedman R. B. Preparation and characterization of dog pancreas microsomal membranes specifically depleted of protein disulphide-isomerase. Biochem J. 1989 Feb 1;257(3):657–663. doi: 10.1042/bj2570657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pease R. J., Harrison G. B., Scott J. Cotranslocational insertion of apolipoprotein B into the inner leaflet of the endoplasmic reticulum. Nature. 1991 Oct 3;353(6343):448–450. doi: 10.1038/353448a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sato R., Imanaka T., Takatsuki A., Takano T. Degradation of newly synthesized apolipoprotein B-100 in a pre-Golgi compartment. J Biol Chem. 1990 Jul 15;265(20):11880–11884. [PubMed] [Google Scholar]
  28. Scow R. O., Blanchette-Mackie E. J. Why fatty acids flow in cell membranes. Prog Lipid Res. 1985;24(3):197–241. doi: 10.1016/0163-7827(85)90002-5. [DOI] [PubMed] [Google Scholar]
  29. Shackelford J. E., Lebherz H. G. Synthesis and secretion of apolipoprotein A1 by chick breast muscle. J Biol Chem. 1983 Jun 10;258(11):7175–7180. [PubMed] [Google Scholar]
  30. Siuta-Mangano P., Howard S. C., Lennarz W. J., Lane M. D. Synthesis, processing, and secretion of apolipoprotein B by the chick liver cell. J Biol Chem. 1982 Apr 25;257(8):4292–4300. [PubMed] [Google Scholar]
  31. Stoffel W., Blobel G., Walter P. Synthesis in vitro and translocation of apolipoprotein AI across microsomal vesicles. Eur J Biochem. 1981 Dec;120(3):519–522. doi: 10.1111/j.1432-1033.1981.tb05730.x. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Vance J. E., Vance D. E. Does rat liver Golgi have the capacity to synthesize phospholipids for lipoprotein secretion? J Biol Chem. 1988 Apr 25;263(12):5898–5909. [PubMed] [Google Scholar]
  34. Walter P., Blobel G. Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol. 1983;96:84–93. doi: 10.1016/s0076-6879(83)96010-x. [DOI] [PubMed] [Google Scholar]
  35. Wong L., Pino R. M. Biogenesis of very-low-density lipoproteins in rat liver. Intracellular distribution of apolipoprotein B. Eur J Biochem. 1987 Apr 15;164(2):357–367. doi: 10.1111/j.1432-1033.1987.tb11066.x. [DOI] [PubMed] [Google Scholar]

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