Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Jul;80(13):3938–3942. doi: 10.1073/pnas.80.13.3938

Subfractionation of rat liver Golgi apparatus: separation of enzyme activities involved in the biosynthesis of the phosphomannosyl recognition marker in lysosomal enzymes.

S L Deutscher, K E Creek, M Merion, C B Hirschberg
PMCID: PMC394174  PMID: 6306653

Abstract

A highly purified Golgi apparatus preparation from rat liver was subfractionated on a Percoll gradient into two major protein peaks of similar size that migrated at densities of 1.028 and 1.051 g/ml. The lighter protein peak contained 70--80% of the total activities of the oligosaccharide-processing enzymes alpha-1,2-mannosidase and mannosidase II and of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyl-1-phosphotransferase (alpha-N-acetylglucosaminylphosphotransferase), an enzyme involved in the biosynthesis of the mannose 6-phosphate recognition marker of lysosomal enzymes. These enzyme activities were enriched 2-fold in specific activity over that of the heavy protein peak. In contrast, 80% of the alpha-N-acetylglucosaminylphosphodiesterase, an enzyme that exposes 6-phosphomonoesters of mannose on the oligosaccharide chains of lysosomal enzymes, migrated in a region of slightly higher density than did the protein peak of density 1.051 g/ml. Sialyltransferase (SiaTase) and galactosyltransferase (Gal-Tase) activities distributed almost equally among the two protein peaks. Controls rule out that the two protein peaks were the result of aggregation/deaggregation and that enzyme activities were altered by Percoll per se. Lysosomal enzyme activities migrated in a region essentially devoid of Golgi apparatus-associated enzyme activities. These results suggest a physical separation within the Golgi apparatus of some of the enzymes involved in the biosynthesis and processing of the oligosaccharides on glycoproteins, including those responsible for the formation of the mannose 6-phosphate recognition marker on lysosomal enzymes.

Full text

PDF
3940

Selected References

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

  1. Andersson G. N., Eriksson L. C. Characterization of UDP-galactosyl:asialo-mucin transferase activity in the Golgi system of rat liver. Biochim Biophys Acta. 1979 Oct 11;570(2):239–247. doi: 10.1016/0005-2744(79)90144-x. [DOI] [PubMed] [Google Scholar]
  2. Bennett G., O'Shaughnessy D. The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N-acetylmannosamine. I. Observations in hepatocytes. J Cell Biol. 1981 Jan;88(1):1–15. doi: 10.1083/jcb.88.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bergeron J. J., Rachubinski R. A., Sikstrom R. A., Posner B. I., Paiement J. Galactose transfer to endogenous acceptors within Golgi fractions of rat liver. J Cell Biol. 1982 Jan;92(1):139–146. doi: 10.1083/jcb.92.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Briles E. B., Li E., Kornfeld S. Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose. J Biol Chem. 1977 Feb 10;252(3):1107–1116. [PubMed] [Google Scholar]
  5. Carey D. J., Hirschberg C. B. Kinetics of glycosylation and intracellular transport of sialoglycoproteins in mouse liver. J Biol Chem. 1980 May 10;255(9):4348–4354. [PubMed] [Google Scholar]
  6. Creek K. E., Sly W. S. Adsorptive pinocytosis of phosphorylated oligosaccharides by human fibroblasts. J Biol Chem. 1982 Sep 10;257(17):9931–9937. [PubMed] [Google Scholar]
  7. Dewald B., Touster O. A new alpha-D-mannosidase occurring in Golgi membranes. J Biol Chem. 1973 Oct 25;248(20):7223–7233. [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. 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]
  10. Emmelot P., Bos C. J., van Hoeven R. P., van Blitterswijk W. J. Isolation of plasma membranes from rat and mouse livers and hepatomas. Methods Enzymol. 1974;31:75–90. doi: 10.1016/0076-6879(74)31008-7. [DOI] [PubMed] [Google Scholar]
  11. Fischer H. D., Gonzalez-Noriega A., Sly W. S., Morré D. J. Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes. J Biol Chem. 1980 Oct 25;255(20):9608–9615. [PubMed] [Google Scholar]
  12. Fleischer B., Zambrano F. Golgi apparatus of rat kidney. Preparation and role in sulfatide formation. J Biol Chem. 1974 Sep 25;249(18):5995–6003. [PubMed] [Google Scholar]
  13. 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]
  14. Griffiths G., Brands R., Burke B., Louvard D., Warren G. Viral membrane proteins acquire galactose in trans Golgi cisternae during intracellular transport. J Cell Biol. 1982 Dec;95(3):781–792. doi: 10.1083/jcb.95.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hess K. A., Morré D. J., Merritt W. D. Lipoprotein secretion by rat liver Golgi apparatus. Lipoprotein particles and lipase activity. Cytobiologie. 1979 Feb;18(3):431–449. [PubMed] [Google Scholar]
  16. 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]
  17. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  18. Kitcher S. A., Siddle K., Luzio J. P. A method for the determination of glucose-6-phosphatase activity in rat liver with [U-14C]glucose 6-phosphate as substrate. Anal Biochem. 1978 Jul 15;88(1):29–36. doi: 10.1016/0003-2697(78)90395-0. [DOI] [PubMed] [Google Scholar]
  19. Merritt W. D., Morré D. J. A glycosyl transferase of high specific activity in secretory vesicles from isolated Golgi apparatus of rat liver. Biochim Biophys Acta. 1973 Apr 28;304(2):397–407. doi: 10.1016/0304-4165(73)90259-6. [DOI] [PubMed] [Google Scholar]
  20. Morré D. J., Ovtracht L. Structure of rat liver Golgi apparatus: relationship to lipoprotein secretion. J Ultrastruct Res. 1981 Mar;74(3):284–295. doi: 10.1016/s0022-5320(81)80119-0. [DOI] [PubMed] [Google Scholar]
  21. NOVIKOFF A. B., GOLDFISCHER S. Nucleosidediphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc Natl Acad Sci U S A. 1961 Jun 15;47:802–810. doi: 10.1073/pnas.47.6.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Natowicz M., Baenziger J. U., Sly W. S. Structural studies of the phosphorylated high mannose-type oligosaccharides on human beta-glucuronidase. J Biol Chem. 1982 Apr 25;257(8):4412–4420. [PubMed] [Google Scholar]
  23. Paiement J., Rachubinski R. A., Ng Ying Kin N. M., Sikstrom R. A., Bergeron J. J. Membrane fusion and glycosylation in the rat hepatic Golgi apparatus. J Cell Biol. 1982 Jan;92(1):147–154. doi: 10.1083/jcb.92.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Ravoet A. M., Amar-Costesec A., Godelaine D., Beaufay H. Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver. J Cell Biol. 1981 Dec;91(3 Pt 1):679–688. doi: 10.1083/jcb.91.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reitman M. L., Kornfeld S. Lysosomal enzyme targeting. N-Acetylglucosaminylphosphotransferase selectively phosphorylates native lysosomal enzymes. J Biol Chem. 1981 Dec 10;256(23):11977–11980. [PubMed] [Google Scholar]
  28. 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]
  29. Rothman J. E., Fries E. Transport of newly synthesized vesicular stomatitis viral glycoprotein to purified Golgi membranes. J Cell Biol. 1981 Apr;89(1):162–168. doi: 10.1083/jcb.89.1.162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rothman J. E. The golgi apparatus: two organelles in tandem. Science. 1981 Sep 11;213(4513):1212–1219. doi: 10.1126/science.7268428. [DOI] [PubMed] [Google Scholar]
  31. Sly W. S., Fischer H. D. The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J Cell Biochem. 1982;18(1):67–85. doi: 10.1002/jcb.1982.240180107. [DOI] [PubMed] [Google Scholar]
  32. Tabas I., Kornfeld S. Purification and characterization of a rat liver Golgi alpha-mannosidase capable of processing asparagine-linked oligosaccharides. J Biol Chem. 1979 Nov 25;254(22):11655–11663. [PubMed] [Google Scholar]
  33. Tulsiani D. R., Hubbard S. C., Robbins P. W., Touster O. alpha-D-Mannosidases of rat liver Golgi membranes. Mannosidase II is the GlcNAcMAN5-cleaving enzyme in glycoprotein biosynthesis and mannosidases Ia and IB are the enzymes converting Man9 precursors to Man5 intermediates. J Biol Chem. 1982 Apr 10;257(7):3660–3668. [PubMed] [Google Scholar]
  34. Tulsiani D. R., Opheim D. J., Touster O. Purification and characterization of alpha-D-mannosidase from rat liver golgi membranes. J Biol Chem. 1977 May 25;252(10):3227–3233. [PubMed] [Google Scholar]
  35. Varki A., Kornfeld S. Identification of a rat liver alpha-N-acetylglucosaminyl phosphodiesterase capable of removing "blocking" alpha-N-acetylglucosamine residues from phosphorylated high mannose oligosaccharides of lysosomal enzymes. J Biol Chem. 1980 Sep 25;255(18):8398–8401. [PubMed] [Google Scholar]
  36. Varki A., Kornfeld S. Purification and characterization of rat liver alpha-N-acetylglucosaminyl phosphodiesterase. J Biol Chem. 1981 Oct 10;256(19):9937–9943. [PubMed] [Google Scholar]
  37. Varki A., Kornfeld S. Structural studies of phosphorylated high mannose-type oligosaccharides. J Biol Chem. 1980 Nov 25;255(22):10847–10858. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES