Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Jun 1;110(6):1923–1933. doi: 10.1083/jcb.110.6.1923

Studies on the mechanisms of autophagy: formation of the autophagic vacuole

PMCID: PMC2116114  PMID: 2351689

Abstract

Autophagic vacuoles form within 15 min of perfusing a liver with amino acid-depleted medium. These vacuoles are bound by a "smooth" double membrane and do not contain acid phosphatase activity. In an attempt to identify the membrane source of these vacuoles, I have used morphological techniques combined with immunological probes to localize specific membrane antigens to the limiting membranes of newly formed or nascent autophagic vacuoles. Antibodies to three integral membrane proteins of the plasma membrane (CE9, HA4, and epidermal growth factor receptor) and one of the Golgi apparatus (sialyltransferase) did not label these vacuoles. Internalized epidermal growth factor and its membrane receptor were not found in nascent autophagic vacuoles but were present in lysosome-like degradative autophagic vacuoles. All these results suggested that autophagic vacuoles were not formed from plasma membrane, Golgi apparatus, or endosome constituents. Antisera prepared against integral membrane proteins (14, 25, and 40 kD) of the RER was found to label the inner and outer limiting membranes of almost all nascent autophagic vacuoles. In addition, ribophorin II was identified at the limiting membranes of many nascent autophagic vacuoles. Finally, secretory proteins, rat serum albumin and alpha 2u- globulin, were localized to the lumen of the RER and to the intramembrane space between the inner and outer membranes of some of these vacuoles. The results were consistent with the formation of autophagic vacuoles from ribosome-free regions of the RER.

Full Text

The Full Text of this article is available as a PDF (4.2 MB).

Selected References

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

  1. Arstila A. U., Trump B. F. Studies on cellular autophagocytosis. The formation of autophagic vacuoles in the liver after glucagon administration. Am J Pathol. 1968 Nov;53(5):687–733. [PMC free article] [PubMed] [Google Scholar]
  2. Barrett A. J., Kirschke H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981;80(Pt 100):535–561. doi: 10.1016/s0076-6879(81)80043-2. [DOI] [PubMed] [Google Scholar]
  3. Bartles J. R., Braiterman L. T., Hubbard A. L. Endogenous and exogenous domain markers of the rat hepatocyte plasma membrane. J Cell Biol. 1985 Apr;100(4):1126–1138. doi: 10.1083/jcb.100.4.1126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown W. J., Farquhar M. G. The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisternae. Cell. 1984 Feb;36(2):295–307. doi: 10.1016/0092-8674(84)90223-x. [DOI] [PubMed] [Google Scholar]
  5. Chiang H. L., Terlecky S. R., Plant C. P., Dice J. F. A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science. 1989 Oct 20;246(4928):382–385. doi: 10.1126/science.2799391. [DOI] [PubMed] [Google Scholar]
  6. De Duve C., Wattiaux R. Functions of lysosomes. Annu Rev Physiol. 1966;28:435–492. doi: 10.1146/annurev.ph.28.030166.002251. [DOI] [PubMed] [Google Scholar]
  7. Deter R. L. Analog modeling of glucagon-induced autophagy in rat liver. I. Conceptual and mathematical model of telolysosome-autophagosome-autolysosome interaction. Exp Cell Res. 1975 Aug;94(1):122–126. doi: 10.1016/0014-4827(75)90538-8. [DOI] [PubMed] [Google Scholar]
  8. Dice J. F. Molecular determinants of protein half-lives in eukaryotic cells. FASEB J. 1987 Nov;1(5):349–357. doi: 10.1096/fasebj.1.5.2824267. [DOI] [PubMed] [Google Scholar]
  9. Dunn W. A., Connolly T. P., Hubbard A. L. Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway. J Cell Biol. 1986 Jan;102(1):24–36. doi: 10.1083/jcb.102.1.24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dunn W. A., Hubbard A. L. Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics. J Cell Biol. 1984 Jun;98(6):2148–2159. doi: 10.1083/jcb.98.6.2148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dunn W. A., Jr Studies on the mechanisms of autophagy: maturation of the autophagic vacuole. J Cell Biol. 1990 Jun;110(6):1935–1945. doi: 10.1083/jcb.110.6.1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dunn W. A., Wall D. A., Hubbard A. L. Use of isolated, perfused liver in studies of receptor-mediated endocytosis. Methods Enzymol. 1983;98:225–241. doi: 10.1016/0076-6879(83)98151-x. [DOI] [PubMed] [Google Scholar]
  13. Ericsson J. L. Studies on induced cellular autophagy. II. Characterization of the membranes bordering autophagosomes in parenchymal liver cells. Exp Cell Res. 1969 Aug;56(2):393–405. doi: 10.1016/0014-4827(69)90030-5. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Gordon P. B., Seglen P. O. Prelysosomal convergence of autophagic and endocytic pathways. Biochem Biophys Res Commun. 1988 Feb 29;151(1):40–47. doi: 10.1016/0006-291x(88)90556-6. [DOI] [PubMed] [Google Scholar]
  16. Hershko A. Ubiquitin-mediated protein degradation. J Biol Chem. 1988 Oct 25;263(30):15237–15240. [PubMed] [Google Scholar]
  17. Hortsch M., Meyer D. I. Immunochemical analysis of rough and smooth microsomes from rat liver. Segregation of docking protein in rough membranes. Eur J Biochem. 1985 Aug 1;150(3):559–564. doi: 10.1111/j.1432-1033.1985.tb09057.x. [DOI] [PubMed] [Google Scholar]
  18. Hubbard A. L., Bartles J. R., Braiterman L. T. Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies. J Cell Biol. 1985 Apr;100(4):1115–1125. doi: 10.1083/jcb.100.4.1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kominami E., Hashida S., Khairallah E. A., Katunuma N. Sequestration of cytoplasmic enzymes in an autophagic vacuole-lysosomal system induced by injection of leupeptin. J Biol Chem. 1983 May 25;258(10):6093–6100. [PubMed] [Google Scholar]
  20. Kreibich G., Debey P., Sabatini D. D. Selective release of content from microsomal vesicles without membrane disassembly. I. Permeability changes induced by low detergent concentrations. J Cell Biol. 1973 Aug;58(2):436–462. doi: 10.1083/jcb.58.2.436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kreibich G., Ulrich B. L., Sabatini D. D. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes. J Cell Biol. 1978 May;77(2):464–487. doi: 10.1083/jcb.77.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lardeux B. R., Mortimore G. E. Amino acid and hormonal control of macromolecular turnover in perfused rat liver. Evidence for selective autophagy. J Biol Chem. 1987 Oct 25;262(30):14514–14519. [PubMed] [Google Scholar]
  23. 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]
  24. Masaki R., Yamamoto A., Tashiro Y. Cytochrome P-450 and NADPH-cytochrome P-450 reductase are degraded in the autolysosomes in rat liver. J Cell Biol. 1987 May;104(5):1207–1215. doi: 10.1083/jcb.104.5.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mortimore G. E., Pösö A. R., Lardeux B. R. Mechanism and regulation of protein degradation in liver. Diabetes Metab Rev. 1989 Feb;5(1):49–70. doi: 10.1002/dmr.5610050105. [DOI] [PubMed] [Google Scholar]
  26. Morton A. J., Goldspink D. F. Protein turnover in the rat uterus during and after pregnancy. Prog Clin Biol Res. 1985;180:641–643. [PubMed] [Google Scholar]
  27. Pfeifer U., Bertling J. A morphometric study of the inhibition of autophagic degradation during restorative growth of liver cells in rats re-fed after starvation. Virchows Arch B Cell Pathol. 1977 Jun 24;24(2):109–120. doi: 10.1007/BF02889272. [DOI] [PubMed] [Google Scholar]
  28. Pfeifer U. Inhibited autophagic degradation of cytoplasm during compensatory growth of liver cells after partial hepatectomy. Virchows Arch B Cell Pathol Incl Mol Pathol. 1979 Jun 29;30(3):313–333. doi: 10.1007/BF02889111. [DOI] [PubMed] [Google Scholar]
  29. Pontremoli S., Melloni E. Extralysosomal protein degradation. Annu Rev Biochem. 1986;55:455–481. doi: 10.1146/annurev.bi.55.070186.002323. [DOI] [PubMed] [Google Scholar]
  30. Pösö A. R., Wert J. J., Jr, Mortimore G. E. Multifunctional control of amino acids of deprivation-induced proteolysis in liver. Role of leucine. J Biol Chem. 1982 Oct 25;257(20):12114–12120. [PubMed] [Google Scholar]
  31. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Reunanen H., Punnonen E. L., Hirsimäki P. Studies on vinblastine-induced autophagocytosis in mouse liver. V. A cytochemical study on the origin of membranes. Histochemistry. 1985;83(6):513–517. doi: 10.1007/BF00492453. [DOI] [PubMed] [Google Scholar]
  33. Robinson J. M. Improved localization of intracellular sites of phosphatases using cerium and cell permeabilization. J Histochem Cytochem. 1985 Aug;33(8):749–754. doi: 10.1177/33.8.2991362. [DOI] [PubMed] [Google Scholar]
  34. Sakai M., Araki N., Ogawa K. Lysosomal movements during heterophagy and autophagy: with special reference to nematolysosome and wrapping lysosome. J Electron Microsc Tech. 1989 Jun;12(2):101–131. doi: 10.1002/jemt.1060120206. [DOI] [PubMed] [Google Scholar]
  35. Schworer C. M., Mortimore G. E. Glucagon-induced autophagy and proteolysis in rat liver: mediation by selective deprivation of intracellular amino acids. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3169–3173. doi: 10.1073/pnas.76.7.3169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schworer C. M., Shiffer K. A., Mortimore G. E. Quantitative relationship between autophagy and proteolysis during graded amino acid deprivation in perfused rat liver. J Biol Chem. 1981 Jul 25;256(14):7652–7658. [PubMed] [Google Scholar]
  37. Scornik O. A., Botbol V. Role of changes in protein degradation in the growth of regenerating livers. J Biol Chem. 1976 May 25;251(10):2891–2897. [PubMed] [Google Scholar]
  38. Shelburne J. D., Arstila A. U., Trump B. F. Studies on cellular autophagocytosis. The relationship of autophagocytosis to protein synthesis and to energy metabolism in rat liver and flounder kidney tubules in vitro. Am J Pathol. 1973 Dec;73(3):641–670. [PMC free article] [PubMed] [Google Scholar]
  39. Stauffer C. E. A linear standard curve for the Folin Lowry determination of protein. Anal Biochem. 1975 Dec;69(2):646–648. doi: 10.1016/0003-2697(75)90172-4. [DOI] [PubMed] [Google Scholar]
  40. Titus D. E., Becker W. M. Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy. J Cell Biol. 1985 Oct;101(4):1288–1299. doi: 10.1083/jcb.101.4.1288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weissmann G. Lysosomes. N Engl J Med. 1965 Nov 18;273(21):1143–concl. doi: 10.1056/NEJM196511182732107. [DOI] [PubMed] [Google Scholar]

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

RESOURCES