Advertisement

Mechanism of Autophagy in Permeabilized Hepatocytes

Evidence for Regulation by GTP Binding Proteins
  • Motoni Kadowaki
  • Rina Venerando
  • Giovanni Miotto
  • Glenn E. Mortimore
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 389)

Abstract

Autophagic vacuoles in hepatocytes are formed from membranes of rough and smooth endoplasmic reticulum (ER) by processes that are under immediate physiologic control by amino acids, insulin, and glucagon (reviewed in 1). Little, though, is known of the molecular steps involved. Microinjection (2) and electropermeabilization (3) have been used to introduce markers into cells and newly formed vacuoles. But because the pores are transient, observations are restricted to events that occur after membrane resealing. In order to gain access to autophagy under steady state conditions, we permeabilized hepatocytes with α-toxin from Staphylococcus aureus, an agent which forms stable ≈1.5 nm channels that limit exchange to molecules of approximately 1000 Da (4). Such pores will admit nucleotides and labeled residualizing probes without loss of cell proteins, a desirable, possibly necessary, condition for evaluating autophagically-mediated proteolysis.

Keywords

Volume Density Autophagic Vacuole Cytoplasmic Sequestration Membrane Reseal Permeabilized Hepatocyte 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Mortimore, G. E., and Kadowaki, M., 1994, Autophagy: Its mechanism and regulation, In: Cellular Proteolytic Systems, (Ciechanover, A. J., and Schwartz, A. L., eds) pp. 65–87, Wiley-Liss, Inc., New York.Google Scholar
  2. 2.
    Hendil, K. B., 1980, Intracellular degradation of hemoglobin transferred into fibroblasts by fusion with red blood cells, J. Cell. Physiol. 105:449–460.PubMedCrossRefGoogle Scholar
  3. 3.
    Seglen, P. O., 1987, Regulation of autophagic protein degradation in isolated liver cells. In: Lysosomes: Their Role in Protein Breakdown (Glaumann, H., and Ballard, F. J., eds) pp. 371–414, Academic Press, London.Google Scholar
  4. 4.
    Bhakdi, S., Weller, U., Walev, I., Martin, E., Jonas, D., and Palmer, M., 1993, A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes, Med. Microbiol. Immunol. 182:167–175.PubMedCrossRefGoogle Scholar
  5. 5.
    Kadowaki, M., Venerando, R., Miotto, G., and Mortimore, G. E., 1994, De novo autophagic vacuole formation in hepatocytes permeabilized by Staphylococcus aureus α-toxin: Inhibition by nonhydrolyzable GTP analogs, J. Biol. Chem. 269,3703–3710.PubMedGoogle Scholar
  6. 6.
    McEwen, B. F., and Arion, W. J., 1985, Permeabilization of rat hepatocytes with Staphylococcus aureus α-toxin, J. Cell Biol. 100:1922–1929.PubMedCrossRefGoogle Scholar
  7. 7.
    Baldini, G., Hohman, R., Charron, M. J., and Lodish, H. F., 1991, Insulin and nonhydrolyzable GTP analogs induce translocation of GLUT 4 to the plasma membrane in α-toxin-permeabilized rat adipose cells, J. Biol. Chem. 266:4037–4040.PubMedGoogle Scholar
  8. 8.
    Seglen, P. O., and Gordon, P. B., 1984, Amino acid control of autophagic sequestration and protein degradation in isolated rat hepatocytes, J. Cell Biol. 99:435–444.PubMedCrossRefGoogle Scholar
  9. 9.
    Surmacz, C. A., Ward, W. F., and Mortimore, G. E., 1982, Distribution of 125l-asialofetuin among liver particles separated on colloidal silica gradients, Biochem. Biophys. Res. Commun. 107:1425–1432.PubMedCrossRefGoogle Scholar
  10. 10.
    Surmacz, C. A., Wert, J. J. Jr., and Mortimore, G. E., 1983, Role of particle interaction on distribution of liver lysosomes in colloidal silica, Am. J. Physiol. 245:C52–C60.PubMedGoogle Scholar
  11. 11.
    Surmacz, C. A., Wert, J. J. Jr., and Mortimore, G. E., 1983, Metabolic alterations and distribution of rat liver lysosomes in colloidal silica, Am. J. Physiol. 245:C61–C67.PubMedGoogle Scholar
  12. 12.
    Gordon, P. B., and Seglen, P. O., 1988, Prelysosomal convergence of autophagic and endocytic pathways, Biochem. Biophys. Res. Commun. 151:40–47.PubMedCrossRefGoogle Scholar
  13. 13.
    Tooze, J., Hollinshead, M., Ludwig, T. Howell, K., Hoflack, B., and Kern, H., 1990. In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome, J. Cell Biol. 111:329–345.PubMedCrossRefGoogle Scholar
  14. 14.
    Zhong, Z.-D., Jadot, M., Wattiaux-De Coninck, S., and Wattiaux, R., 1992, Uptake of tyramine cellobiose by rat liver, Biochim. Biophys. Acta 1106,3 11–316.Google Scholar
  15. 15.
    Mortimore, G. E., Lardeux, B. R., and Adams, C. A., 1988, Regulation of microautophagy and basal protein turnover in rat liver: Effects of short-term starvation, J. Biol. Chem. 263:2506–2512.PubMedGoogle Scholar
  16. 16.
    Sai, Y., and Ohkuma, S., 1992, Small GTP-binding proteins on rat liver lysosomal membranes, Cell Struc. Func. 17:363–369.CrossRefGoogle Scholar
  17. 17.
    Sai, Y, Arai, K., and Ohkuma, S., 1994, Cytosol treated with GTPyS disintegrates lysosomes in vitro, Biochem. Biophys. Res. Commun. 198:869–877.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1996

Authors and Affiliations

  • Motoni Kadowaki
    • 1
  • Rina Venerando
    • 2
  • Giovanni Miotto
    • 2
  • Glenn E. Mortimore
    • 3
  1. 1.Department of Applied BiochemistryNiigata UniversityNiigataJapan
  2. 2.Dipartimento di Chimica BiologicaUniversità Degli Studi di PadovaPadovaItaly
  3. 3.Department of Cellular and Molecular PhysiologyThe Pennsylvania State UniversityHersheyUSA

Personalised recommendations