Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Jun 2;113(6):1305–1312. doi: 10.1083/jcb.113.6.1305

Inhibition of autophagic-lysosomal delivery and autophagic lactolysis by asparagine

PMCID: PMC2289037  PMID: 1904444

Abstract

Overall autophagy was measured in isolated hepatocytes as the sequestration and lysosomal hydrolysis of electroinjected [14C]lactose, using HPLC to separate the degradation product [14C]glucose from undegraded lactose. In addition, the sequestration step was measured separately as the transfer from cytosol to sedimentable cell structures of electroinjected [3H]raffinose or endogenous lactate dehydrogenase (LDH; in the presence of leupeptin to inhibit lysosomal proteolysis). Inhibitor effects at postsequestrational steps could be detected as the accumulation of autophaged lactose (which otherwise is degraded intralysosomally), or of LDH in the absence of leupeptin. Asparagine, previously shown to inhibit autophagic but not endocytic protein breakdown, strongly suppressed the autophagic hydrolysis of electroinjected lactose. Vinblastine, which inhibits both types of degradation, likewise suppressed lactose hydrolysis. Asparagine had little or no effect on sequestration, but caused an accumulation of autophaged LDH and lactose, indicating inhibition at a postsequestrational step. Neither asparagine nor vinblastine affected the degradation of intralysosomal lactose preaccumulated in the presence of the reversible lysosome inhibitor propylamine. However, if lactose was preaccumulated in the presence of asparagine, both asparagine and vinblastine suppressed its subsequent degradation. The data thus indicate that autophagic-lysosomal delivery, i.e., the transfer of autophaged material from prelysosomal vacuoles to lysosomes, is inhibited selectively by asparagine and non-selectively by vinblastine.

Full Text

The Full Text of this article is available as a PDF (813.6 KB).

Selected References

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

  1. Berg T., Kindberg G. M., Ford T., Blomhoff R. Intracellular transport of asialoglycoproteins in rat hepatocytes. Evidence for two subpopulations of lysosomes. Exp Cell Res. 1985 Dec;161(2):285–296. doi: 10.1016/0014-4827(85)90086-2. [DOI] [PubMed] [Google Scholar]
  2. Beynon R. J., Bond J. S. Catabolism of intracellular protein: molecular aspects. Am J Physiol. 1986 Aug;251(2 Pt 1):C141–C152. doi: 10.1152/ajpcell.1986.251.2.C141. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Fuchs R., Schmid S., Mellman I. A possible role for Na+,K+-ATPase in regulating ATP-dependent endosome acidification. Proc Natl Acad Sci U S A. 1989 Jan;86(2):539–543. doi: 10.1073/pnas.86.2.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gordon P. B., Høyvik H., Seglen P. O. Sequestration and hydrolysis of electroinjected [14C]lactose as a means of investigating autophagosome-lysosome fusion in isolated rat hepatocytes. Prog Clin Biol Res. 1985;180:475–477. [PubMed] [Google Scholar]
  6. Gordon P. B., Kisen G. O., Kovács A. L., Seglen P. O. Experimental characterization of the autophagic-lysosomal pathway in isolated rat hepatocytes. Biochem Soc Symp. 1989;55:129–143. [PubMed] [Google Scholar]
  7. Gordon P. B., Seglen P. O. Autophagic sequestration of [14C]sucrose, introduced into rat hepatocytes by reversible electro-permeabilization. Exp Cell Res. 1982 Nov;142(1):1–14. doi: 10.1016/0014-4827(82)90402-5. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Gordon P. B., Tolleshaug H., Seglen P. O. Autophagic sequestration of [14C]sucrose introduced into isolated rat hepatocytes by electrical and non-electrical methods. Exp Cell Res. 1985 Oct;160(2):449–458. doi: 10.1016/0014-4827(85)90192-2. [DOI] [PubMed] [Google Scholar]
  10. Gordon P. B., Tolleshaug H., Seglen P. O. Use of digitonin extraction to distinguish between autophagic-lysosomal sequestration and mitochondrial uptake of [14C]sucrose in hepatocytes. Biochem J. 1985 Dec 15;232(3):773–780. doi: 10.1042/bj2320773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Griffiths G., Hoflack B., Simons K., Mellman I., Kornfeld S. The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell. 1988 Feb 12;52(3):329–341. doi: 10.1016/s0092-8674(88)80026-6. [DOI] [PubMed] [Google Scholar]
  12. Grinde B., Seglen P. O. Leucine inhibition of autophagic vacuole formation in isolated rat hepatocytes. Exp Cell Res. 1981 Jul;134(1):33–39. doi: 10.1016/0014-4827(81)90460-2. [DOI] [PubMed] [Google Scholar]
  13. Grinde B., Seglen P. O. Role of microtubuli in the lysosomal degradation of endogenous and exogenous protein in isolated rat hepatocytes. Hoppe Seylers Z Physiol Chem. 1981 May;362(5):549–556. doi: 10.1515/bchm2.1981.362.1.549. [DOI] [PubMed] [Google Scholar]
  14. Gruenberg J., Griffiths G., Howell K. E. Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J Cell Biol. 1989 Apr;108(4):1301–1316. doi: 10.1083/jcb.108.4.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henell F., Glaumann H. Effect of leupeptin on the autophagic vacuolar system of rat hepatocytes. Correlation between ultrastructure and degradation of membrane and cytosolic proteins. Lab Invest. 1984 Jul;51(1):46–56. [PubMed] [Google Scholar]
  16. Høyvik H., Gordon P. B., Seglen P. O. Use of a hydrolysable probe, [14C]lactose, to distinguish between pre-lysosomal and lysosomal steps in the autophagic pathway. Exp Cell Res. 1986 Sep;166(1):1–14. doi: 10.1016/0014-4827(86)90503-3. [DOI] [PubMed] [Google Scholar]
  17. Ishikawa T., Furuno K., Kato K. Ultrastructural studies on autolysosomes in rat hepatocytes after leupeptin treatment. Exp Cell Res. 1983 Mar;144(1):15–24. doi: 10.1016/0014-4827(83)90436-6. [DOI] [PubMed] [Google Scholar]
  18. Kolset S. O., Tolleshaug H., Berg T. The effects of colchicine and cytochalasin B on uptake and degradation of asialo-glycoproteins in isolated rat hepatocytes. Exp Cell Res. 1979 Aug;122(1):159–167. doi: 10.1016/0014-4827(79)90570-6. [DOI] [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. Kopitz J., Kisen G. O., Gordon P. B., Bohley P., Seglen P. O. Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes. J Cell Biol. 1990 Sep;111(3):941–953. doi: 10.1083/jcb.111.3.941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kovács A. L., Grinde B., Seglen P. O. Inhibition of autophagic vacuole formation and protein degradation by amino acids in isolated hepatocytes. Exp Cell Res. 1981 Jun;133(2):431–436. doi: 10.1016/0014-4827(81)90336-0. [DOI] [PubMed] [Google Scholar]
  22. Kovács A. L., Reith A., Seglen P. O. Accumulation of autophagosomes after inhibition of hepatocytic protein degradation by vinblastine, leupeptin or a lysosomotropic amine. Exp Cell Res. 1982 Jan;137(1):191–201. doi: 10.1016/0014-4827(82)90020-9. [DOI] [PubMed] [Google Scholar]
  23. Mortimore G. E., Schworer C. M. Induction of autophagy by amino-acid deprivation in perfused rat liver. Nature. 1977 Nov 10;270(5633):174–176. doi: 10.1038/270174a0. [DOI] [PubMed] [Google Scholar]
  24. Schwarze E., Tolleshaug H., Seglen O. Uptake and degradation of asialo-orosomucoid in hepatocytes from carcinogen-treated rats. Carcinogenesis. 1985 May;6(5):777–782. doi: 10.1093/carcin/6.5.777. [DOI] [PubMed] [Google Scholar]
  25. Seglen P. O., Gordon P. B. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1889–1892. doi: 10.1073/pnas.79.6.1889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Seglen P. O., Gordon P. B. Amino acid control of autophagic sequestration and protein degradation in isolated rat hepatocytes. J Cell Biol. 1984 Aug;99(2):435–444. doi: 10.1083/jcb.99.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Seglen P. O., Gordon P. B., Høyvik H. Radiolabelled sugars as probes of hepatocytic autophagy. Biomed Biochim Acta. 1986;45(11-12):1647–1656. [PubMed] [Google Scholar]
  28. Seglen P. O., Gordon P. B., Poli A. Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes. Biochim Biophys Acta. 1980 Jun 5;630(1):103–118. doi: 10.1016/0304-4165(80)90141-5. [DOI] [PubMed] [Google Scholar]
  29. Seglen P. O., Gordon P. B., Tolleshaug H., Høyvik H. Use of [3H]raffinose as a specific probe of autophagic sequestration. Exp Cell Res. 1986 Jan;162(1):273–277. doi: 10.1016/0014-4827(86)90446-5. [DOI] [PubMed] [Google Scholar]
  30. Seglen P. O., Grinde B., Solheim A. E. Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur J Biochem. 1979 Apr 2;95(2):215–225. doi: 10.1111/j.1432-1033.1979.tb12956.x. [DOI] [PubMed] [Google Scholar]
  31. Seglen P. O. Inhibitors of lysosomal function. Methods Enzymol. 1983;96:737–764. doi: 10.1016/s0076-6879(83)96063-9. [DOI] [PubMed] [Google Scholar]
  32. Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  33. Tolleshaug H., Berg T. The effect of leupeptin on intracellular digestion of asialofetuin in rat hepatocytes. Exp Cell Res. 1981 Jul;134(1):207–217. doi: 10.1016/0014-4827(81)90478-x. [DOI] [PubMed] [Google Scholar]
  34. Tolleshaug H., Seglen P. O. Autophagic-lysosomal and mitochondrial sequestration of [14C]sucrose. Density gradient distribution of sequestered radioactivity. Eur J Biochem. 1985 Dec 2;153(2):223–229. doi: 10.1111/j.1432-1033.1985.tb09290.x. [DOI] [PubMed] [Google Scholar]
  35. Tooze J., Hollinshead M., Ludwig T., Howell K., Hoflack B., Kern H. In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome. J Cell Biol. 1990 Aug;111(2):329–345. doi: 10.1083/jcb.111.2.329. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES