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. 1990 Aug 1;111(2):329–345. doi: 10.1083/jcb.111.2.329

In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome

PMCID: PMC2116176  PMID: 2166050

Abstract

Intracisternal granules (ICGs) are insoluble aggregates of pancreatic digestive enzymes and proenzymes that develop within the lumen of the rough endoplasmic reticulum of exocrine pancreatic cells, especially in guinea pigs. These ICGs are eliminated by autophagy. By morphological criteria, we identified three distinct and sequential classes of autophagic compartments, which we refer to as phagophores, Type I autophagic vacuoles, and Type II autophagic vacuoles. Lobules of guinea pig pancreas were incubated in media containing HRP for periods of 5- 120 min to determine the relationship between the endocytic and autophagic pathways. Incubations with HRP of 15 min or less labeled early endosomes at the cell periphery that were not involved in autophagy of ICGs, but after these short incubations none of the autophagic compartments were HRP positive. After 30-min incubation with HRP, early endosomes at the cell periphery, late endosomes in the pericentriolar region, and, in addition, Type I autophagic vacuoles containing ICGs were all labeled by the tracer. Type II autophagic vacuoles were not labeled after 30-min incubation with HRP but were labeled after incubations of 60-120 min. Phagophores did not receive HRP even after 120 min incubations. We concluded that the autophagic and endocytic pathways converge immediately after the early endosome level and that Type I autophagic vacuoles precede Type II autophagic vacuoles on the endocytic pathway. We studied the distribution of acid phosphatase, lysosomal proteases and cation-independent-mannose-6- phosphate receptor (CI-M6PR) in the three classes of autophagic compartments by histochemical and immunocytochemical methods. Phagophores, the earliest autophagic compartment, contained none of these markers. Type I autophagic vacuoles contained acid phosphatase but, at most, only very low levels of cathepsin D and CI-M6PR. Type II autophagic vacuoles, by contrast, are enriched for acid phosphatase, cathepsin D, and other lysosomal enzymes, and they are also enriched for CI-M6PR. Moreover, soluble fragments of bovine CI-M6PR conjugated to colloidal gold particles heavily labeled Type II but not Type I autophagic vacuoles, and this labeling was specifically blocked by mannose-6-phosphate. This indicates that the lysosomal enzymes present in Type II autophagic vacuoles carry mannose-6-phosphate monoester residues. Using 3-C2, 4-dinitroanilino-3'-amino-N-methyldipropylamine (DAMP), we showed that Type II autophagic vacuoles are acidic. We interpret these findings as indicating that Type II autophagic vacuoles are a prelysosomal compartment in which the already combined endocytic and autophagic pathways meet the delivery pathway of lysosomal enzymes.

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Selected References

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

  1. Amara J. F., Lederkremer G., Lodish H. F. Intracellular degradation of unassembled asialoglycoprotein receptor subunits: a pre-Golgi, nonlysosomal endoproteolytic cleavage. J Cell Biol. 1989 Dec;109(6 Pt 2):3315–3324. doi: 10.1083/jcb.109.6.3315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. G., Falck J. R., Goldstein J. L., Brown M. S. Visualization of acidic organelles in intact cells by electron microscopy. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4838–4842. doi: 10.1073/pnas.81.15.4838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson R. G., Pathak R. K. Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments. Cell. 1985 Mar;40(3):635–643. doi: 10.1016/0092-8674(85)90212-0. [DOI] [PubMed] [Google Scholar]
  4. Bonifacino J. S., Suzuki C. K., Klausner R. D. A peptide sequence confers retention and rapid degradation in the endoplasmic reticulum. Science. 1990 Jan 5;247(4938):79–82. doi: 10.1126/science.2294595. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  7. Chen C., Bonifacino J. S., Yuan L. C., Klausner R. D. Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment. J Cell Biol. 1988 Dec;107(6 Pt 1):2149–2161. doi: 10.1083/jcb.107.6.2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Geuze H. J., Slot J. W. Disproportional immunostaining patterns of two secretory proteins in guinea pig and rat exocrine pancreatic cells. An immunoferritin and fluorescence study. Eur J Cell Biol. 1980 Apr;21(1):93–100. [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Griffiths G., Matteoni R., Back R., Hoflack B. Characterization of the cation-independent mannose 6-phosphate receptor-enriched prelysosomal compartment in NRK cells. J Cell Sci. 1990 Mar;95(Pt 3):441–461. doi: 10.1242/jcs.95.3.441. [DOI] [PubMed] [Google Scholar]
  12. Griffiths G., McDowall A., Back R., Dubochet J. On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res. 1984 Oct;89(1):65–78. doi: 10.1016/s0022-5320(84)80024-6. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Gruenberg J., Howell K. E. Membrane traffic in endocytosis: insights from cell-free assays. Annu Rev Cell Biol. 1989;5:453–481. doi: 10.1146/annurev.cb.05.110189.002321. [DOI] [PubMed] [Google Scholar]
  15. Herzog V., Reggio H. Pathways of endocytosis from luminal plasma membrane in rat exocrine pancreas. Eur J Cell Biol. 1980 Jun;21(2):141–150. [PubMed] [Google Scholar]
  16. Kern H. F., Kern D. Elektronenmikroskopische Untersuchungen über die Wirkung von Kobaltchlorid auf das exokrine Pankreasgewebe des Meerschweinchens. Virchows Arch B Cell Pathol. 1969;4(1):54–70. [PubMed] [Google Scholar]
  17. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  18. Lippincott-Schwartz J., Bonifacino J. S., Yuan L. C., Klausner R. D. Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins. Cell. 1988 Jul 15;54(2):209–220. doi: 10.1016/0092-8674(88)90553-3. [DOI] [PubMed] [Google Scholar]
  19. Marsh M., Griffiths G., Dean G. E., Mellman I., Helenius A. Three-dimensional structure of endosomes in BHK-21 cells. Proc Natl Acad Sci U S A. 1986 May;83(9):2899–2903. doi: 10.1073/pnas.83.9.2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McDowall A., Gruenberg J., Römisch K., Griffiths G. The structure of organelles of the endocytic pathway in hydrated cryosections of cultured cells. Eur J Cell Biol. 1989 Aug;49(2):281–294. [PubMed] [Google Scholar]
  21. Novikoff A. B., Mori M., Quintana N., Yam A. Studies of the secretory process in the mammalian exocrine pancreas. I. The condensing vacuoles. J Cell Biol. 1977 Oct;75(1):148–165. doi: 10.1083/jcb.75.1.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. PALADE G. E. Intracisternal granules in the exocrine cells of the pancreas. J Biophys Biochem Cytol. 1956 Jul 25;2(4):417–422. doi: 10.1083/jcb.2.4.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Scheele G. A., Palade G. E. Studies on the guinea pig pancreas. Parallel discharge of exocrine enzyme activities. J Biol Chem. 1975 Apr 10;250(7):2660–2670. [PubMed] [Google Scholar]
  24. Scheele G. A. Two-dimensional gel analysis of soluble proteins. Charaterization of guinea pig exocrine pancreatic proteins. J Biol Chem. 1975 Jul 25;250(14):5375–5385. [PubMed] [Google Scholar]
  25. Schick J., Kern H., Scheele G. Hormonal stimulation in the exocrine pancreas results in coordinate and anticoordinate regulation of protein synthesis. J Cell Biol. 1984 Nov;99(5):1569–1574. doi: 10.1083/jcb.99.5.1569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Steinberg T. H., Swanson J. A., Silverstein S. C. A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages. J Cell Biol. 1988 Sep;107(3):887–896. doi: 10.1083/jcb.107.3.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  28. Tooze J., Kern H. F., Fuller S. D., Howell K. E. Condensation-sorting events in the rough endoplasmic reticulum of exocrine pancreatic cells. J Cell Biol. 1989 Jul;109(1):35–50. doi: 10.1083/jcb.109.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wileman T., Carson G. R., Concino M., Ahmed A., Terhorst C. The gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors. J Cell Biol. 1990 Apr;110(4):973–986. doi: 10.1083/jcb.110.4.973. [DOI] [PMC free article] [PubMed] [Google Scholar]

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