Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
J Cell Biol. 1987 Aug 1; 105(2): 691–703.
doi: 10.1083/jcb.105.2.691
PMCID: PMC2114780
PMID: 3305521

Identification of an endosomal antigen specific to absorptive cells of suckling rat ileum

Copyright and License information PMC Disclaimer

Abstract

A membrane fraction enriched in apical endosomal tubules was isolated from absorptive cells of suckling rat ileum and used as an immunogen to generate anti-endosome monoclonal antibodies. By immunofluorescence, one of these antibodies bound exclusively to the region of the apical endocytic complex in ileal absorptive cells, but not to other cell types. Immunoblot analysis showed the antigen as a diffuse 55-61-kD band which was highly enriched in the endosome fraction over whole-cell homogenate. The antigen appears to be an intramembrane glycoprotein: it partitioned primarily in the detergent phase after TX-114 extraction, and shifted to 44 kD after chemical deglycosylation. EM immunocytochemistry showed that the antibody bound to the luminal side of endosomal tubule membranes, a portion of endosomal vesicle membranes, and in endocytic pits of apical plasma membranes. However, it did not bind to multivesicular bodies, the giant lysosome, or other organelles. Immunocytochemistry after uptake with adsorbed or soluble tracer proteins showed that the antigen labeled portions of both prelysosomal pathways previously described in these cells (Gonnella, P.A., and M. R. Neutra, 1984, J. Cell Biol., 99:909-917). The function of this glycoprotein is not known, but inasmuch as it has been detected only in absorptive cells of suckling rat ileum, it may serve a function specific to these cells. Nevertheless, this endosomal antigen, designated glycoprotein (gp) 55-61, will serve as a useful marker for exploring membrane dynamics in early stages of the endocytic pathway.

Full Text

The Full Text of this article is available as a PDF (5.4M).

Selected References

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

  • Baenziger JU, Fiete D. Separation of two populations of endocytic vesicles involved in receptor-ligand sorting in rat hepatocytes. J Biol Chem. 1986 Jun 5;261(16):7445–7454. [PubMed] [Google Scholar]
  • Balch WE, Rothman JE. Characterization of protein transport between successive compartments of the Golgi apparatus: asymmetric properties of donor and acceptor activities in a cell-free system. Arch Biochem Biophys. 1985 Jul;240(1):413–425. [PubMed] [Google Scholar]
  • Bartles JR, Braiterman LT, Hubbard AL. Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane. J Biol Chem. 1985 Oct 15;260(23):12792–12802. [PubMed] [Google Scholar]
  • Bartles JR, Braiterman LT, Hubbard AL. Endogenous and exogenous domain markers of the rat hepatocyte plasma membrane. J Cell Biol. 1985 Apr;100(4):1126–1138. [PMC free article] [PubMed] [Google Scholar]
  • Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  • Bradford MM. 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. [PubMed] [Google Scholar]
  • Brown WJ, Goodhouse J, Farquhar MG. Mannose-6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes. J Cell Biol. 1986 Oct;103(4):1235–1247. [PMC free article] [PubMed] [Google Scholar]
  • Chen JW, Murphy TL, Willingham MC, Pastan I, August JT. Identification of two lysosomal membrane glycoproteins. J Cell Biol. 1985 Jul;101(1):85–95. [PMC free article] [PubMed] [Google Scholar]
  • Christensen EI. Rapid membrane recycling in renal proximal tubule cells. Eur J Cell Biol. 1982 Nov;29(1):43–49. [PubMed] [Google Scholar]
  • Cornell R, Padykula HA. A cytological study of intestinal absorption in the suckling rat. Am J Anat. 1969 Jul;125(3):291–315. [PubMed] [Google Scholar]
  • Dickson RB, Beguinot L, Hanover JA, Richert ND, Willingham MC, Pastan I. Isolation and characterization of a highly enriched preparation of receptosomes (endosomes) from a human cell line. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5335–5339. [PMC free article] [PubMed] [Google Scholar]
  • Dunn WA, Connolly TP, Hubbard AL. Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway. J Cell Biol. 1986 Jan;102(1):24–36. [PMC free article] [PubMed] [Google Scholar]
  • Dunn WA, LaBadie JH, Aronson NN., Jr Inhibition of 125I-asialofetuin catabolism by leupeptin in the perfused rat liver and in vivo. J Biol Chem. 1979 May 25;254(10):4191–4196. [PubMed] [Google Scholar]
  • Edge AS, Faltynek CR, Hof L, Reichert LE, Jr, Weber P. Deglycosylation of glycoproteins by trifluoromethanesulfonic acid. Anal Biochem. 1981 Nov 15;118(1):131–137. [PubMed] [Google Scholar]
  • Geuze HJ, Slot JW, Strous GJ, Peppard J, von Figura K, Hasilik A, Schwartz AL. Intracellular receptor sorting during endocytosis: comparative immunoelectron microscopy of multiple receptors in rat liver. Cell. 1984 May;37(1):195–204. [PubMed] [Google Scholar]
  • Gonnella PA, Neutra MR. Membrane-bound and fluid-phase macromolecules enter separate prelysosomal compartments in absorptive cells of suckling rat ileum. J Cell Biol. 1984 Sep;99(3):909–917. [PMC free article] [PubMed] [Google Scholar]
  • Gonnella PA, Neutra MR. Glycoconjugate distribution and mobility on apical membranes of absorptive cells of suckling rat ileum in vivo. Anat Rec. 1985 Dec;213(4):520–528. [PubMed] [Google Scholar]
  • Gonnella PA, Siminoski K, Murphy RA, Neutra MR. Transepithelial transport of epidermal growth factor by absorptive cells of suckling rat ileum. J Clin Invest. 1987 Jul;80(1):22–32. [PMC free article] [PubMed] [Google Scholar]
  • Hoppe CA, Connolly TP, Hubbard AL. Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway. J Cell Biol. 1985 Dec;101(6):2113–2123. [PMC free article] [PubMed] [Google Scholar]
  • Hubbard AL, Wall DA, Ma A. Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains. J Cell Biol. 1983 Jan;96(1):217–229. [PMC free article] [PubMed] [Google Scholar]
  • Jakoi ER, Kempe K, Gaston SM. Ligatin binds phosphohexose residues on acidic hydrolases. J Supramol Struct Cell Biochem. 1981;16(2):139–153. [PubMed] [Google Scholar]
  • Jakoi ER, Zampighi G, Robertson JD. Regular structures in unit membranes. II. Morphological and biochemical characterization of two water-soluble membrane proteins isolated from the suckling rat ileum. J Cell Biol. 1976 Jul;70(1):97–111. [PMC free article] [PubMed] [Google Scholar]
  • Khan MN, Savoie S, Bergeron JJ, Posner BI. Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures. J Biol Chem. 1986 Jun 25;261(18):8462–8472. [PubMed] [Google Scholar]
  • Kerjaschki D, Noronha-Blob L, Sacktor B, Farquhar MG. Microdomains of distinctive glycoprotein composition in the kidney proximal tubule brush border. J Cell Biol. 1984 Apr;98(4):1505–1513. [PMC free article] [PubMed] [Google Scholar]
  • Knutton S, Limbrick AR, Robertson JD. Regular structures in membranes. I. Membranes in the endocytic complex of ileal epithelial cells. J Cell Biol. 1974 Sep;62(3):679–694. [PMC free article] [PubMed] [Google Scholar]
  • Koldovský O, Palmieri M. Cortisone-evoked decrease of acid -galactosidase, -glucuronidase, N-acetyl- -glucosaminidase and arylsulphatase in the ileum of suckling rats. Biochem J. 1971 Dec;125(3):697–701. [PMC free article] [PubMed] [Google Scholar]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed] [Google Scholar]
  • Lewis V, Green SA, Marsh M, Vihko P, Helenius A, Mellman I. Glycoproteins of the lysosomal membrane. J Cell Biol. 1985 Jun;100(6):1839–1847. [PMC free article] [PubMed] [Google Scholar]
  • Lippincott-Schwartz J, Fambrough DM. Lysosomal membrane dynamics: structure and interorganellar movement of a major lysosomal membrane glycoprotein. J Cell Biol. 1986 May;102(5):1593–1605. [PMC free article] [PubMed] [Google Scholar]
  • 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. [PMC free article] [PubMed] [Google Scholar]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  • Marsh M, Griffiths G, Dean GE, 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. [PMC free article] [PubMed] [Google Scholar]
  • McLean IW, Nakane PK. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. [PubMed] [Google Scholar]
  • Mellman IS. Endocytosis, membrane recycling and Fc receptor function. Ciba Found Symp. 1982;(92):35–58. [PubMed] [Google Scholar]
  • Merion M, Sly WS. The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts. J Cell Biol. 1983 Mar;96(3):644–650. [PMC free article] [PubMed] [Google Scholar]
  • Mueller SC, Hubbard AL. Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes. J Cell Biol. 1986 Mar;102(3):932–942. [PMC free article] [PubMed] [Google Scholar]
  • Muller WA, Steinman RM, Cohn ZA. Membrane proteins of the vacuolar system. III. Further studies on the composition and recycling of endocytic vacuole membrane in cultured macrophages. J Cell Biol. 1983 Jan;96(1):29–36. [PMC free article] [PubMed] [Google Scholar]
  • Neutra MR, Ciechanover A, Owen LS, Lodish HF. Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis. J Histochem Cytochem. 1985 Nov;33(11):1134–1144. [PubMed] [Google Scholar]
  • Quaroni A. Pre- and postnatal development of differentiated functions in rat intestinal epithelial cells. Dev Biol. 1985 Oct;111(2):280–292. [PubMed] [Google Scholar]
  • Quaroni A, Isselbacher KJ. Study of intestinal cell differentiation with monoclonal antibodies to intestinal cell surface components. Dev Biol. 1985 Oct;111(2):267–279. [PubMed] [Google Scholar]
  • Quintart J, Courtoy PJ, Baudhuin P. Receptor-mediated endocytosis in rat liver: purification and enzymic characterization of low density organelles involved in uptake of galactose-exposing proteins. J Cell Biol. 1984 Mar;98(3):877–884. [PMC free article] [PubMed] [Google Scholar]
  • Reggio H, Bainton D, Harms E, Coudrier E, Louvard D. Antibodies against lysosomal membranes reveal a 100,000-mol-wt protein that cross-reacts with purified H+,K+ ATPase from gastric mucosa. J Cell Biol. 1984 Oct;99(4 Pt 1):1511–1526. [PMC free article] [PubMed] [Google Scholar]
  • Rodewald R, Abrahamson DR. Receptor-mediated transport of IgG across the intestinal epithelium of the neonatal rat. Ciba Found Symp. 1982;(92):209–232. [PubMed] [Google Scholar]
  • Seglen PO. Inhibitors of lysosomal function. Methods Enzymol. 1983;96:737–764. [PubMed] [Google Scholar]
  • Shibata Y, Arima T, Arima T, Yamamoto T. Regular structures on the microvillar surface membrane of ileal epithelial cells in suckling rat intestine. J Ultrastruct Res. 1983 Oct;85(1):70–81. [PubMed] [Google Scholar]
  • Siminoski K, Gonnella P, Bernanke J, Owen L, Neutra M, Murphy RA. Uptake and transepithelial transport of nerve growth factor in suckling rat ileum. J Cell Biol. 1986 Nov;103(5):1979–1990. [PMC free article] [PubMed] [Google Scholar]
  • Slot JW, Geuze HJ. A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol. 1985 Jul;38(1):87–93. [PubMed] [Google Scholar]
  • Steinman RM, Mellman IS, Muller WA, Cohn ZA. Endocytosis and the recycling of plasma membrane. J Cell Biol. 1983 Jan;96(1):1–27. [PMC free article] [PubMed] [Google Scholar]
  • Titus DE, Becker WM. Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy. J Cell Biol. 1985 Oct;101(4):1288–1299. [PMC free article] [PubMed] [Google Scholar]
  • van Renswoude J, Bridges KR, Harford JB, Klausner RD. Receptor-mediated endocytosis of transferrin and the uptake of fe in K562 cells: identification of a nonlysosomal acidic compartment. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6186–6190. [PMC free article] [PubMed] [Google Scholar]
  • Wall DA, Hubbard AL. Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments. J Cell Biol. 1985 Dec;101(6):2104–2112. [PMC free article] [PubMed] [Google Scholar]
  • Widnell CC, Unkeless JC. Partial purification of a lipoprotein with 5'-nucleotidase activity from membranes of rat liver cells. Proc Natl Acad Sci U S A. 1968 Nov;61(3):1050–1057. [PMC free article] [PubMed] [Google Scholar]
  • Wissig SL, Graney DO. Membrane modifications in the apical endocytic complex of ileal epithelial cells. J Cell Biol. 1968 Dec;39(3):564–579. [PMC free article] [PubMed] [Google Scholar]
  • Woodward MP, Young WW, Jr, Bloodgood RA. Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J Immunol Methods. 1985 Apr 8;78(1):143–153. [PubMed] [Google Scholar]
  • Zeitlin PL, Hubbard AL. Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures. J Cell Biol. 1982 Mar;92(3):634–647. [PMC free article] [PubMed] [Google Scholar]
Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press
-