Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1986 Jul 1;103(1):287–297. doi: 10.1083/jcb.103.1.287

Subpopulations of liver coated vesicles resolved by preparative agarose gel electrophoresis

PMCID: PMC2113801  PMID: 2941442

Abstract

Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis (Kedersha, N. L., and L. H. Rome, 1986, Anal. Biochem., in press), the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from different populations appeared identical on SDS PAGE, and triskelions stripped from the different populations showed the same mobility on the agarose gel, suggesting that the mobility differences observed in intact CVs were due to differences in the surface charge of underlying vesicles rather than to variations in their clathrin coats. Several non-coat polypeptides appeared to segregate exclusively with different populations as resolved by two-dimensional electrophoresis. Stripped CVs also exhibited considerable heterogeneity when analyzed by Western blotting: the fast-migrating population was enriched in the mannose 6- phosphate receptor, secretory acetylcholine esterase, and an Mr 195,000 glycoprotein. The slow-migrating population of CVs was enriched in the asialoglycoprotein receptor, and it appeared to contain all detectable concanavalin A-binding polypeptides as well as the bulk of detectable WGA-binding proteins. When CVs were prepared from 125I- asialoorosomucoid-perfused rat liver, ligand was found in the slow- migrating CVs, suggesting that these were endocytic in origin. Morphological differences were also observed: the fast-migrating population was enriched in smaller CVs, whereas the slow-migrating population exhibited an enrichment in larger CVs. As liver consists largely of hepatocytes, these subpopulations appear to originate from the same cell type and probably represent CVs of different intracellular origin and destination.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Bartles J. R., Hubbard A. L. 125I-wheat germ agglutinin blotting: increased sensitivity with polyvinylpyrrolidone quenching and periodate oxidation/reductive phenylamination. Anal Biochem. 1984 Jul;140(1):284–292. doi: 10.1016/0003-2697(84)90166-0. [DOI] [PubMed] [Google Scholar]
  2. Blitz A. L., Fine R. E., Toselli P. A. Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum. J Cell Biol. 1977 Oct;75(1):135–147. doi: 10.1083/jcb.75.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bretscher M. S., Thomson J. N., Pearse B. M. Coated pits act as molecular filters. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4156–4159. doi: 10.1073/pnas.77.7.4156. [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. Campbell C. H., Rome L. H. Coated vesicles from rat liver and calf brain contain lysosomal enzymes bound to mannose 6-phosphate receptors. J Biol Chem. 1983 Nov 10;258(21):13347–13352. [PubMed] [Google Scholar]
  6. Childs J. D. Effect of hoc protein on the electrophoretic mobility of intact bacteriophage T4D particles in polyacrylamide gel electrophoresis. J Mol Biol. 1980 Aug 5;141(2):163–173. doi: 10.1016/0022-2836(80)90383-6. [DOI] [PubMed] [Google Scholar]
  7. Croze E. M., Morré D. J., Morré D. M., Kartenbeck J., Franke W. W. Distribution of clathrin and spiny-coated vesicles on membranes within mature Golgi apparatus elements of mouse liver. Eur J Cell Biol. 1982 Aug;28(1):130–138. [PubMed] [Google Scholar]
  8. 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]
  9. Farquhar M. G. Intracellular membrane traffic: pathways, carriers, and sorting devices. Methods Enzymol. 1983;98:1–13. doi: 10.1016/0076-6879(83)98134-x. [DOI] [PubMed] [Google Scholar]
  10. Friend D. S., Farquhar M. G. Functions of coated vesicles during protein absorption in the rat vas deferens. J Cell Biol. 1967 Nov;35(2):357–376. doi: 10.1083/jcb.35.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Geuze H. J., Slot J. W., Strous G. J., Hasilik A., Von Figura K. Ultrastructural localization of the mannose 6-phosphate receptor in rat liver. J Cell Biol. 1984 Jun;98(6):2047–2054. doi: 10.1083/jcb.98.6.2047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Geuze H. J., Slot J. W., Strous G. J., Lodish H. F., Schwartz A. L. Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell. 1983 Jan;32(1):277–287. doi: 10.1016/0092-8674(83)90518-4. [DOI] [PubMed] [Google Scholar]
  13. Geuze H. J., Slot J. W., Strous G. J., Peppard J., von Figura K., Hasilik A., Schwartz A. L. Intracellular receptor sorting during endocytosis: comparative immunoelectron microscopy of multiple receptors in rat liver. Cell. 1984 May;37(1):195–204. doi: 10.1016/0092-8674(84)90315-5. [DOI] [PubMed] [Google Scholar]
  14. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  15. Gottlieb M. H., Steer C. J., Steven A. C., Chrambach A. Applicability of agarose gel electrophoresis to the physical characterization of clathrin-coated vesicles. Anal Biochem. 1985 Jun;147(2):353–363. doi: 10.1016/0003-2697(85)90282-9. [DOI] [PubMed] [Google Scholar]
  16. Hawkes R. Identification of concanavalin A-binding proteins after sodium dodecyl sulfate--gel electrophoresis and protein blotting. Anal Biochem. 1982 Jun;123(1):143–146. doi: 10.1016/0003-2697(82)90634-0. [DOI] [PubMed] [Google Scholar]
  17. Helmy S., Porter-Jordan K., Dawidowicz E. A., Pilch P., Schwartz A. L., Fine R. E. Separation of endocytic from exocytic coated vesicles using a novel cholinesterase mediated density shift technique. Cell. 1986 Feb 14;44(3):497–506. doi: 10.1016/0092-8674(86)90471-x. [DOI] [PubMed] [Google Scholar]
  18. Johnson C. D., Russell R. L. A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations. Anal Biochem. 1975 Mar;64(1):229–238. doi: 10.1016/0003-2697(75)90423-6. [DOI] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Markwell M. A., Fox C. F. Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril. Biochemistry. 1978 Oct 31;17(22):4807–4817. doi: 10.1021/bi00615a031. [DOI] [PubMed] [Google Scholar]
  21. Merril C. R., Goldman D., Van Keuren M. L. Silver staining methods for polyacrylamide gel electrophoresis. Methods Enzymol. 1983;96:230–239. doi: 10.1016/s0076-6879(83)96021-4. [DOI] [PubMed] [Google Scholar]
  22. Nandi P. K., Irace G., Van Jaarsveld P. P., Lippoldt R. E., Edelhoch H. Instability of coated vesicles in concentrated sucrose solutions. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5881–5885. doi: 10.1073/pnas.79.19.5881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  24. Pearse B. M. Assembly of the mannose-6-phosphate receptor into reconstituted clathrin coats. EMBO J. 1985 Oct;4(10):2457–2460. doi: 10.1002/j.1460-2075.1985.tb03956.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pearse B. M., Bretscher M. S. Membrane recycling by coated vesicles. Annu Rev Biochem. 1981;50:85–101. doi: 10.1146/annurev.bi.50.070181.000505. [DOI] [PubMed] [Google Scholar]
  26. Pfeffer S. R., Kelly R. B. The subpopulation of brain coated vesicles that carries synaptic vesicle proteins contains two unique polypeptides. Cell. 1985 Apr;40(4):949–957. doi: 10.1016/0092-8674(85)90355-1. [DOI] [PubMed] [Google Scholar]
  27. Rothman J. E., Fries E., Dunphy W. G., Urbani L. J. The Golgi apparatus, coated vesicles, and the sorting problem. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):797–805. doi: 10.1101/sqb.1982.046.01.075. [DOI] [PubMed] [Google Scholar]
  28. Rubenstein J. L., Fine R. E., Luskey B. D., Rothman J. E. Purification of coated vesicles by agarose gel electrophoresis. J Cell Biol. 1981 May;89(2):357–361. doi: 10.1083/jcb.89.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schook W., Puszkin S. Dissociation and reassociation of clathrin. Methods Enzymol. 1983;98:350–358. doi: 10.1016/0076-6879(83)98163-6. [DOI] [PubMed] [Google Scholar]
  30. Steven A. C., Hainfeld J. F., Wall J. S., Steer C. J. Mass distributions of coated vesicles isolated from liver and brain: analysis by scanning transmission electron microscopy. J Cell Biol. 1983 Dec;97(6):1714–1723. doi: 10.1083/jcb.97.6.1714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Walter P., Ibrahimi I., Blobel G. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):545–550. doi: 10.1083/jcb.91.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wiedenmann B., Franke W. W. Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell. 1985 Jul;41(3):1017–1028. doi: 10.1016/s0092-8674(85)80082-9. [DOI] [PubMed] [Google Scholar]
  34. Wiedenmann B., Lawley K., Grund C., Branton D. Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100. J Cell Biol. 1985 Jul;101(1):12–18. doi: 10.1083/jcb.101.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Woodward M. P., Roth T. F. Coated vesicles: characterization, selective dissociation, and reassembly. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4394–4398. doi: 10.1073/pnas.75.9.4394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yamaguchi Y., Yanagida M. Head shell protein hoc alters the surface charge of bacteriophage T4. Composite slab gel electrophoresis of phage T4 and related particles. J Mol Biol. 1980 Aug 5;141(2):175–193. doi: 10.1016/0022-2836(80)90384-8. [DOI] [PubMed] [Google Scholar]

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

RESOURCES