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. 1984 Oct 1;99(4):1296–1308. doi: 10.1083/jcb.99.4.1296

A single mutation in Chinese hamster ovary cells impairs both Golgi and endosomal functions

PMCID: PMC2113302  PMID: 6480694

Abstract

A Chinese hamster ovary cell mutant DTG 1-5-4, was selected for pleiotropic defects in receptor-mediated endocytosis by methods previously described (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071). DTG 1-5-4 exhibited increased resistance to modeccin, Pseudomonas toxin, diphtheria toxin, Sindbis virus, and vesicular stomatitis virus, as well as decreased uptake via the mannose 6-phosphate receptor. Fluorescein-dextran-labeled endosomes isolated from DTG 1-5-4 were deficient in ATP-dependent acidification in vitro. Endocytosis and endosome acidification were both restored in revertants of DTG 1-5-4 and in hybrids of DTG 1-5-4 with DTF 1-5-1, another endocytosis mutant exhibiting decreased ATP-dependent endosome acidification. Both DTG 1-5-4 and DTF 1-5-1 were blocked at two stages of infection with Sindbis virus: at low multiplicities of infecting virus, resistance reflected a block in viral penetration into the cytoplasm, but at higher multiplicities of infection the block was in virus release. Like endocytosis, release of Sindbis virus was increased in revertants of DTG 1-5-4 and in DTG 1-5-4 X DTF 1-5-1 hybrids. Decreased release of virus from DTG 1-5-4 correlated with defects in some of the Golgi apparatus-associated steps of Sindbis glycoprotein maturation: proteolytic processing of the precursor pE2, galactosylation, and transport to the cell surface all were inhibited. In contrast, mannosylation, fucosylation, and acylation of the Sindbis glycoproteins, and galactosylation of vesicular stomatitis virus and cellular glycoproteins occurred to similar respective extents in mutant and parent. Electron microscopic examination of Sindbis-infected DTG 1- 5-4 showed a remarkable accumulation of nucleocapsids bound to cisternae adjacent to the Golgi apparatus; virions were observed in the lumina of some of these cisternae. That the alterations in both endocytosis and Golgi-associated steps of viral maturation result from a single genetic lesion indicates that these processes are dependent on a common biochemical mechanism. We suggest that endocytic and secretory pathways may share a common component involved in ion transport.

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Selected References

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  1. Apps D. K., Pryde J. G., Sutton R. The H+-translocating adenosine triphosphatase of chromaffin granule membranes. Ann N Y Acad Sci. 1982;402:134–145. doi: 10.1111/j.1749-6632.1982.tb25737.x. [DOI] [PubMed] [Google Scholar]
  2. Basu S. K., Goldstein J. L., Anderson R. G., Brown M. S. Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts. Cell. 1981 May;24(2):493–502. doi: 10.1016/0092-8674(81)90340-8. [DOI] [PubMed] [Google Scholar]
  3. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  4. Bracha M., Schlesinger M. J. Defects in RNA+ temperature-sensitive mutants of Sindbis virus and evidence for a complex of PE2-E1 viral glycoproteins. Virology. 1976 Oct 15;74(2):441–449. doi: 10.1016/0042-6822(76)90350-0. [DOI] [PubMed] [Google Scholar]
  5. Brown M. S., Anderson R. G., Goldstein J. L. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell. 1983 Mar;32(3):663–667. doi: 10.1016/0092-8674(83)90052-1. [DOI] [PubMed] [Google Scholar]
  6. Dautry-Varsat A., Ciechanover A., Lodish H. F. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2258–2262. doi: 10.1073/pnas.80.8.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Draper R. K., Simon M. I. The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement. J Cell Biol. 1980 Dec;87(3 Pt 1):849–854. doi: 10.1083/jcb.87.3.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eidels L., Proia R. L., Hart D. A. Membrane receptors for bacterial toxins. Microbiol Rev. 1983 Dec;47(4):596–620. doi: 10.1128/mr.47.4.596-620.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Galloway C. J., Dean G. E., Marsh M., Rudnick G., Mellman I. Acidification of macrophage and fibroblast endocytic vesicles in vitro. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3334–3338. doi: 10.1073/pnas.80.11.3334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Glickman J., Croen K., Kelly S., Al-Awqati Q. Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance. J Cell Biol. 1983 Oct;97(4):1303–1308. doi: 10.1083/jcb.97.4.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gonzalez-Noriega A., Grubb J. H., Talkad V., Sly W. S. Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling. J Cell Biol. 1980 Jun;85(3):839–852. doi: 10.1083/jcb.85.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Griffiths G., Quinn P., Warren G. Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus. J Cell Biol. 1983 Mar;96(3):835–850. doi: 10.1083/jcb.96.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harikumar P., Reeves J. P. The lysosomal proton pump is electrogenic. J Biol Chem. 1983 Sep 10;258(17):10403–10410. [PubMed] [Google Scholar]
  14. Harms E., Kern H., Schneider J. A. Human lysosomes can be purified from diploid skin fibroblasts by free-flow electrophoresis. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6139–6143. doi: 10.1073/pnas.77.10.6139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Helenius A., Kartenbeck J., Simons K., Fries E. On the entry of Semliki forest virus into BHK-21 cells. J Cell Biol. 1980 Feb;84(2):404–420. doi: 10.1083/jcb.84.2.404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hudgin R. L., Pricer W. E., Jr, Ashwell G., Stockert R. J., Morell A. G. The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins. J Biol Chem. 1974 Sep 10;249(17):5536–5543. [PubMed] [Google Scholar]
  17. Johnson D. C., Schlesinger M. J. Vesicular stomatitis virus and sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores. Virology. 1980 Jun;103(2):407–424. doi: 10.1016/0042-6822(80)90200-7. [DOI] [PubMed] [Google Scholar]
  18. Kim K., Groman N. B. Mode of inhibition of diphtheria toxin by ammonium chloride. J Bacteriol. 1965 Dec;90(6):1557–1562. doi: 10.1128/jb.90.6.1557-1562.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Klausner R. D., Ashwell G., van Renswoude J., Harford J. B., Bridges K. R. Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2263–2266. doi: 10.1073/pnas.80.8.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klausner R. D., van Renswoude J., Kempf C., Rao K., Bateman J. L., Robbins A. R. Failure to release iron from transferrin in a Chinese hamster ovary cell mutant pleiotropically defective in endocytosis. J Cell Biol. 1984 Mar;98(3):1098–1101. doi: 10.1083/jcb.98.3.1098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Krag S. S. A concanavalin A-resistant Chinese hamster ovary cell line is deficient in the synthesis of [3H]glucosyl oligosaccharide-lipid. J Biol Chem. 1979 Sep 25;254(18):9167–9177. [PubMed] [Google Scholar]
  22. Krag S. S., Robbins P. W. Sindbis envelope proteins as endogenous acceptors in reactions of guanosine diphosphate-[14C]Mannose with preparations of infected chicken embryo fibroblasts. J Biol Chem. 1977 Apr 25;252(8):2621–2629. [PubMed] [Google Scholar]
  23. Käriäinen L., Hashimoto K., Saraste J., Virtanen I., Penttinen K. Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins. J Cell Biol. 1980 Dec;87(3 Pt 1):783–791. doi: 10.1083/jcb.87.3.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. 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]
  26. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  27. Marsh M., Bolzau E., Helenius A. Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell. 1983 Mar;32(3):931–940. doi: 10.1016/0092-8674(83)90078-8. [DOI] [PubMed] [Google Scholar]
  28. Merion M., Schlesinger P., Brooks R. M., Moehring J. M., Moehring T. J., Sly W. S. Defective acidification of endosomes in Chinese hamster ovary cell mutants "cross-resistant" to toxins and viruses. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5315–5319. doi: 10.1073/pnas.80.17.5315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moehring J. M., Moehring T. J. Strains of CHO-K1 cells resistant to Pseudomonas exotoxin A and cross-resistant to diphtheria toxin and viruses. Infect Immun. 1983 Sep;41(3):998–1009. doi: 10.1128/iai.41.3.998-1009.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moehring T. J., Moehring J. M. Response of cultured mammalian cells to diphtheria toxin. V. Concurrent resistance to ribonucleic acid viruses in diphtheria toxin-resistant KB cell strains. Infect Immun. 1972 Oct;6(4):493–500. doi: 10.1128/iai.6.4.493-500.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morrison M. The determination of the exposed proteins on membranes by the use of lactoperoxidase. Methods Enzymol. 1974;32:103–109. doi: 10.1016/0076-6879(74)32013-7. [DOI] [PubMed] [Google Scholar]
  32. Ohkuma S., Moriyama Y., Takano T. Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence. Proc Natl Acad Sci U S A. 1982 May;79(9):2758–2762. doi: 10.1073/pnas.79.9.2758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ohkuma S., Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327–3331. doi: 10.1073/pnas.75.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Olsnes S., Haylett T., Refsnes K. Purification and characterization of the highly toxic lectin modeccin. J Biol Chem. 1978 Jul 25;253(14):5069–5073. [PubMed] [Google Scholar]
  35. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rice C. M., Strauss J. H. Association of sindbis virion glycoproteins and their precursors. J Mol Biol. 1982 Jan 15;154(2):325–348. doi: 10.1016/0022-2836(82)90067-5. [DOI] [PubMed] [Google Scholar]
  37. Robbins A. R. Isolation of lysosomal alpha-mannosidase mutants of Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1911–1915. doi: 10.1073/pnas.76.4.1911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Robbins A. R., Peng S. S., Marshall J. L. Mutant Chinese hamster ovary cells pleiotropically defective in receptor-mediated endocytosis. J Cell Biol. 1983 Apr;96(4):1064–1071. doi: 10.1083/jcb.96.4.1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sahagian G. G., Neufeld E. F. Biosynthesis and turnover of the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells. J Biol Chem. 1983 Jun 10;258(11):7121–7128. [PubMed] [Google Scholar]
  40. Sandvig K., Olsnes S. Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol. 1980 Dec;87(3 Pt 1):828–832. doi: 10.1083/jcb.87.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schlesinger S., Gottlieb C., Feil P., Gelb N., Kornfeld S. Growth of enveloped RNA viruses in a line of chinese hamster ovary cells with deficient N-acetylglucosaminyltransferase activity. J Virol. 1975 Jan;17(1):239–246. doi: 10.1128/jvi.17.1.239-246.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schneider D. L. ATP-dependent acidification of intact and disrupted lysosomes. Evidence for an ATP-driven proton pump. J Biol Chem. 1981 Apr 25;256(8):3858–3864. [PubMed] [Google Scholar]
  43. Schneiderman S., Farber J. L., Baserga R. A simple method for decreasing the toxicity of polyethylene glycol in mammalian cell hybridization. Somatic Cell Genet. 1979 Mar;5(2):263–269. doi: 10.1007/BF01539165. [DOI] [PubMed] [Google Scholar]
  44. Spurr A. R. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res. 1969 Jan;26(1):31–43. doi: 10.1016/s0022-5320(69)90033-1. [DOI] [PubMed] [Google Scholar]
  45. Stanley P., Sudo T., Carver J. P. Differential involvement of cell surface sialic acid residues in wheat germ agglutinin binding to parental and wheat germ agglutinin-resistant Chinese hamster ovary cells. J Cell Biol. 1980 Apr;85(1):60–69. doi: 10.1083/jcb.85.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stone D. K., Xie X. S., Racker E. An ATP-driven proton pump in clathrin-coated vesicles. J Biol Chem. 1983 Apr 10;258(7):4059–4062. [PubMed] [Google Scholar]
  47. TREVELYAN W. E., PROCTER D. P., HARRISON J. S. Detection of sugars on paper chromatograms. Nature. 1950 Sep 9;166(4219):444–445. doi: 10.1038/166444b0. [DOI] [PubMed] [Google Scholar]
  48. Thompson L. H., Baker R. M. Isolation of mutants of cultured mammalian cells. Methods Cell Biol. 1973;6:209–281. doi: 10.1016/s0091-679x(08)60052-7. [DOI] [PubMed] [Google Scholar]
  49. Tietze C., Schlesinger P., Stahl P. Mannose-specific endocytosis receptor of alveolar macrophages: demonstration of two functionally distinct intracellular pools of receptor and their roles in receptor recycling. J Cell Biol. 1982 Feb;92(2):417–424. doi: 10.1083/jcb.92.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tycko B., Maxfield F. R. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 1982 Mar;28(3):643–651. doi: 10.1016/0092-8674(82)90219-7. [DOI] [PubMed] [Google Scholar]
  51. Van Lenten L., Ashwell G. Studies on the chemical and enzymatic modification of glycoproteins. A general method for the tritiation of sialic acid-containing glycoproteins. J Biol Chem. 1971 Mar 25;246(6):1889–1894. [PubMed] [Google Scholar]
  52. White J., Kartenbeck J., Helenius A. Fusion of Semliki forest virus with the plasma membrane can be induced by low pH. J Cell Biol. 1980 Oct;87(1):264–272. doi: 10.1083/jcb.87.1.264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  54. Zhang F., Schneider D. L. The bioenergetics of Golgi apparatus function: evidence for an ATP-dependent proton pump. Biochem Biophys Res Commun. 1983 Jul 29;114(2):620–625. doi: 10.1016/0006-291x(83)90825-2. [DOI] [PubMed] [Google Scholar]
  55. van Renswoude J., Bridges K. R., Harford J. B., Klausner R. D. 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. doi: 10.1073/pnas.79.20.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]

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