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. 1991 Apr 2;113(2):235–244. doi: 10.1083/jcb.113.2.235

Transport of exogenous fluorescent phosphatidylserine analogue to the Golgi apparatus in cultured fibroblasts

PMCID: PMC2288936  PMID: 2010461

Abstract

We have examined intracellular transport and metabolism of the fluorescent analogue of phosphatidylserine, 1-palmitoyl-2-(N-[12[(7- nitrobenz-2-oxa-1,3-diazole-4-yl)amino] dodecanoyl])-phosphatidylserine ([palmitoyl-C12-NBD]-PS) in cultured fibroblasts. When monolayer cultures were incubated with liposomes containing (palmitoyl-C12-NBD)- PS at 37 degrees C, fluorescent PS was transported to the Golgi apparatus. NBD-containing analogues of phosphatidylcholine, phosphatidylethanolamine (PE), or phosphatidic acid did not accumulate in the Golgi apparatus under the same experimental conditions. We suggest that the transport is not due to endocytosis, but is the result of incorporation and trans-bilayer movement of the (palmitoyl-C12-NBD)- PS at the plasma membrane followed by translocation of the lipid from plasma membrane to the Golgi apparatus via nonvesicular mechanisms. Uptake of fluorescent PS was inhibited by depletion of cellular ATP and was blocked by structural analogues of the lipid or by pretreatment of cells with glutaraldehyde or N-ethylmaleimide. After incorporation into the cell, fluorescent PS was metabolized to fluorescent PE. The intracellular distribution of fluorescence changed during the conversion. In addition to the Golgi apparatus, mitochondria also became labeled.

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Selected References

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  1. Allan V. J., Kreis T. E. A microtubule-binding protein associated with membranes of the Golgi apparatus. J Cell Biol. 1986 Dec;103(6 Pt 1):2229–2239. doi: 10.1083/jcb.103.6.2229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allen T. M., Williamson P., Schlegel R. A. Phosphatidylserine as a determinant of reticuloendothelial recognition of liposome models of the erythrocyte surface. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8067–8071. doi: 10.1073/pnas.85.21.8067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  4. Balch W. E., Dunphy W. G., Braell W. A., Rothman J. E. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell. 1984 Dec;39(2 Pt 1):405–416. doi: 10.1016/0092-8674(84)90019-9. [DOI] [PubMed] [Google Scholar]
  5. Bankaitis V. A., Aitken J. R., Cleves A. E., Dowhan W. An essential role for a phospholipid transfer protein in yeast Golgi function. Nature. 1990 Oct 11;347(6293):561–562. doi: 10.1038/347561a0. [DOI] [PubMed] [Google Scholar]
  6. Berlin R. D., Oliver J. M. Surface functions during mitosis. II. Quantitation of pinocytosis and kinetic characterization of the mitotic cycle with a new fluorescence technique. J Cell Biol. 1980 Jun;85(3):660–671. doi: 10.1083/jcb.85.3.660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Berlin R. D., Oliver J. M., Walter R. J. Surface functions during Mitosis I: phagocytosis, pinocytosis and mobility of surface-bound Con A. Cell. 1978 Oct;15(2):327–341. doi: 10.1016/0092-8674(78)90002-8. [DOI] [PubMed] [Google Scholar]
  8. Bishop W. R., Bell R. M. Assembly of phospholipids into cellular membranes: biosynthesis, transmembrane movement and intracellular translocation. Annu Rev Cell Biol. 1988;4:579–610. doi: 10.1146/annurev.cb.04.110188.003051. [DOI] [PubMed] [Google Scholar]
  9. Davey J., Hurtley S. M., Warren G. Reconstitution of an endocytic fusion event in a cell-free system. Cell. 1985 Dec;43(3 Pt 2):643–652. doi: 10.1016/0092-8674(85)90236-3. [DOI] [PubMed] [Google Scholar]
  10. Dennis E. A., Kennedy E. P. Intracellular sites of lipid synthesis and the biogenesis of mitochondria. J Lipid Res. 1972 Mar;13(2):263–267. [PubMed] [Google Scholar]
  11. Devaux P. F. Phospholipid flippases. FEBS Lett. 1988 Jul 4;234(1):8–12. doi: 10.1016/0014-5793(88)81291-2. [DOI] [PubMed] [Google Scholar]
  12. FAWCETT D. W. SURFACE SPECIALIZATIONS OF ABSORBING CELLS. J Histochem Cytochem. 1965 Feb;13:75–91. doi: 10.1177/13.2.75. [DOI] [PubMed] [Google Scholar]
  13. Featherstone C., Griffiths G., Warren G. Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells. J Cell Biol. 1985 Dec;101(6):2036–2046. doi: 10.1083/jcb.101.6.2036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hasegawa K., Kuge O., Nishijima M., Akamatsu Y. Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis. J Biol Chem. 1989 Nov 25;264(33):19887–19892. [PubMed] [Google Scholar]
  15. Jin M., Sahagian G. G., Jr, Snider M. D. Transport of surface mannose 6-phosphate receptor to the Golgi complex in cultured human cells. J Biol Chem. 1989 May 5;264(13):7675–7680. [PubMed] [Google Scholar]
  16. Kaplan M. R., Simoni R. D. Intracellular transport of phosphatidylcholine to the plasma membrane. J Cell Biol. 1985 Aug;101(2):441–445. doi: 10.1083/jcb.101.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Klevecz R. R. Automated cell cycle analysis. Methods Cell Biol. 1975;10:157–172. doi: 10.1016/s0091-679x(08)60735-9. [DOI] [PubMed] [Google Scholar]
  18. Kobayashi T., Okamoto H., Yamada J., Setaka M., Kwan T. Vesiculation of platelet plasma membranes. Dilauroylglycerophosphocholine-induced shedding of a platelet plasma membrane fraction enriched in acetylcholinesterase activity. Biochim Biophys Acta. 1984 Nov 21;778(1):210–218. doi: 10.1016/0005-2736(84)90464-4. [DOI] [PubMed] [Google Scholar]
  19. Kobayashi T., Pagano R. E. ATP-dependent fusion of liposomes with the Golgi apparatus of perforated cells. Cell. 1988 Dec 2;55(5):797–805. doi: 10.1016/0092-8674(88)90135-3. [DOI] [PubMed] [Google Scholar]
  20. Kobayashi T., Pagano R. E. Lipid transport during mitosis. Alternative pathways for delivery of newly synthesized lipids to the cell surface. J Biol Chem. 1989 Apr 5;264(10):5966–5973. [PubMed] [Google Scholar]
  21. Kok J. W., Eskelinen S., Hoekstra K., Hoekstra D. Salvage of glucosylceramide by recycling after internalization along the pathway of receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9896–9900. doi: 10.1073/pnas.86.24.9896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Koval M., Pagano R. E. Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts. J Cell Biol. 1989 Jun;108(6):2169–2181. doi: 10.1083/jcb.108.6.2169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koval M., Pagano R. E. Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts. J Cell Biol. 1990 Aug;111(2):429–442. doi: 10.1083/jcb.111.2.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kremer J. M., Esker M. W., Pathmamanoharan C., Wiersema P. H. Vesicles of variable diameter prepared by a modified injection method. Biochemistry. 1977 Aug 23;16(17):3932–3935. doi: 10.1021/bi00636a033. [DOI] [PubMed] [Google Scholar]
  25. Labarca C., Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem. 1980 Mar 1;102(2):344–352. doi: 10.1016/0003-2697(80)90165-7. [DOI] [PubMed] [Google Scholar]
  26. Lipsky N. G., Pagano R. E. A vital stain for the Golgi apparatus. Science. 1985 May 10;228(4700):745–747. doi: 10.1126/science.2581316. [DOI] [PubMed] [Google Scholar]
  27. Lipsky N. G., Pagano R. E. Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane. J Cell Biol. 1985 Jan;100(1):27–34. doi: 10.1083/jcb.100.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lipsky N. G., Pagano R. E. Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue. Proc Natl Acad Sci U S A. 1983 May;80(9):2608–2612. doi: 10.1073/pnas.80.9.2608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Martin O. C., Pagano R. E. Transbilayer movement of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine at the plasma membrane of cultured cells. Evidence for a protein-mediated and ATP-dependent process(es). J Biol Chem. 1987 Apr 25;262(12):5890–5898. [PubMed] [Google Scholar]
  30. Nichols J. W., Pagano R. E. Resonance energy transfer assay of protein-mediated lipid transfer between vesicles. J Biol Chem. 1983 May 10;258(9):5368–5371. [PubMed] [Google Scholar]
  31. Nichols J. W., Pagano R. E. Use of resonance energy transfer to study the kinetics of amphiphile transfer between vesicles. Biochemistry. 1982 Apr 13;21(8):1720–1726. doi: 10.1021/bi00537a003. [DOI] [PubMed] [Google Scholar]
  32. Nishijima M., Kuge O., Akamatsu Y. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation. J Biol Chem. 1986 May 5;261(13):5784–5789. [PubMed] [Google Scholar]
  33. Nishikawa K., Arai H., Inoue K. Scavenger receptor-mediated uptake and metabolism of lipid vesicles containing acidic phospholipids by mouse peritoneal macrophages. J Biol Chem. 1990 Mar 25;265(9):5226–5231. [PubMed] [Google Scholar]
  34. Pagano R. E. Lipid traffic in eukaryotic cells: mechanisms for intracellular transport and organelle-specific enrichment of lipids. Curr Opin Cell Biol. 1990 Aug;2(4):652–663. doi: 10.1016/0955-0674(90)90107-p. [DOI] [PubMed] [Google Scholar]
  35. Pagano R. E., Longmuir K. J. Phosphorylation, transbilayer movement, and facilitated intracellular transport of diacylglycerol are involved in the uptake of a fluorescent analog of phosphatidic acid by cultured fibroblasts. J Biol Chem. 1985 Feb 10;260(3):1909–1916. [PubMed] [Google Scholar]
  36. Pagano R. E., Martin O. C., Schroit A. J., Struck D. K. Formation of asymmetric phospholipid membranes via spontaneous transfer of fluorescent lipid analogues between vesicle populations. Biochemistry. 1981 Aug 18;20(17):4920–4927. doi: 10.1021/bi00520a018. [DOI] [PubMed] [Google Scholar]
  37. Rouser G., Siakotos A. N., Fleischer S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids. 1966 Jan;1(1):85–86. doi: 10.1007/BF02668129. [DOI] [PubMed] [Google Scholar]
  38. Schroit A. J., Madsen J. W., Tanaka Y. In vivo recognition and clearance of red blood cells containing phosphatidylserine in their plasma membranes. J Biol Chem. 1985 Apr 25;260(8):5131–5138. [PubMed] [Google Scholar]
  39. Schroit A. J., Tanaka Y., Madsen J., Fidler I. J. The recognition of red blood cells by macrophages: role of phosphatidylserine and possible implications of membrane phospholipid asymmetry. Biol Cell. 1984;51(2):227–238. doi: 10.1111/j.1768-322x.1984.tb00303.x. [DOI] [PubMed] [Google Scholar]
  40. Simons K., Virta H. Perforated MDCK cells support intracellular transport. EMBO J. 1987 Aug;6(8):2241–2247. doi: 10.1002/j.1460-2075.1987.tb02496.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sleight R. G., Abanto M. N. Differences in intracellular transport of a fluorescent phosphatidylcholine analog in established cell lines. J Cell Sci. 1989 Jun;93(Pt 2):363–374. doi: 10.1242/jcs.93.2.363. [DOI] [PubMed] [Google Scholar]
  42. Sleight R. G. Intracellular lipid transport in eukaryotes. Annu Rev Physiol. 1987;49:193–208. doi: 10.1146/annurev.ph.49.030187.001205. [DOI] [PubMed] [Google Scholar]
  43. Sleight R. G., Pagano R. E. Rapid appearance of newly synthesized phosphatidylethanolamine at the plasma membrane. J Biol Chem. 1983 Aug 10;258(15):9050–9058. [PubMed] [Google Scholar]
  44. Sleight R. G., Pagano R. E. Transbilayer movement of a fluorescent phosphatidylethanolamine analogue across the plasma membranes of cultured mammalian cells. J Biol Chem. 1985 Jan 25;260(2):1146–1154. [PubMed] [Google Scholar]
  45. Sleight R. G., Pagano R. E. Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus. J Cell Biol. 1984 Aug;99(2):742–751. doi: 10.1083/jcb.99.2.742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Struck D. K., Hoekstra D., Pagano R. E. Use of resonance energy transfer to monitor membrane fusion. Biochemistry. 1981 Jul 7;20(14):4093–4099. doi: 10.1021/bi00517a023. [DOI] [PubMed] [Google Scholar]
  47. Struck D. K., Pagano R. E. Insertion of fluorescent phospholipids into the plasma membrane of a mammalian cell. J Biol Chem. 1980 Jun 10;255(11):5404–5410. [PubMed] [Google Scholar]
  48. Tanaka Y., Schroit A. J. Insertion of fluorescent phosphatidylserine into the plasma membrane of red blood cells. Recognition by autologous macrophages. J Biol Chem. 1983 Sep 25;258(18):11335–11343. [PubMed] [Google Scholar]
  49. Tartakoff A. M. Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell. 1983 Apr;32(4):1026–1028. doi: 10.1016/0092-8674(83)90286-6. [DOI] [PubMed] [Google Scholar]
  50. Ting A. E., Pagano R. E. Detection of a phosphatidylinositol-specific phospholipase C at the surface of Swiss 3T3 cells and its potential role in the regulation of cell growth. J Biol Chem. 1990 Apr 5;265(10):5337–5340. [PubMed] [Google Scholar]
  51. Voelker D. R. Disruption of phosphatidylserine translocation to the mitochondria in baby hamster kidney cells. J Biol Chem. 1985 Nov 25;260(27):14671–14676. [PubMed] [Google Scholar]
  52. Voelker D. R., Frazier J. L. Isolation and characterization of a Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for growth and exhibiting defective phosphatidylserine synthase activity. J Biol Chem. 1986 Jan 25;261(3):1002–1008. [PubMed] [Google Scholar]
  53. Yaffe M. P., Kennedy E. P. Intracellular phospholipid movement and the role of phospholipid transfer proteins in animal cells. Biochemistry. 1983 Mar 15;22(6):1497–1507. doi: 10.1021/bi00275a026. [DOI] [PubMed] [Google Scholar]
  54. Zieve G. W., Turnbull D., Mullins J. M., McIntosh J. R. Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole. Nocodazole accumulated mitotic cells. Exp Cell Res. 1980 Apr;126(2):397–405. doi: 10.1016/0014-4827(80)90279-7. [DOI] [PubMed] [Google Scholar]
  55. van 't Hof W., van Meer G. Generation of lipid polarity in intestinal epithelial (Caco-2) cells: sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane. J Cell Biol. 1990 Sep;111(3):977–986. doi: 10.1083/jcb.111.3.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. van Golde L. M., Raben J., Batenburg J. J., Fleischer B., Zambrano F., Fleischer S. Biosynthesis of lipids in Golgi complex and other subcellular fractions from rat liver. Biochim Biophys Acta. 1974 Aug 22;360(2):179–192. doi: 10.1016/0005-2760(74)90168-4. [DOI] [PubMed] [Google Scholar]
  57. van Meer G. Lipid traffic in animal cells. Annu Rev Cell Biol. 1989;5:247–275. doi: 10.1146/annurev.cb.05.110189.001335. [DOI] [PubMed] [Google Scholar]
  58. van Meer G., Stelzer E. H., Wijnaendts-van-Resandt R. W., Simons K. Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells. J Cell Biol. 1987 Oct;105(4):1623–1635. doi: 10.1083/jcb.105.4.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]

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