Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1993 Mar 15;290(Pt 3):707–715. doi: 10.1042/bj2900707

Differential targeting of glucose transporter isoforms heterologously expressed in Xenopus oocytes.

H M Thomas 1, J Takeda 1, G W Gould 1
PMCID: PMC1132338  PMID: 8457198

Abstract

We have examined the subcellular distribution of three members of the human glucose transporter family expressed in oocytes from Xenopus laevis. Following injection of in vitro-transcribed mRNA encoding the transporter isoform to be studied, we have determined the subcellular localization of the expressed protein by immunofluorescence and by subcellular fractionation coupled with immunoblotting using specific anti-peptide antibodies. We have shown that both the liver-type (GLUT 2) and brain-type (GLUT 3) glucose transporters are expressed predominantly in the plasma membranes of oocytes, and in both cases high levels of glucose transport activity are exhibited. In contrast, the insulin-regulatable glucose transporter (GLUT 4) is localized predominantly to an intracellular membrane pool, and the levels of transport activity recorded in oocytes expressing GLUT 4 are correspondingly lower. The localization of the different transporter isoforms to distinct subcellular fractions mirrors the situation observed in their native cell type and thus demonstrates that oocytes may prove to be a useful system with which to study the targeting signals for this important class of membrane proteins. In addition, the determination of the amounts of the transporters expressed per oocyte together with a knowledge of their Km values has allowed us to estimate the turnover numbers of these transporters. Insulin was without effect on glucose transport in oocytes expressing any of these transporter isoforms. Microinjection of guanosine 5'-[gamma-thio]triphosphate into oocytes expressing GLUT 4 was also without effect on the transport rate.

Full text

PDF
707

Images in this article

710Figure 1
on p.710
711Figure 2
on p.711

Selected References

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

  1. Baldini G., Hohman R., Charron M. J., Lodish H. F. Insulin and nonhydrolyzable GTP analogs induce translocation of GLUT 4 to the plasma membrane in alpha-toxin-permeabilized rat adipose cells. J Biol Chem. 1991 Mar 5;266(7):4037–4040. [PubMed] [Google Scholar]
  2. Baldwin J. M., Gorga J. C., Lienhard G. E. The monosaccharide transporter of the human erythrocyte. Transport activity upon reconstitution. J Biol Chem. 1981 Apr 25;256(8):3685–3689. [PubMed] [Google Scholar]
  3. Bell G. I., Kayano T., Buse J. B., Burant C. F., Takeda J., Lin D., Fukumoto H., Seino S. Molecular biology of mammalian glucose transporters. Diabetes Care. 1990 Mar;13(3):198–208. doi: 10.2337/diacare.13.3.198. [DOI] [PubMed] [Google Scholar]
  4. Birnbaum M. J., Haspel H. C., Rosen O. M. Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5784–5788. doi: 10.1073/pnas.83.16.5784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnbaum M. J. Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell. 1989 Apr 21;57(2):305–315. doi: 10.1016/0092-8674(89)90968-9. [DOI] [PubMed] [Google Scholar]
  6. Bloch R. Inhibition of glucose transport in the human erythrocyte by cytochalasin B. Biochemistry. 1973 Nov 6;12(23):4799–4801. doi: 10.1021/bi00747a036. [DOI] [PubMed] [Google Scholar]
  7. Brant A. M., Gibbs E. M., Gould G. W., Thomas H. M. Immunological identification of five members of the human facilitative glucose transporter family. Biochem Soc Trans. 1992 Aug;20(3):236S–236S. doi: 10.1042/bst020236s. [DOI] [PubMed] [Google Scholar]
  8. Brown S. J., Gould G. W., Davies A., Baldwin S. A., Lienhard G. E., Gibbs E. M. Characterization of vesicles containing insulin-responsive intracellular glucose transporters isolated from 3T3-L1 adipocytes by an improved procedure. Biochim Biophys Acta. 1988 Oct 7;971(3):339–350. doi: 10.1016/0167-4889(88)90150-4. [DOI] [PubMed] [Google Scholar]
  9. Burant C. F., Takeda J., Brot-Laroche E., Bell G. I., Davidson N. O. Fructose transporter in human spermatozoa and small intestine is GLUT5. J Biol Chem. 1992 Jul 25;267(21):14523–14526. [PubMed] [Google Scholar]
  10. Calderhead D. M., Kitagawa K., Tanner L. I., Holman G. D., Lienhard G. E. Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes. J Biol Chem. 1990 Aug 15;265(23):13801–13808. [PubMed] [Google Scholar]
  11. Charron M. J., Brosius F. C., 3rd, Alper S. L., Lodish H. F. A glucose transport protein expressed predominately in insulin-responsive tissues. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2535–2539. doi: 10.1073/pnas.86.8.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Colman A., Lane C. D., Craig R., Boulton A., Mohun T., Morser J. The influence of topology and glycosylation on the fate of heterologous secretory proteins made in Xenopus oocytes. Eur J Biochem. 1981 Jan;113(2):339–348. doi: 10.1111/j.1432-1033.1981.tb05072.x. [DOI] [PubMed] [Google Scholar]
  13. Colman A., Morser J. Export of proteins from oocytes of Xenopus laevis. Cell. 1979 Jul;17(3):517–526. doi: 10.1016/0092-8674(79)90260-5. [DOI] [PubMed] [Google Scholar]
  14. Colville C. A., Seatter M. J., Jess T. J., Gould G. W., Thomas H. M. Kinetic analysis of the liver-type (GLUT2) and brain-type (GLUT3) glucose transporters in Xenopus oocytes: substrate specificities and effects of transport inhibitors. Biochem J. 1993 Mar 15;290(Pt 3):701–706. doi: 10.1042/bj2900701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cushman S. W., Wardzala L. J. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem. 1980 May 25;255(10):4758–4762. [PubMed] [Google Scholar]
  16. Flier J. S., Mueckler M., McCall A. L., Lodish H. F. Distribution of glucose transporter messenger RNA transcripts in tissues of rat and man. J Clin Invest. 1987 Feb;79(2):657–661. doi: 10.1172/JCI112864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fukumoto H., Kayano T., Buse J. B., Edwards Y., Pilch P. F., Bell G. I., Seino S. Cloning and characterization of the major insulin-responsive glucose transporter expressed in human skeletal muscle and other insulin-responsive tissues. J Biol Chem. 1989 May 15;264(14):7776–7779. [PubMed] [Google Scholar]
  18. Fukumoto H., Seino S., Imura H., Seino Y., Bell G. I. Characterization and expression of human HepG2/erythrocyte glucose-transporter gene. Diabetes. 1988 May;37(5):657–661. doi: 10.2337/diab.37.5.657. [DOI] [PubMed] [Google Scholar]
  19. Fukumoto H., Seino S., Imura H., Seino Y., Eddy R. L., Fukushima Y., Byers M. G., Shows T. B., Bell G. I. Sequence, tissue distribution, and chromosomal localization of mRNA encoding a human glucose transporter-like protein. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5434–5438. doi: 10.1073/pnas.85.15.5434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Garcia J. C., Strube M., Leingang K., Keller K., Mueckler M. M. Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG2 (Glut1) glucose transporter inhibit transport activity and targeting to the plasma membrane in Xenopus oocytes. J Biol Chem. 1992 Apr 15;267(11):7770–7776. [PubMed] [Google Scholar]
  21. Geering K., Theulaz I., Verrey F., Häuptle M. T., Rossier B. C. A role for the beta-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. Am J Physiol. 1989 Nov;257(5 Pt 1):C851–C858. doi: 10.1152/ajpcell.1989.257.5.C851. [DOI] [PubMed] [Google Scholar]
  22. Gerhart D. Z., Broderius M. A., Borson N. D., Drewes L. R. Neurons and microvessels express the brain glucose transporter protein GLUT3. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):733–737. doi: 10.1073/pnas.89.2.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gibbs E. M., Calderhead D. M., Holman G. D., Gould G. W. Phorbol ester only partially mimics the effects of insulin on glucose transport and glucose-transporter distribution in 3T3-L1 adipocytes. Biochem J. 1991 Apr 1;275(Pt 1):145–150. doi: 10.1042/bj2750145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gould G. W., Bell G. I. Facilitative glucose transporters: an expanding family. Trends Biochem Sci. 1990 Jan;15(1):18–23. doi: 10.1016/0968-0004(90)90125-u. [DOI] [PubMed] [Google Scholar]
  25. Gould G. W., Brant A. M., Kahn B. B., Shepherd P. R., McCoid S. C., Gibbs E. M. Expression of the brain-type glucose transporter is restricted to brain and neuronal cells in mice. Diabetologia. 1992 Apr;35(4):304–309. doi: 10.1007/BF00401196. [DOI] [PubMed] [Google Scholar]
  26. Gould G. W., Lienhard G. E. Expression of a functional glucose transporter in Xenopus oocytes. Biochemistry. 1989 Nov 28;28(24):9447–9452. doi: 10.1021/bi00450a030. [DOI] [PubMed] [Google Scholar]
  27. Gould G. W., Thomas H. M., Jess T. J., Bell G. I. Expression of human glucose transporters in Xenopus oocytes: kinetic characterization and substrate specificities of the erythrocyte, liver, and brain isoforms. Biochemistry. 1991 May 28;30(21):5139–5145. doi: 10.1021/bi00235a004. [DOI] [PubMed] [Google Scholar]
  28. Harris D. S., Slot J. W., Geuze H. J., James D. E. Polarized distribution of glucose transporter isoforms in Caco-2 cells. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7556–7560. doi: 10.1073/pnas.89.16.7556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hitchcock M. J., Ginns E. I., Marcus-Sekura C. J. Microinjection into Xenopus oocytes: equipment. Methods Enzymol. 1987;152:276–284. doi: 10.1016/0076-6879(87)52031-6. [DOI] [PubMed] [Google Scholar]
  30. Holman G. D., Kozka I. J., Clark A. E., Flower C. J., Saltis J., Habberfield A. D., Simpson I. A., Cushman S. W. Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. J Biol Chem. 1990 Oct 25;265(30):18172–18179. [PubMed] [Google Scholar]
  31. Hudson A. W., Ruiz M., Birnbaum M. J. Isoform-specific subcellular targeting of glucose transporters in mouse fibroblasts. J Cell Biol. 1992 Feb;116(3):785–797. doi: 10.1083/jcb.116.3.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hughes S. D., Johnson J. H., Quaade C., Newgard C. B. Engineering of glucose-stimulated insulin secretion and biosynthesis in non-islet cells. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):688–692. doi: 10.1073/pnas.89.2.688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. James D. E., Brown R., Navarro J., Pilch P. F. Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein. Nature. 1988 May 12;333(6169):183–185. doi: 10.1038/333183a0. [DOI] [PubMed] [Google Scholar]
  34. James D. E., Lederman L., Pilch P. F. Purification of insulin-dependent exocytic vesicles containing the glucose transporter. J Biol Chem. 1987 Aug 25;262(24):11817–11824. [PubMed] [Google Scholar]
  35. James D. E., Strube M., Mueckler M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature. 1989 Mar 2;338(6210):83–87. doi: 10.1038/338083a0. [DOI] [PubMed] [Google Scholar]
  36. Janicot M., Lane M. D. Activation of glucose uptake by insulin and insulin-like growth factor I in Xenopus oocytes. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2642–2646. doi: 10.1073/pnas.86.8.2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kaestner K. H., Christy R. J., McLenithan J. C., Braiterman L. T., Cornelius P., Pekala P. H., Lane M. D. Sequence, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3-L1 adipocytes. Proc Natl Acad Sci U S A. 1989 May;86(9):3150–3154. doi: 10.1073/pnas.86.9.3150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kayano T., Burant C. F., Fukumoto H., Gould G. W., Fan Y. S., Eddy R. L., Byers M. G., Shows T. B., Seino S., Bell G. I. Human facilitative glucose transporters. Isolation, functional characterization, and gene localization of cDNAs encoding an isoform (GLUT5) expressed in small intestine, kidney, muscle, and adipose tissue and an unusual glucose transporter pseudogene-like sequence (GLUT6). J Biol Chem. 1990 Aug 5;265(22):13276–13282. [PubMed] [Google Scholar]
  39. Kayano T., Fukumoto H., Eddy R. L., Fan Y. S., Byers M. G., Shows T. B., Bell G. I. Evidence for a family of human glucose transporter-like proteins. Sequence and gene localization of a protein expressed in fetal skeletal muscle and other tissues. J Biol Chem. 1988 Oct 25;263(30):15245–15248. [PubMed] [Google Scholar]
  40. Keller K., Strube M., Mueckler M. Functional expression of the human HepG2 and rat adipocyte glucose transporters in Xenopus oocytes. Comparison of kinetic parameters. J Biol Chem. 1989 Nov 15;264(32):18884–18889. [PubMed] [Google Scholar]
  41. Krupka R. M. Inhibition of sugar transport in erythrocytes by fluorodinitrobenzene. Biochemistry. 1971 Mar 30;10(7):1148–1153. doi: 10.1021/bi00783a008. [DOI] [PubMed] [Google Scholar]
  42. Maher F., Vannucci S., Takeda J., Simpson I. A. Expression of mouse-GLUT3 and human-GLUT3 glucose transporter proteins in brain. Biochem Biophys Res Commun. 1992 Jan 31;182(2):703–711. doi: 10.1016/0006-291x(92)91789-s. [DOI] [PubMed] [Google Scholar]
  43. Matthews J. A., Brown J. W., Hall T. C. Phaseolin mRNA is translated to yield glycosylated polypeptides in Xenopus oocytes. Nature. 1981 Nov 12;294(5837):175–176. doi: 10.1038/294175a0. [DOI] [PubMed] [Google Scholar]
  44. Melton D. A. Translation of messenger RNA in injected frog oocytes. Methods Enzymol. 1987;152:288–296. doi: 10.1016/0076-6879(87)52033-x. [DOI] [PubMed] [Google Scholar]
  45. Mueckler M., Caruso C., Baldwin S. A., Panico M., Blench I., Morris H. R., Allard W. J., Lienhard G. E., Lodish H. F. Sequence and structure of a human glucose transporter. Science. 1985 Sep 6;229(4717):941–945. doi: 10.1126/science.3839598. [DOI] [PubMed] [Google Scholar]
  46. Mueckler M. Family of glucose-transporter genes. Implications for glucose homeostasis and diabetes. Diabetes. 1990 Jan;39(1):6–11. doi: 10.2337/diacare.39.1.6. [DOI] [PubMed] [Google Scholar]
  47. Orci L., Thorens B., Ravazzola M., Lodish H. F. Localization of the pancreatic beta cell glucose transporter to specific plasma membrane domains. Science. 1989 Jul 21;245(4915):295–297. doi: 10.1126/science.2665080. [DOI] [PubMed] [Google Scholar]
  48. Permutt M. A., Koranyi L., Keller K., Lacy P. E., Scharp D. W., Mueckler M. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8688–8692. doi: 10.1073/pnas.86.22.8688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Piper R. C., Tai C., Slot J. W., Hahn C. S., Rice C. M., Huang H., James D. E. The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus. J Cell Biol. 1992 May;117(4):729–743. doi: 10.1083/jcb.117.4.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sadler S. E., Maller J. L. A similar pool of cyclic AMP phosphodiesterase in Xenopus oocytes is stimulated by insulin, insulin-like growth factor 1, and [Val12,Thr59]Ha-ras protein. J Biol Chem. 1989 Jan 15;264(2):856–861. [PubMed] [Google Scholar]
  51. Shepherd P. R., Gibbs E. M., Wesslau C., Gould G. W., Kahn B. B. Human small intestine facilitative fructose/glucose transporter (GLUT5) is also present in insulin-responsive tissues and brain. Investigation of biochemical characteristics and translocation. Diabetes. 1992 Oct;41(10):1360–1365. doi: 10.2337/diab.41.10.1360. [DOI] [PubMed] [Google Scholar]
  52. Shepherd P. R., Gould G. W., Colville C. A., McCoid S. C., Gibbs E. M., Kahn B. B. Distribution of GLUT3 glucose transporter protein in human tissues. Biochem Biophys Res Commun. 1992 Oct 15;188(1):149–154. doi: 10.1016/0006-291x(92)92362-2. [DOI] [PubMed] [Google Scholar]
  53. Shibasaki Y., Asano T., Lin J. L., Tsukuda K., Katagiri H., Ishihara H., Yazaki Y., Oka Y. Two glucose transporter isoforms are sorted differentially and are expressed in distinct cellular compartments. Biochem J. 1992 Feb 1;281(Pt 3):829–834. doi: 10.1042/bj2810829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Slot J. W., Geuze H. J., Gigengack S., James D. E., Lienhard G. E. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7815–7819. doi: 10.1073/pnas.88.17.7815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Slot J. W., Geuze H. J., Gigengack S., Lienhard G. E., James D. E. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol. 1991 Apr;113(1):123–135. doi: 10.1083/jcb.113.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Thorens B., Cheng Z. Q., Brown D., Lodish H. F. Liver glucose transporter: a basolateral protein in hepatocytes and intestine and kidney cells. Am J Physiol. 1990 Dec;259(6 Pt 1):C279–C285. doi: 10.1152/ajpcell.1990.259.2.C279. [DOI] [PubMed] [Google Scholar]
  57. Thorens B., Lodish H. F., Brown D. Differential localization of two glucose transporter isoforms in rat kidney. Am J Physiol. 1990 Dec;259(6 Pt 1):C286–C294. doi: 10.1152/ajpcell.1990.259.2.C286. [DOI] [PubMed] [Google Scholar]
  58. Thorens B., Sarkar H. K., Kaback H. R., Lodish H. F. Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and beta-pancreatic islet cells. Cell. 1988 Oct 21;55(2):281–290. doi: 10.1016/0092-8674(88)90051-7. [DOI] [PubMed] [Google Scholar]
  59. Yang J., Clark A. E., Kozka I. J., Cushman S. W., Holman G. D. Development of an intracellular pool of glucose transporters in 3T3-L1 cells. J Biol Chem. 1992 May 25;267(15):10393–10399. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES