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. 1999 Apr 15;339(Pt 2):299–307.

Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein.

A L Kruckeberg 1, L Ye 1, J A Berden 1, K van Dam 1
PMCID: PMC1220158  PMID: 10191260

Abstract

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4x10(5) Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 degrees C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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Selected References

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

  1. Berkower C., Loayza D., Michaelis S. Metabolic instability and constitutive endocytosis of STE6, the a-factor transporter of Saccharomyces cerevisiae. Mol Biol Cell. 1994 Nov;5(11):1185–1198. doi: 10.1091/mbc.5.11.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bisson L. F. Derepression of high-affinity glucose uptake requires a functional secretory system in Saccharomyces cerevisiae. J Bacteriol. 1988 Jun;170(6):2654–2658. doi: 10.1128/jb.170.6.2654-2658.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boles E., Hollenberg C. P. The molecular genetics of hexose transport in yeasts. FEMS Microbiol Rev. 1997 Aug;21(1):85–111. doi: 10.1111/j.1574-6976.1997.tb00346.x. [DOI] [PubMed] [Google Scholar]
  4. Chalfie M. Green fluorescent protein. Photochem Photobiol. 1995 Oct;62(4):651–656. doi: 10.1111/j.1751-1097.1995.tb08712.x. [DOI] [PubMed] [Google Scholar]
  5. Chirio M. C., Brèthes D., Napias C., Grandier-Vazeille X., Rakotomanana F., Chevallier J. Photoaffinity labelling of the purine-cytosine permease of Saccharomyces cerevisiae. Eur J Biochem. 1990 Nov 26;194(1):293–299. doi: 10.1111/j.1432-1033.1990.tb19456.x. [DOI] [PubMed] [Google Scholar]
  6. Chvatchko Y., Howald I., Riezman H. Two yeast mutants defective in endocytosis are defective in pheromone response. Cell. 1986 Aug 1;46(3):355–364. doi: 10.1016/0092-8674(86)90656-2. [DOI] [PubMed] [Google Scholar]
  7. Cirillo V. P. Sugar transport in normal and mutant yeast cells. Methods Enzymol. 1989;174:617–622. doi: 10.1016/0076-6879(89)74040-4. [DOI] [PubMed] [Google Scholar]
  8. DeRisi J. L., Iyer V. R., Brown P. O. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science. 1997 Oct 24;278(5338):680–686. doi: 10.1126/science.278.5338.680. [DOI] [PubMed] [Google Scholar]
  9. Dobson S. P., Livingstone C., Gould G. W., Tavaré J. M. Dynamics of insulin-stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein. FEBS Lett. 1996 Sep 16;393(2-3):179–184. doi: 10.1016/0014-5793(96)00879-4. [DOI] [PubMed] [Google Scholar]
  10. Egner R., Mahé Y., Pandjaitan R., Kuchler K. Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae. Mol Cell Biol. 1995 Nov;15(11):5879–5887. doi: 10.1128/mcb.15.11.5879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  12. Gould G. W., Holman G. D. The glucose transporter family: structure, function and tissue-specific expression. Biochem J. 1993 Oct 15;295(Pt 2):329–341. doi: 10.1042/bj2950329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hampton R. Y., Koning A., Wright R., Rine J. In vivo examination of membrane protein localization and degradation with green fluorescent protein. Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):828–833. doi: 10.1073/pnas.93.2.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hebert D. N., Carruthers A. Cholate-solubilized erythrocyte glucose transporters exist as a mixture of homodimers and homotetramers. Biochemistry. 1991 May 14;30(19):4654–4658. doi: 10.1021/bi00233a003. [DOI] [PubMed] [Google Scholar]
  15. Heim R., Tsien R. Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol. 1996 Feb 1;6(2):178–182. doi: 10.1016/s0960-9822(02)00450-5. [DOI] [PubMed] [Google Scholar]
  16. Hein C., Springael J. Y., Volland C., Haguenauer-Tsapis R., André B. NPl1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol Microbiol. 1995 Oct;18(1):77–87. doi: 10.1111/j.1365-2958.1995.mmi_18010077.x. [DOI] [PubMed] [Google Scholar]
  17. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  18. Holcomb C. L., Hansen W. J., Etcheverry T., Schekman R. Secretory vesicles externalize the major plasma membrane ATPase in yeast. J Cell Biol. 1988 Mar;106(3):641–648. doi: 10.1083/jcb.106.3.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Horak J., Wolf D. H. Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination, endocytosis, and degradation in the vacuole. J Bacteriol. 1997 Mar;179(5):1541–1549. doi: 10.1128/jb.179.5.1541-1549.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klingenberg M. The ADP-ATP translocation in mitochondria, a membrane potential controlled transport. J Membr Biol. 1980 Sep 30;56(2):97–105. doi: 10.1007/BF01875961. [DOI] [PubMed] [Google Scholar]
  21. Klip A., Pâquet M. R. Glucose transport and glucose transporters in muscle and their metabolic regulation. Diabetes Care. 1990 Mar;13(3):228–243. doi: 10.2337/diacare.13.3.228. [DOI] [PubMed] [Google Scholar]
  22. Kruckeberg A. L., Bisson L. F. The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport. Mol Cell Biol. 1990 Nov;10(11):5903–5913. doi: 10.1128/mcb.10.11.5903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kruckeberg A. L. The hexose transporter family of Saccharomyces cerevisiae. Arch Microbiol. 1996 Nov;166(5):283–292. doi: 10.1007/s002030050385. [DOI] [PubMed] [Google Scholar]
  24. Kölling R., Hollenberg C. P. The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J. 1994 Jul 15;13(14):3261–3271. doi: 10.1002/j.1460-2075.1994.tb06627.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lai K., Bolognese C. P., Swift S., McGraw P. Regulation of inositol transport in Saccharomyces cerevisiae involves inositol-induced changes in permease stability and endocytic degradation in the vacuole. J Biol Chem. 1995 Feb 10;270(6):2525–2534. doi: 10.1074/jbc.270.6.2525. [DOI] [PubMed] [Google Scholar]
  26. Leucine-zipper motif update. Nature. 1989 Jul 13;340(6229):103–104. doi: 10.1038/340103a0. [DOI] [PubMed] [Google Scholar]
  27. Maher F., Davies-Hill T. M., Simpson I. A. Substrate specificity and kinetic parameters of GLUT3 in rat cerebellar granule neurons. Biochem J. 1996 May 1;315(Pt 3):827–831. doi: 10.1042/bj3150827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Medintz I., Jiang H., Han E. K., Cui W., Michels C. A. Characterization of the glucose-induced inactivation of maltose permease in Saccharomyces cerevisiae. J Bacteriol. 1996 Apr;178(8):2245–2254. doi: 10.1128/jb.178.8.2245-2254.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nakamoto R. K., Rao R., Slayman C. W. Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles. A new strategy for directed mutagenesis. J Biol Chem. 1991 Apr 25;266(12):7940–7949. [PubMed] [Google Scholar]
  30. Nehlin J. O., Carlberg M., Ronne H. Yeast galactose permease is related to yeast and mammalian glucose transporters. Gene. 1989 Dec 28;85(2):313–319. doi: 10.1016/0378-1119(89)90423-x. [DOI] [PubMed] [Google Scholar]
  31. Niedenthal R. K., Riles L., Johnston M., Hegemann J. H. Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast. Yeast. 1996 Jun 30;12(8):773–786. doi: 10.1002/(SICI)1097-0061(19960630)12:8%3C773::AID-YEA972%3E3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  32. Novick P., Field C., Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. doi: 10.1016/0092-8674(80)90128-2. [DOI] [PubMed] [Google Scholar]
  33. Ozcan S., Dover J., Johnston M. Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae. EMBO J. 1998 May 1;17(9):2566–2573. doi: 10.1093/emboj/17.9.2566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ozcan S., Dover J., Rosenwald A. G., Wölfl S., Johnston M. Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression. Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12428–12432. doi: 10.1073/pnas.93.22.12428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Patterson G. H., Knobel S. M., Sharif W. D., Kain S. R., Piston D. W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J. 1997 Nov;73(5):2782–2790. doi: 10.1016/S0006-3495(97)78307-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Prescott M., Lourbakos A., Bateson M., Boyle G., Nagley P., Devenish R. J. A novel fluorescent marker for assembled mitochondria ATP synthase of yeast. OSCP subunit fused to green fluorescent protein is assembled into the complex in vivo. FEBS Lett. 1997 Jul 7;411(1):97–101. doi: 10.1016/s0014-5793(97)00670-4. [DOI] [PubMed] [Google Scholar]
  37. Raths S., Rohrer J., Crausaz F., Riezman H. end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J Cell Biol. 1993 Jan;120(1):55–65. doi: 10.1083/jcb.120.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reifenberger E., Boles E., Ciriacy M. Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. Eur J Biochem. 1997 Apr 15;245(2):324–333. doi: 10.1111/j.1432-1033.1997.00324.x. [DOI] [PubMed] [Google Scholar]
  39. Reifenberger E., Freidel K., Ciriacy M. Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on glycolytic flux. Mol Microbiol. 1995 Apr;16(1):157–167. doi: 10.1111/j.1365-2958.1995.tb02400.x. [DOI] [PubMed] [Google Scholar]
  40. Riballo E., Herweijer M., Wolf D. H., Lagunas R. Catabolite inactivation of the yeast maltose transporter occurs in the vacuole after internalization by endocytosis. J Bacteriol. 1995 Oct;177(19):5622–5627. doi: 10.1128/jb.177.19.5622-5627.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Riballo E., Lagunas R. Involvement of endocytosis in catabolite inactivation of the K+ and glucose transport systems in Saccharomyces cerevisiae. FEMS Microbiol Lett. 1994 Aug 1;121(1):77–80. doi: 10.1111/j.1574-6968.1994.tb07078.x. [DOI] [PubMed] [Google Scholar]
  42. Roca J., Gartenberg M. R., Oshima Y., Wang J. C. A hit-and-run system for targeted genetic manipulations in yeast. Nucleic Acids Res. 1992 Sep 11;20(17):4671–4672. doi: 10.1093/nar/20.17.4671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schneppenheim R., Budde U., Dahlmann N., Rautenberg P. Luminography--a new, highly sensitive visualization method for electrophoresis. Electrophoresis. 1991 May;12(5):367–372. doi: 10.1002/elps.1150120508. [DOI] [PubMed] [Google Scholar]
  44. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  45. Tschopp J. F., Emr S. D., Field C., Schekman R. GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae. J Bacteriol. 1986 Apr;166(1):313–318. doi: 10.1128/jb.166.1.313-318.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tschopp J., Esmon P. C., Schekman R. Defective plasma membrane assembly in yeast secretory mutants. J Bacteriol. 1984 Dec;160(3):966–970. doi: 10.1128/jb.160.3.966-970.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Volland C., Urban-Grimal D., Géraud G., Haguenauer-Tsapis R. Endocytosis and degradation of the yeast uracil permease under adverse conditions. J Biol Chem. 1994 Apr 1;269(13):9833–9841. [PubMed] [Google Scholar]
  48. Walsh M. C., Smits H. P., Scholte M., van Dam K. Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose. J Bacteriol. 1994 Feb;176(4):953–958. doi: 10.1128/jb.176.4.953-958.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Walworth N. C., Goud B., Ruohola H., Novick P. J. Fractionation of yeast organelles. Methods Cell Biol. 1989;31:335–356. doi: 10.1016/s0091-679x(08)61618-0. [DOI] [PubMed] [Google Scholar]
  50. Wendell D. L., Bisson L. F. Expression of high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae is both repressed and induced by glucose and appears to be regulated posttranslationally. J Bacteriol. 1994 Jun;176(12):3730–3737. doi: 10.1128/jb.176.12.3730-3737.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wendell D. L., Bisson L. F. Physiological characterization of putative high-affinity glucose transport protein Hxt2 of Saccharomyces cerevisiae by use of anti-synthetic peptide antibodies. J Bacteriol. 1993 Dec;175(23):7689–7696. doi: 10.1128/jb.175.23.7689-7696.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wohlrab H. Molecular aspects of inorganic phosphate transport in mitochondria. Biochim Biophys Acta. 1986;853(2):115–134. doi: 10.1016/0304-4173(86)90007-8. [DOI] [PubMed] [Google Scholar]

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