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. 1993 Dec;175(23):7689–7696. doi: 10.1128/jb.175.23.7689-7696.1993

Physiological characterization of putative high-affinity glucose transport protein Hxt2 of Saccharomyces cerevisiae by use of anti-synthetic peptide antibodies.

D L Wendell 1, L F Bisson 1
PMCID: PMC206927  PMID: 8244939

Abstract

Characterization and quantification of the Hxt2 (hexose transport) protein of Saccharomyces cerevisiae indicate that it is one of a set of differentially expressed high-affinity glucose transporters. The protein product of the HXT2 gene was specifically detected by antibodies raised against a synthetic peptide encompassing the 13 carboxyl-terminal amino acids predicted by the HXT2 gene sequence. Hxt2 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band or closely spaced doublet with an average M(r) of 47,000. Hxt2 cofractionated with the plasma membrane ATPase, Pma1, indicating that it is a plasma membrane protein. Hxt2 was not solubilized by high pH or urea but was solublized by detergents, which is characteristic of an integral membrane protein. Expression of the Hxt2 protein was measured under two different conditions that produce expression of high-affinity glucose transport: a medium shift from a high (2.0%) to a low (0.05%) glucose concentration (referred to below as high and low glucose) and growth from high to low glucose. Hxt2 as measured by immunoblotting increased 20-fold upon a shift from high-glucose to low-glucose medium, and the high-affinity glucose transport expressed had a strong HXT2-dependent component. Surprisingly, Hxt2 was not detectable when S. cerevisiae growing in high glucose approached glucose exhaustion, and the high-affinity glucose transport expressed under these conditions did not have an HXT2-dependent component. The role of Hxt2 in growth during aerobic batch culture in low-glucose medium was examined. An hxt2 null mutant grew and consumed glucose significantly more slowly than the wild type, and this phenotype correlated directly with appearance of the Hxt2 protein.

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Selected References

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

  1. 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]
  2. Bisson L. F., Coons D. M., Kruckeberg A. L., Lewis D. A. Yeast sugar transporters. Crit Rev Biochem Mol Biol. 1993;28(4):259–308. doi: 10.3109/10409239309078437. [DOI] [PubMed] [Google Scholar]
  3. Bisson L. F., Fraenkel D. G. Expression of kinase-dependent glucose uptake in Saccharomyces cerevisiae. J Bacteriol. 1984 Sep;159(3):1013–1017. doi: 10.1128/jb.159.3.1013-1017.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bisson L. F., Fraenkel D. G. Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1730–1734. doi: 10.1073/pnas.80.6.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bisson L. F., Neigeborn L., Carlson M., Fraenkel D. G. The SNF3 gene is required for high-affinity glucose transport in Saccharomyces cerevisiae. J Bacteriol. 1987 Apr;169(4):1656–1662. doi: 10.1128/jb.169.4.1656-1662.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blumer K. J., Reneke J. E., Thorner J. The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae. J Biol Chem. 1988 Aug 5;263(22):10836–10842. [PubMed] [Google Scholar]
  7. Carrasco N., Herzlinger D., Mitchell R., DeChiara S., Danho W., Gabriel T. F., Kaback H. R. Intramolecular dislocation of the COOH terminus of the lac carrier protein in reconstituted proteoliposomes. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4672–4676. doi: 10.1073/pnas.81.15.4672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davies A., Meeran K., Cairns M. T., Baldwin S. A. Peptide-specific antibodies as probes of the orientation of the glucose transporter in the human erythrocyte membrane. J Biol Chem. 1987 Jul 5;262(19):9347–9352. [PubMed] [Google Scholar]
  9. Dunbar B. S., Schwoebel E. D. Preparation of polyclonal antibodies. Methods Enzymol. 1990;182:663–670. doi: 10.1016/0076-6879(90)82051-3. [DOI] [PubMed] [Google Scholar]
  10. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  11. Fuhrmann G. F., Völker B. Misuse of graphical analysis in nonlinear sugar transport kinetics by Eadie-Hofstee plots. Biochim Biophys Acta. 1993 Jan 18;1145(1):180–182. doi: 10.1016/0005-2736(93)90396-h. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Haspel H. C., Rosenfeld M. G., Rosen O. M. Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein. J Biol Chem. 1988 Jan 5;263(1):398–403. [PubMed] [Google Scholar]
  14. Ko C. H., Liang H., Gaber R. F. Roles of multiple glucose transporters in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Jan;13(1):638–648. doi: 10.1128/mcb.13.1.638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Kubota S., Yoshida Y., Kumaoka H., Furumichi A. Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase. J Biochem. 1977 Jan;81(1):197–205. doi: 10.1093/oxfordjournals.jbchem.a131436. [DOI] [PubMed] [Google Scholar]
  17. Lerner R. A., Sutcliffe J. G., Shinnick T. M. Antibodies to chemically synthesized peptides predicted from DNA sequences as probes of gene expression. Cell. 1981 Feb;23(2):309–310. doi: 10.1016/0092-8674(81)90126-4. [DOI] [PubMed] [Google Scholar]
  18. Lewis D. A., Bisson L. F. The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters. Mol Cell Biol. 1991 Jul;11(7):3804–3813. doi: 10.1128/mcb.11.7.3804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  21. Rizzolo L. J., Maire M., Reynolds J. A., Tanford C. Molecular weights and hydrophobicity of the polypeptide chain of sarcoplasmic reticulum calcium(II) adenosine triphosphatase and of its primary tryptic fragments. Biochemistry. 1976 Aug 10;15(16):3433–3437. doi: 10.1021/bi00661a006. [DOI] [PubMed] [Google Scholar]
  22. Rosevear P., VanAken T., Baxter J., Ferguson-Miller S. Alkyl glycoside detergents: a simpler synthesis and their effects on kinetic and physical properties of cytochrome c oxidase. Biochemistry. 1980 Aug 19;19(17):4108–4115. doi: 10.1021/bi00558a032. [DOI] [PubMed] [Google Scholar]
  23. Seckler R., Möröy T., Wright J. K., Overath P. Anti-peptide antibodies and proteases as structural probes for the lactose/H+ transporter of Escherichia coli: a loop around amino acid residue 130 faces the cytoplasmic side of the membrane. Biochemistry. 1986 May 6;25(9):2403–2409. doi: 10.1021/bi00357a016. [DOI] [PubMed] [Google Scholar]
  24. Serrano R. Characterization of the plasma membrane ATPase of Saccharomyces cerevisiae. Mol Cell Biochem. 1978 Nov 30;22(1):51–63. doi: 10.1007/BF00241470. [DOI] [PubMed] [Google Scholar]
  25. Serrano R. H+-ATPase from plasma membranes of Saccharomyces cerevisiae and Avena sativa roots: purification and reconstitution. Methods Enzymol. 1988;157:533–544. doi: 10.1016/0076-6879(88)57102-1. [DOI] [PubMed] [Google Scholar]
  26. Serrano R. In vivo glucose activation of the yeast plasma membrane ATPase. FEBS Lett. 1983 May 30;156(1):11–14. doi: 10.1016/0014-5793(83)80237-3. [DOI] [PubMed] [Google Scholar]
  27. Sutcliffe J. G., Shinnick T. M., Green N., Liu F. T., Niman H. L., Lerner R. A. Chemical synthesis of a polypeptide predicted from nucleotide sequence allows detection of a new retroviral gene product. Nature. 1980 Oct 30;287(5785):801–805. doi: 10.1038/287801a0. [DOI] [PubMed] [Google Scholar]
  28. Tsang V. C., Wilkins P. P. Optimum dissociating condition for immunoaffinity and preferential isolation of antibodies with high specific activity. J Immunol Methods. 1991 Apr 25;138(2):291–299. doi: 10.1016/0022-1759(91)90178-i. [DOI] [PubMed] [Google Scholar]
  29. Walter G., Scheidtmann K. H., Carbone A., Laudano A. P., Doolittle R. F. Antibodies specific for the carboxy- and amino-terminal regions of simian virus 40 large tumor antigen. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5197–5200. doi: 10.1073/pnas.77.9.5197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wright J. K., Seckler R. The lactose/H+ carrier of Escherichia coli: lac YUN mutation decreases the rate of active transport and mimics an energy-uncoupled phenotype. Biochem J. 1985 Apr 1;227(1):287–297. doi: 10.1042/bj2270287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yanagisawa K., Resnick D., Abeijon C., Robbins P. W., Hirschberg C. B. A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae. Purification and characterization. J Biol Chem. 1990 Nov 5;265(31):19351–19355. [PubMed] [Google Scholar]
  32. Yi C. K., Charalambous B. M., Emery V. C., Baldwin S. A. Characterization of functional human erythrocyte-type glucose transporter (GLUT1) expressed in insect cells using a recombinant baculovirus. Biochem J. 1992 May 1;283(Pt 3):643–646. doi: 10.1042/bj2830643. [DOI] [PMC free article] [PubMed] [Google Scholar]

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