Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Jul;169(7):2926–2931. doi: 10.1128/jb.169.7.2926-2931.1987

Glucose transport in vesicles reconstituted from Saccharomyces cerevisiae membranes and liposomes.

R Ongjoco, K Szkutnicka, V P Cirillo
PMCID: PMC212328  PMID: 2954946

Abstract

Glucose transport activity was reconstituted into liposomes by the freeze-thaw-sonication procedure from unextracted Saccharomyces cerevisiae membranes and preformed phospholipid liposomes. Fluorescence-dequenching measurements with octadecylrhodamine B chloride (R18)-labeled membranes showed that the yeast membrane lipids are diluted by the liposome lipids after the freeze-thaw-sonication procedure. At lipid-to-protein ratios greater than 75:1, vesicles with single transporters were formed. Reconstituted specific activity was increased at least twofold if the liposomes contained 50 mol% cholesterol. A further increase in specific activity, from 3- to 10-fold, was achieved by fractionation of the membranes on a Renografin gradient before reconstitution. Examination of the fractions from the Renografin gradient by sodium dodecyl sulfate-gel electrophoresis showed a parallel enrichment of glucose transport activity and a number of proteins including one with an apparent Mr of ca. 60,000, which might be the glucose transporter. Finally, preliminary kinetic analysis of glucose transport activity in vesicles reconstituted at a high lipid-to-protein ratio gave a Vmax of ca. 2.8 mumol/mg of protein per min at 23 degrees C and a Km of ca. 8 mM. The latter value corresponds to the kinase-independent, low-affinity component of glucose transport observed in wild-type cells.

Full text

PDF
2928

Images in this article

2930Image
on p.2930

Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. Citovsky V., Blumenthal R., Loyter A. Fusion of Sendai virions with phosphatidylcholine-cholesterol liposomes reflects the viral activity required for fusion with biological membranes. FEBS Lett. 1985 Dec 2;193(2):135–140. doi: 10.1016/0014-5793(85)80137-x. [DOI] [PubMed] [Google Scholar]
  5. Franzusoff A. J., Cirillo V. P. Glucose transport activity in isolated plasma membrane vesicles from Saccharomyces cerevisiae. J Biol Chem. 1983 Mar 25;258(6):3608–3614. [PubMed] [Google Scholar]
  6. Franzusoff A., Cirillo V. P. Uptake and phosphorylation of 2-deoxy-D-glucose by wild-type and single-kinase strains of Saccharomyces cerevisiae. Biochim Biophys Acta. 1982 Jun 14;688(2):295–304. doi: 10.1016/0005-2736(82)90340-6. [DOI] [PubMed] [Google Scholar]
  7. Fuhrmann G. F., Boehm C., Theuvenet A. P. Sugar transport and potassium permeability in yeast plasma membrane vesicles. Biochim Biophys Acta. 1976 May 21;433(3):583–596. doi: 10.1016/0005-2736(76)90283-2. [DOI] [PubMed] [Google Scholar]
  8. Haspel H. C., Wilk E. W., Birnbaum M. J., Cushman S. W., Rosen O. M. Glucose deprivation and hexose transporter polypeptides of murine fibroblasts. J Biol Chem. 1986 May 25;261(15):6778–6789. [PubMed] [Google Scholar]
  9. Kasahara M., Hinkle P. C. Reconstitution and purification of the D-glucose transporter from human erythrocytes. J Biol Chem. 1977 Oct 25;252(20):7384–7390. [PubMed] [Google Scholar]
  10. Kuo S. C., Cirillo V. P. Galactose transport in Saccharomyces cerevisiae. 3. Characteristics of galactose uptake in transferaseless cells: evidence against transport-associated phosphorylation. J Bacteriol. 1970 Sep;103(3):679–685. doi: 10.1128/jb.103.3.679-685.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Malpartida F., Serrano R. Purification of the yeast plasma membrane ATPase solubilized with a novel zwitterionic detergent. FEBS Lett. 1980 Feb 25;111(1):69–72. doi: 10.1016/0014-5793(80)80763-0. [DOI] [PubMed] [Google Scholar]
  13. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Penefsky H. S. Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1977 May 10;252(9):2891–2899. [PubMed] [Google Scholar]
  16. Pick U. Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures. Arch Biochem Biophys. 1981 Nov;212(1):186–194. doi: 10.1016/0003-9861(81)90358-1. [DOI] [PubMed] [Google Scholar]
  17. Serrano R., Delafuente G. Regulatory properties of the constitutive hexose transport in Saccharomyces cerevisiae. Mol Cell Biochem. 1974 Dec 20;5(3):161–171. doi: 10.1007/BF01731379. [DOI] [PubMed] [Google Scholar]
  18. Serrano R., Kielland-Brandt M. C., Fink G. R. Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases. Nature. 1986 Feb 20;319(6055):689–693. doi: 10.1038/319689a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Tschopp J., Schekman R. Two distinct subfractions in isolated Saccharomyces cerevisiae plasma membranes. J Bacteriol. 1983 Oct;156(1):222–229. doi: 10.1128/jb.156.1.222-229.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Weber J., Warden D. A., Semenza G., Diedrich D. F. Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane. J Cell Biochem. 1985;27(2):83–96. doi: 10.1002/jcb.240270203. [DOI] [PubMed] [Google Scholar]
  22. Wheeler T. J. Reconstitution of glucose transport activity from erythrocyte membranes without detergent and its use in studying effects of ATP depletion. Biochim Biophys Acta. 1986 Jul 24;859(2):180–188. doi: 10.1016/0005-2736(86)90213-0. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES