Characterization of functional human erythrocyte-type glucose transporter (GLUT1) expressed in insect cells using a recombinant baculovirus

Yi, Chong-Kee; Charalambous, Bambos M.; Emery, V. C.; Baldwin, Stephen A.

doi:10.1042/bj2830643

Cited by 17 publications

(11 citation statements)

References 18 publications

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“…Using the human erythrocyte GLUTI as a standard to quantify the amounts of expressed protein, it was found that the level of GLUT 1 produced reached a maximum of -150 pmol/mg of membrane protein after 48 h. All of the expressed protein could be sedimented by centrifugation at 100000 gmax., while I 80 % of this protein was sedimented at 16000 gmax . These results suggest that a large proportion of the expressed GLUTI protein is targeted to the plasma membrane in this system [14]. Truncated GLUT1 constructs were prepared in which the N-domain (from b 98 to b 940 and corresponding to amino acids 1-272; Nl) and the C-domain (from b 883 to b 1602 and corresponding to amino acids 254-492; R12) were also expressed in the Sf9 cells and, surprisingly, were found to be targeted with very high efficiency to the plasma membrane.…”

Section: Resultsmentioning

confidence: 68%

Domain assembly of the GLUT1 glucose transporter

Cope

Holman

Baldwin

et al. 1994

Biochemical Journal

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A full-length construct of the glucose transporter isoform GLUT1 has been expressed in Sf9 (Spodoptera frugiperida Clone 9) insect cells, and a photolabelling approach has been used to show that the expressed protein binds the bismannose compound 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine (ATB-BMPA) and cytochalasin B at its exofacial and endofacial binding sites respectively. Constructs of GLUT1 which produce either the N-terminal (amino acids 1-272) or C-terminal (amino acids 254-492) halves are expressed at levels in the plasma membrane which are similar to that of the full-length GLUT1 (approximately 200 pmol/mg of membrane protein), but do not bind either ATB-BMPA or cytochalasin B. When Sf9 cells are doubly infected with virus constructs producing both the C- and N-terminal halves of GLUT1, then the ligand labelling is restored. Only the C-terminal half is labelled, and, therefore, the labelling of this domain is dependent on the presence of the N-terminal half of the protein. These results suggest that the two halves of GLUT1 can assemble to form a stable complex and support the concept of a bilobular structure for the intact glucose transporters in which separate C- and N-domain halves pack together to produce a ligand-binding conformation.

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Section: Resultsmentioning

confidence: 68%

Domain assembly of the GLUT1 glucose transporter

Cope

Holman

Baldwin

et al. 1994

Biochemical Journal

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“…The second is the existence of intrinsic glucose transporters in these systems that hindered an unequivocal interpretation [9]. The third is the difficulty in estimating the proportion of transporter contributing to actual transport : the retention of a considerable portion of GLUT1 in intracellular sites [9][10][11] or production of inactive transporter [12] are described. In order to challenge these problems, we introduced rat GLUT1 in yeast.…”

Section: Introductionmentioning

confidence: 99%

Complete Sequence, Subunit Structure, and Complexes with Pancreatic -Amylase of an -Amylase Inhibitor from Phaseolus vulgaris White Kidney Beans

Kasahara

Hayashi

Arakawa

et al. 1996

Journal of Biochemistry

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We expressed the rat GLUT1 facilitative glucose transporter in the yeast Saccharomyces cerevisiae with the use of a galactose-inducible expression system. Confocal immunofluorescence microscopy indicated that a majority of this protein is retained in an intracellular structure that probably corresponds to endoplasmic reticulum. Yeast cells expressing GLUT1 exhibited little increase in glucose-transport activity. We prepared a crude membrane fraction from these cells and made liposomes with this fraction using the freeze-thaw/sonication method. In this reconstituted system, D-glucose-transport activity was observed with a Km for D-glucose of 3.4 +/- 0.2 mM (mean +/- S.E.M.) and was inhibited by cytochalasin B (IC50= 0.44 +/- 0.03 microM), HgCl2 (IC50)= 3.5 +/- 0.5 microM), phloretin (IC50= 49 +/- 12 microM) and phloridzin (IC50= 355 +/- 67 microM). To compare these properties with native GLUT1 we made reconstituted liposomes with a membrane fraction prepared from human erythrocytes, in which the Km of D-glucose transport and ICs of these inhibitors were approximately equal to those obtained with GLUT1 made by yeast. When the relative amounts of GLUT1 in the crude membrane fractions were measured by quantitative immunoblotting, the specific activity of the yeast-made GLUT1 was 110% of erythrocyte GLUT1, indicating that GLUT1 expressed in yeast is fully active in glucose transport.

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“…We have confirmed that the GLUT1 antibody is highly suitable for immunohistochemistry as it recognizes GLUT1 across a diverse number of mammalian species including human, canine, rat, mouse, marmot, bovine, ovine, equine and elephant. There are numerous publications in the literature that report successful application of this polyclonal antibody in studies of human and animal tissues (Brown et al, 1988;Davies et al, 1987Davies et al, , 1990Froehner et al, 1988;Kasanicki et al, 1987;Takakura et al, 1991;Yi et al, 1992). Rabbit polyclonal antibodies against human GLUT9 (Augustin et al, 2004;Carayannopoulos et al, 2004) Although the GLUT9 antisera are less well characterized, they have recently been affinity purified and successfully used to study GLUT9 expression (Augustin et al, 2004).…”

Section: Antibodies To Glut1 and Glut9mentioning

confidence: 99%

Expression of the GLUT1 and GLUT9 facilitative glucose transporters in embryonic chondroblasts and mature chondrocytes in ovine articular cartilage

Mobasheri

Dobson

Mason

et al. 2005

Cell Biology International

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Glucose transport across the chondrocyte membrane is essential for chondrogenesis and the development of the skeletal system. We have previously used RT-PCR to show that fully developed human articular chondrocytes express transcripts for the GLUT1 and GLUT9 glucose transporters. In this study we report on the expression and immunohistochemical localization of the GLUT1 and GLUT9 proteins in embryonic and mature ovine cartilage. We also provide Western blot evidence for GLUT1 and GLUT9 expression in mature ovine chondrocytes. Ovine embryos (developmental stages E32 to E36 and E42 to E45) were obtained from pregnant ewes humanely killed by injection with sodium pentobarbitone. Embryos were fixed and processed for immunohistochemistry. Polyclonal antibodies to GLUT1 and GLUT9 revealed that both transporters are expressed in developing chondrocytes in ovine embryos and in the superficial, middle and deep layers of ovine cartilage from mature animals. GLUT1 expression was observed in erythrocytes and organs including heart, liver, and kidney. GLUT9 was also found in heart, kidney and liver. Western blotting confirmed the presence of the GLUT1 protein which migrated between the 50 and 64 kDa markers and two specific GLUT9 bands migrating under the 50 and 60 kDa markers, respectively. The presence of GLUT1 and GLUT9 in developing joints of ovine embryos suggests that these proteins may be important in glucose delivery to developing chondroblasts. Expression of these GLUT isoforms may be an important bioenergetic adaptation for chondrocytes in the extracellular matrix of developing cartilage.

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Characterization of functional human erythrocyte-type glucose transporter (GLUT1) expressed in insect cells using a recombinant baculovirus

Cited by 17 publications

References 18 publications

Domain assembly of the GLUT1 glucose transporter

Domain assembly of the GLUT1 glucose transporter

Complete Sequence, Subunit Structure, and Complexes with Pancreatic -Amylase of an -Amylase Inhibitor from Phaseolus vulgaris White Kidney Beans

Expression of the GLUT1 and GLUT9 facilitative glucose transporters in embryonic chondroblasts and mature chondrocytes in ovine articular cartilage

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