title: Determination of GLUT1 Oligomerization Parameters using Bioluminescent Förster Resonance Energy Transfer.
abstract: The facilitated glucose transporter GLUT1 (SLC2A1) is an important mediator of glucose homeostasis in humans. Though it is found in most cell types to some extent, the level of GLUT1 expression across different cell types can vary dramatically. Prior studies in erythrocytes-which express particularly high levels of GLUT1-have suggested that GLUT1 is able to form tetrameric complexes with enhanced transport activity. Whether dynamic aggregation of GLUT1 also occurs in cell types with more modest expression of GLUT1, however, is unclear. To address this question, we developed a genetically encoded bioluminescent Förster resonance energy transfer (BRET) assay using the luminescent donor Nanoluciferase and fluorescent acceptor mCherry. By tethering these proteins to the N-terminus of GLUT1 and performing saturation BRET analysis, we were able to demonstrate the formation of multimeric complexes in live cells. Parallel use of flow cytometry and immunoblotting further enabled us to estimate the density of GLUT1 proteins required for spontaneous oligomerization. These data provide new insights into the physiological relevance of GLUT1 multimerization as well as a new variant of BRET assay that is useful for measuring the interactions among other cell membrane proteins in live cells.
authors.affiliation: Calvin College, Department of Chemistry &Biochemistry, 3201 Burton St SE, Grand Rapids, MI, 49546, USA.
authors.forename: Brendan
authors.initials: B
authors.lastname: Looyenga
authors.affiliation: Calvin College, Department of Chemistry &Biochemistry, 3201 Burton St SE, Grand Rapids, MI, 49546, USA.
authors.forename: Calvin
authors.initials: C
authors.lastname: VanOpstall
authors.affiliation: Calvin College, Department of Chemistry &Biochemistry, 3201 Burton St SE, Grand Rapids, MI, 49546, USA.
authors.forename: Zion
authors.initials: Z
authors.lastname: Lee
authors.affiliation: Calvin College, Department of Chemistry &Biochemistry, 3201 Burton St SE, Grand Rapids, MI, 49546, USA.
authors.forename: Jed
authors.initials: J
authors.lastname: Bell
authors.affiliation: Calvin College, Department of Chemistry &Biochemistry, 3201 Burton St SE, Grand Rapids, MI, 49546, USA.
authors.forename: Evans
authors.initials: E
authors.lastname: Lodge
authors.affiliation: Calvin College, Department of Chemistry &Biochemistry, 3201 Burton St SE, Grand Rapids, MI, 49546, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Wrobel
authors.affiliation: Calvin College, Department of Chemistry &Biochemistry, 3201 Burton St SE, Grand Rapids, MI, 49546, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Arnoys
authors.affiliation: Calvin College, Department of Chemistry &Biochemistry, 3201 Burton St SE, Grand Rapids, MI, 49546, USA.
authors.forename: Larry
authors.initials: L
authors.lastname: Louters
mesh_terms: D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D051272:Glucose Transporter Type 1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D057809:HEK293 Cells; D006801:Humans; D008163:Luminescent Measurements; D066329:Protein Aggregates / Q000235:genetics*; D055503:Protein Multimerization / Q000235:genetics
chemical_list: D051272:Glucose Transporter Type 1; D066329:Protein Aggregates; C495677:SLC2A1 protein, human