Determination of GLUT1 Oligomerization Parameters using Bioluminescent Förster Resonance Energy Transfer

• DOI: 10.1038/srep29130
• Publication Date: 2016-06-30T09:40:22Z
• Authors: Brendan Looyenga; Calvin VanOpstall; Zion Lee; Jed Bell; Evans Lodge; Katherine Wrobel; Eric Arnoys; Larry Louters
• Keywords: mCherry

Abstract The facilitated glucose transporter GLUT1 (SLC2A1) is an important mediator of homeostasis in humans. Though it found most cell types to some extent, the level expression across different can vary dramatically. Prior studies erythrocytes—which express particularly high levels GLUT1—have suggested that able form tetrameric complexes with enhanced transport activity. Whether dynamic aggregation also occurs more modest GLUT1, however, unclear. To address this question, we developed a genetically encoded bioluminescent Förster resonance energy transfer (BRET) assay using luminescent donor Nanoluciferase and fluorescent acceptor mCherry. By tethering these proteins N-terminus performing saturation BRET analysis, were demonstrate formation multimeric live cells. Parallel use flow cytometry immunoblotting further enabled us estimate density required for spontaneous oligomerization. These data provide new insights into physiological relevance multimerization as well variant useful measuring interactions among other membrane