Calvin College, Department of Chemistry &Biochemistry, 3201 Burton St SE, Grand Rapids, MI, 49546, USA.
Sci Rep. 2016 Jun 30;6:29130. doi: 10.1038/srep29130.
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.
易化葡萄糖转运蛋白 GLUT1（SLC2A1）是人类葡萄糖稳态的重要介质。尽管它在大多数细胞类型中都有一定程度的表达，但不同细胞类型中 GLUT1 的表达水平差异很大。先前在红细胞中的研究——红细胞中 GLUT1 的表达水平特别高——表明 GLUT1 能够形成具有增强转运活性的四聚体复合物。然而，在 GLUT1 表达水平较低的细胞类型中，GLUT1 是否也会发生动态聚集尚不清楚。为了解决这个问题，我们使用发光供体 Nanoluciferase 和荧光受体 mCherry 开发了一种遗传编码的生物发光荧光共振能量转移（BRET）测定法。通过将这些蛋白质连接到 GLUT1 的 N 端，并进行饱和 BRET 分析，我们能够在活细胞中证明多聚体复合物的形成。平行使用流式细胞术和免疫印迹进一步使我们能够估计自发寡聚化所需的 GLUT1 蛋白密度。这些数据为 GLUT1 多聚化的生理相关性提供了新的见解，以及一种新的 BRET 测定法变体，该变体可用于测量活细胞中其他细胞膜蛋白之间的相互作用。
