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.
Highlights
Nanoluc Signal** 0.06 0.06 0.11 0.14 0.34 0.39 0.43 1.21 1.70 2.11 4.54(*the peak emission shift was calculated by subtracting the Nluc emission maximum [450 nm] from the acceptor protein emission maximum; **the theoretical background bioluminescent Förster resonance energy transfer (BRET) ratio was calculated by taking the ratio of light emitted by Nluc alone at the acceptor emission maxima divided by the measured donor wavelength [410 nm]).BRET ratio without dampening due to high background noise (Supplemental Figure 1)
The quantitative principles governing BRET reactions are based upon equations developed by Theodor Förster for determining resonance energy transfer between two fluorophores (FRET)[16,23]
For the purposes of determining the degree of association between molecules in the two-dimensional plane of a biological membrane, the two key factors of interest are the efficiency of resonance energy transfer (E) between a given donor/acceptor pair, and the distance at which this efficiency is at 50%, defined as the Förster radius (R0)[24]
Summary
Nanoluc Signal** 0.06 0.06 0.11 0.14 0.34 0.39 0.43 1.21 1.70 2.11 4.54(*the peak emission shift was calculated by subtracting the Nluc emission maximum [450 nm] from the acceptor protein emission maximum; **the theoretical background BRET ratio was calculated by taking the ratio of light emitted by Nluc alone at the acceptor emission maxima divided by the measured donor wavelength [410 nm]).BRET ratio without dampening due to high background noise (Supplemental Figure 1). The hexapeptide linker series we created produces data that fit well in the linear portion of the Förster equation (equation 1), allowing us to produce a standard curve by which the distance between Nluc and mCherry can be calculated using linkers of unknown dimensions (Fig. 2c). With this principle in mind, we fused mCherry to the N-terminus of the glucose transporter GLUT1 and Nluc to the C-terminus to create a fusion protein in which the donor and acceptor interact at a fixed distance in transfected cells (Fig. 2a). Our data suggest an average distance of 28.28 +/− 7.61 angstroms typically separates these two domains when GLUT1 is inserted into the membrane of cells (Fig. 2b), which is a reasonable estimate based on the crystal structure and homology model of human GLUT127,28
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