Supplementary Materialsao0c01054_si_001. However, a significant upsurge in reddish colored punctate fluorescence (mCherry can be resistant to acidic pH), however, not green fluorescence (EGFP can be quenched by acidic pH), was noticed upon blood sugar deprivation, indicating that the mCherry-EGFP-GLUT1 practical proteins was trafficked towards the acidic endolysosomal program. Besides, we could actually calculate the comparative percentage of mCherry to EGFP by quantification from the translocation L-Thyroxine coefficient, which may be used like a readout for GLUT1 internalization and following lysosomal degradation. Two mutants, mCherry-EGFP-GLUT1-C4 and mCherry-EGFP-GLUT1-S226D, were constructed also, which verified the specificity of mCherry-EGFP-GLUT1 for monitoring GLUT1 trafficking indirectly. With a group of endosomal (Rab5, Rab7, and Rab11) and lysosomal markers, we could actually define a style of GLUT1 trafficking in live cells where upon blood sugar deprivation, GLUT1 dissociates through the PM and experiences a pH gradient from 6.8C6.1 in the early endosomes to 6.0C4.8 in the late endosomes and finally pH 4.5 in lysosomes, which is appropriate for degradation. In addition, our proof-of-concept study indicated that the pmCherry-EGFP-GLUT1 tracing system can accurately reflect endogenous changes in GLUT1 in response to treatment with the small molecule, andrographolide. Since targeting GLUT1 expression and GLUT1-dependent glucose metabolism is a promising therapeutic strategy for diverse types of cancers and certain other glucose addiction diseases, our study herein indicates that pmCherry-EGFP-GLUT1 can be utilized as a biosensor for GLUT1-dependent functional studies and potential small molecule screening. Introduction Glucose is the most fundamental source for the production of energy and subsequent metabolic processes in all organisms; consequently, the fine-tuning of glucose uptake is critical for maintaining glucose homeostasis and metabolic balance.1 At the cellular level, the transportation of glucose across the plasma membrane (PM) is the first rate-limiting step for glucose metabolism and is facilitated by a series of glucose transporter/solute carrier 2A (GLUT/SLC2A) family members.2 Glucose transporter 1 (GLUT1) is one of the most NF-ATC intensively studied GLUT family members due to its ubiquitous role in glucose uptake in various cell types.3 Increasing evidence suggests that the dysregulation of GLUT1 plays a key role in a series of diseases.4,5 For instance, the impairment of GLUT1-dependent glucose transport has been reported to lead to a novel group of disorders named GLUT1 deficiency syndrome.4 In contrast, the overexpression of GLUT1 has been observed in several cancers, thereby facilitating glucose uptake and increasing glycolytic flux, which in turn L-Thyroxine facilitates cancer cell dependence on glucose and/or survival.5,6 Therefore, exploring the regulation of GLUT1 at the cellular level has become a hot topic, especially in the metabolic field. Several mechanisms have been revealed to be involved in the regulation of GLUT1 expression: (i) several perturbations, such as decreases in oxygen or nutrient availability, have got been proven to raise the basal transcription of GLUT17 typically,8 and (ii) the subcellular trafficking of GLUT1 between internal vesicular compartments and the cell surface is usually another major form of GLUT1 regulation. GLUT1 can undergo internalization via early endosomes (EE)-late endosomes (LE) and then traffic into the lysosome for degradation via the endolysosomal pathway; however, GLUT1 can also be recycled back to the PM by recycling endosomes (RE) through the actions of multiple GTPases.9?11 In addition, mutations within the GLUT1 functional domain name can also affect its cellular trafficking, e.g., S226 phosphorylation of GLUT1 promotes its membrane localization, whereas the deletion of the PDZ-binding motif impairs GLUT1 cell surface trafficking.12,13 Studies L-Thyroxine aimed at determining GLUT1 trafficking provide insight into glucose uptake. Although immunofluorescence and immunohistochemistry, as well as biochemical fractionation, can be used to access the endogenous GLUT1 expression, they cannot reflect the real-time changes in GLUT1. In addition, GLUT1, as a transmembrane protein, preferentially forms clusters or dimeric or tetrameric complexes, which may cause troubles in GLUT1 functional studies, e.g., fuzzy positioning, nonspecific coloring, difficult separation, long experimental cycles, and expense.14,15 At the live cell level, GLUT1 trafficking can be observed by microscopy using proteins tagged with single fluorescence, yet it is difficult to quantify the kinematics of GLUT1 since it can be either degraded or recycled.9,13,16 A recent study exhibited that a genetically encoded bioluminescent.