Dataset for: Real Time Imaging of Sodium Glucose Transporter (SGLT1) Trafficking and Activity in Single Cells.

The processes controlling targeting of glucose transporters to apical and basolateral membranes of polarized cells are complex and not-well understood. We have engineered SGLT1 and GLUT4 constructs linked to fluorescent proteins to highlight the differences in transporter expression and trafficking, in real time, in different cell types. Activity was assessed in parallel using a FRET glucose sensor. In COS cells and HEK cells, SGLT1 was distributed between the plasma membrane and intracellular compartments, but there was little expression in CHO cells. Trafficking was investigated using the lysosome inhibitors NH4Cl (10 mM) and chloroquine (150 M) and the proteasome inhibitors MG-262 (1 µM) and Lactacystin (5 µM). Lysosome inhibitors caused SGLT1 accumulation into intracellular bodies, whereas proteasome inhibitors induced SGLT1 accumulation in the plasma membrane, even in CHO cells. Our data suggest that a fraction of SGLT1 is rapidly degraded by lysosomes and never reached the plasma membrane; another fraction reaches the membrane and is subsequently degraded by lysosomes following internalization. The latter process is regulated by the ubiquitin/proteasome pathway, acting at a late stage of the lysosomal pathway. Using the cholesterol inhibitor MβCD (3 mM), a dominant negative dynamin (K44A) and caveolin, we showed that SGLT1 internalization is lipid raft-mediated, but caveolin-independent. In contrast, GLUT4 internalization is dynamin-dependent, but cholesterol-independent. The physiological relevance of these data is discussed in terms of differential membrane compartmentalization of the transporters and expression under stress conditions.

调控葡萄糖转运蛋白靶向极化细胞顶膜与基底侧膜的过程极为复杂，目前尚未得到充分阐明。我们构建了与荧光蛋白融合的SGLT1与GLUT4重组载体，以实时观测不同细胞类型中转运蛋白的表达差异与转运过程。同时采用荧光共振能量转移（FRET）葡萄糖传感器对转运活性进行平行评估。在COS细胞与HEK细胞中，SGLT1分布于质膜与细胞内区室之间，但CHO细胞中几乎无SGLT1表达。我们分别使用溶酶体抑制剂NH4Cl（10 mM）、氯喹（150 μM），以及蛋白酶体抑制剂MG-262（1 μM）与乳胞素（Lactacystin，5 μM），对SGLT1的转运过程进行了研究。实验结果显示，溶酶体抑制剂可诱导SGLT1在细胞内囊泡中积累，而蛋白酶体抑制剂甚至可在CHO细胞中促使SGLT1在质膜上聚集。我们的数据表明，部分SGLT1会通过溶酶体途径快速降解，且从未抵达质膜；另一部分抵达质膜后经内吞作用被溶酶体降解。该后续降解过程受泛素-蛋白酶体通路调控，其作用位点位于溶酶体通路的下游阶段。通过使用胆固醇抑制剂MβCD（3 mM）、显性负突变发动蛋白（dynamin，K44A）以及小窝蛋白（caveolin），我们证实SGLT1的内吞过程依赖脂筏结构，但不依赖小窝蛋白。与之形成对比的是，GLUT4的内吞依赖发动蛋白，但不依赖胆固醇。本文最后从转运蛋白的差异化膜区室化特征以及应激条件下的表达模式两个维度，探讨了上述实验数据的生理学相关性。