Haney P M, Levy M A, Strube M S, Mueckler M
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Cell Biol. 1995 May;129(3):641-58. doi: 10.1083/jcb.129.3.641.
The GLUT4 glucose transporter appears to be targeted to a unique insulin-sensitive intracellular membrane compartment in fat and muscle cells. Insulin stimulates glucose transport in these cell types by mediating the partial redistribution of GLUT4 from this intracellular compartment to the plasma membrane. The structural basis for the unique targeting behavior of GLUT4 was investigated in the insulin-sensitive L6 myoblast cell line. Analysis of immunogold-labeled cells of independent clonal lines by electron microscopy indicated that 51-53% of GLUT1 was present in the plasma membrane in the basal state. Insulin did not significantly affect this distribution. In contrast, only 4.2-6.1% of GLUT4 was present in the plasma membrane of basal L6 cells and insulin increased this percentage by 3.7-6.1-fold. Under basal conditions and after insulin treatment, GLUT4 was detected in tubulovesicular structures, often clustered near Golgi stacks, and in endosome-like vesicles. Analysis of 25 chimeric transporters consisting of reciprocal domains of GLUT1 and GLUT4 by confocal immunofluorescence microscopy indicated that only the final 25 amino acids of the COOH-terminal cytoplasmic tail of GLUT4 were both necessary and sufficient for the targeting pattern observed for GLUT4. A dileucine motif present in the COOH-terminal tail of GLUT4 was found to be necessary, but not sufficient, for intracellular targeting. Contrary to previous studies, the NH2 terminus of GLUT4 did not affect the subcellular distribution of chimeras. Analysis of a chimera containing the COOH-terminal tail of GLUT4 by immunogold electron microscopy indicated that its subcellular distribution in basal cells was very similar to that of wild-type GLUT4 and that its content in the plasma membrane increased 6.8-10.5-fold in the presence of insulin. Furthermore, only the chimera containing the COOH terminus of GLUT4 enhanced insulin responsive 2-deoxyglucose uptake. GLUT1 and two other chimeras lacking the COOH terminus of GLUT4 were studied by immunogold electron microscopy and did not demonstrate insulin-mediated changes in subcellular distribution. The NH2-terminal cytoplasmic tail of GLUT4 did not confer intracellular sequestration and did not cause altered subcellular distribution in the presence of insulin. Intracellular targeting of one chimera to non-insulin-sensitive compartments was also observed. We conclude that the COOH terminus of GLUT4 is both necessary and sufficient to confer insulin-sensitive subcellular targeting of chimeric glucose transporters in L6 myoblasts.
葡萄糖转运蛋白4（GLUT4）似乎定位于脂肪和肌肉细胞中一个独特的对胰岛素敏感的细胞内膜区室。胰岛素通过介导GLUT4从这个细胞内区室向质膜的部分重新分布，刺激这些细胞类型中的葡萄糖转运。在对胰岛素敏感的L6成肌细胞系中研究了GLUT4独特靶向行为的结构基础。通过电子显微镜对独立克隆系的免疫金标记细胞进行分析表明，在基础状态下，51% - 53%的GLUT1存在于质膜中。胰岛素对这种分布没有显著影响。相比之下，在基础状态的L6细胞的质膜中，仅4.2% - 6.1%的GLUT4存在，胰岛素使这一百分比增加了3.7 - 6.1倍。在基础条件下和胰岛素处理后，在管状小泡结构中检测到GLUT4，这些结构通常聚集在高尔基体堆栈附近，以及在类似内体的小泡中。通过共聚焦免疫荧光显微镜对由GLUT1和GLUT4的相互结构域组成的25种嵌合转运蛋白进行分析表明，对于GLUT4所观察到的靶向模式，仅GLUT4羧基末端胞质尾的最后25个氨基酸是必需且足够的。发现GLUT4羧基末端尾巴中存在的双亮氨酸基序对于细胞内靶向是必需的，但不是足够的。与先前的研究相反，GLUT4的氨基末端不影响嵌合体的亚细胞分布。通过免疫金电子显微镜对含有GLUT4羧基末端尾巴的嵌合体进行分析表明，其在基础细胞中的亚细胞分布与野生型GLUT4非常相似，并且在胰岛素存在下其在质膜中的含量增加了6.8 - 10.5倍。此外，只有含有GLUT4羧基末端的嵌合体增强了胰岛素反应性2 - 脱氧葡萄糖摄取。通过免疫金电子显微镜研究了GLUT1和另外两种缺乏GLUT4羧基末端的嵌合体，它们没有显示出胰岛素介导的亚细胞分布变化。GLUT4的氨基末端胞质尾没有赋予细胞内隔离，并且在胰岛素存在下也没有导致亚细胞分布改变。还观察到一种嵌合体向非胰岛素敏感区室的细胞内靶向。我们得出结论，GLUT4的羧基末端对于赋予L6成肌细胞中嵌合葡萄糖转运蛋白胰岛素敏感的亚细胞靶向是必需且足够的。
