Haspel H C, Mynarcik D C, Ortiz P A, Honkanen R A, Rosenfeld M G
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
Mol Endocrinol. 1991 Jan;5(1):61-72. doi: 10.1210/mend-5-1-61.
Antibody to the carboxyl-terminal of hexose transporter protein GLUT-1 was used to localize this carrier in normal rat kidney (NRK) cells during D-glucose (Glc) deprivation. Glc-deprivation of NRK cells induces increased hexose transport, inhibits the glycosylation of GLUT-1, and increases the content of both native, 55,000 apparent mol wt (Mr) and aglyco, 38,000 Mr GLUT-1 polypeptides. The distribution of GLUT-1 protein in subcellular fractions isolated from Glc-fed NRK cells shows that the 55,000 Mr polypeptide is most abundant in intracellular membrane fractions. Glc-fed cells that have been tunicamycin treated contain principally the 38,000 Mr GLUT-1 polypeptide, which is found predominantly in intracellular membrane fractions. In Glc-deprived cells the 55,000 Mr GLUT-1 polypeptide localizes predominantly in the Golgi and plasma membrane fractions, whereas the more abundant 38,000 Mr GLUT-1 polypeptide is distributed throughout all membrane fractions. In Glc-deprived but fructose-fed cells only the 55,000 Mr GLUT-1 polypeptide is detected, and it is found predominantly in the plasma membrane and Golgi fractions. The localization of GLUT-1 protein was directly and specifically visualized in NRK cells by immunofluorescence microscopy. Glc-fed cells show little labeling of cell borders and a small punctate juxtanuclear pattern suggestive of localization to the Golgi and, perhaps, endoplasmic reticulum. Glc-fed cells that have been tunicamycin treated show large punctate intracellular accumulations suggestive of localization to distended Golgi and perhaps endoplasmic reticulum. Glc-deprived cells exhibited intense labeling of cell borders as well as intracellular accumulations. Glc-deprived but fructose-fed cells show fewer intracellular accumulations, and the labeling is, in general, limited to the cell borders. Our results suggest that Glc deprivation induces the selective accumulation of GLUT-1 in the plasma membrane of NRK cells.
在D - 葡萄糖（Glc）缺乏期间，利用针对己糖转运蛋白GLUT - 1羧基末端的抗体，将该载体定位在正常大鼠肾（NRK）细胞中。NRK细胞的Glc缺乏会诱导己糖转运增加，抑制GLUT - 1的糖基化，并增加天然的、表观分子量为55,000（Mr）和无糖基的、分子量为38,000 Mr的GLUT - 1多肽的含量。从喂食Glc的NRK细胞中分离得到的亚细胞组分中GLUT - 1蛋白的分布表明，55,000 Mr的多肽在细胞内膜组分中最为丰富。用衣霉素处理过的喂食Glc的细胞主要含有38,000 Mr的GLUT - 1多肽，该多肽主要存在于细胞内膜组分中。在Glc缺乏的细胞中，55,000 Mr的GLUT - 1多肽主要定位于高尔基体和质膜组分，而含量更丰富的38,000 Mr的GLUT - 1多肽则分布在所有膜组分中。在Glc缺乏但喂食果糖的细胞中，仅检测到55,000 Mr的GLUT - 1多肽，且主要存在于质膜和高尔基体组分中。通过免疫荧光显微镜在NRK细胞中直接且特异性地观察到了GLUT - 1蛋白的定位。喂食Glc的细胞显示细胞边界几乎没有标记，并且有一个小的点状核周模式，提示定位于高尔基体，或许还有内质网。用衣霉素处理过的喂食Glc的细胞显示细胞内有大量点状聚集物，提示定位于扩张的高尔基体，或许还有内质网。Glc缺乏的细胞表现出细胞边界以及细胞内聚集物的强烈标记。Glc缺乏但喂食果糖的细胞显示细胞内聚集物较少，并且标记通常仅限于细胞边界。我们的结果表明，Glc缺乏诱导GLUT - 1在NRK细胞质膜中选择性积累。
