Due A D, Qu Z C, Thomas J M, Buchs A, Powers A C, May J M
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 27232-2230, USA.
Biochemistry. 1995 Apr 25;34(16):5462-71. doi: 10.1021/bi00016a017.
Structural determinants for the glucose transport kinetics of the erythrocyte glucose transporter have not been established. In this work the role of the cytosolic carboxy-terminal tail in the expression and function of the human GLUT1 isoform in Xenopus oocytes was investigated. Oocyte plasma membrane expression of GLUT1 was a saturable function of the amount of mRNA injected. Transport activity increased as a linear function of the amount of immunoreactive transporter in the plasma membrane. Transport kinetics of human GLUT1 expressed in oocytes resembled those of human erythrocyte GLUT1. Addition of up to 31 extra amino acids to the carboxy-terminal tail of GLUT1 was without effect on its function in oocytes. Removal of the carboxy-terminal 21 amino acids also did not affect GLUT1 expression or transport kinetics in oocytes. Removal of the entire carboxy-terminal tail to Phe-450 resulted in a transporter that had moderately decreased plasma membrane expression compared to that of GLUT1. However, transport activity of this construct was less than 5% of that of GLUT1, and was associated with loss of its outward-facing inhibitor binding site. When the carboxy-terminal 29 amino acids of GLUT1 were replaced with the corresponding region of GLUT4, transporter expression in the plasma membrane and the transport Vmax fell to low levels, similar to those of native GLUT4. When the carboxy-terminal 29 or 73 amino acids of GLUT1 were swapped into the corresponding region of GLUT4, the transport Vmax markedly increased to about one-third to one-half that of GLUT1, although the affinity for substrate was halved. These results show that the carboxy-terminal tail of the GLUT1 is not critical for targeting of the protein to the plasma membrane, but that this region is an important determinant of transport function.
红细胞葡萄糖转运蛋白葡萄糖转运动力学的结构决定因素尚未明确。在这项研究中，我们研究了胞质羧基末端尾巴在非洲爪蟾卵母细胞中人类GLUT1亚型的表达和功能中的作用。GLUT1在卵母细胞质膜上的表达是所注射mRNA量的饱和函数。转运活性随质膜中免疫反应性转运蛋白量的线性增加而增加。卵母细胞中表达的人类GLUT1的转运动力学与人类红细胞GLUT1相似。在GLUT1的羧基末端尾巴上添加多达31个额外氨基酸对其在卵母细胞中的功能没有影响。去除羧基末端的21个氨基酸也不影响GLUT1在卵母细胞中的表达或转运动力学。去除整个羧基末端尾巴至Phe-450会产生一种转运蛋白，与GLUT1相比，其质膜表达适度降低。然而，这种构建体的转运活性不到GLUT1的5%，并且与其外向抑制剂结合位点的丧失有关。当GLUT1的羧基末端29个氨基酸被GLUT4的相应区域取代时，质膜中的转运蛋白表达和转运Vmax降至低水平，类似于天然GLUT4。当GLUT1的羧基末端29个或73个氨基酸被交换到GLUT4的相应区域时，转运Vmax显著增加至GLUT1的约三分之一至二分之一，尽管对底物的亲和力减半。这些结果表明，GLUT1的羧基末端尾巴对于蛋白质靶向质膜并不关键，但该区域是转运功能的重要决定因素。
