Levine Kara B, Robichaud Trista K, Hamill Stephanie, Sultzman Lisa A, Carruthers Anthony
Department of Biochemistry and Molecular Pharmacology, UMass Medical School, 364 Plantation Street, Worcester, Massachusetts 01605, USA.
Biochemistry. 2005 Apr 19;44(15):5606-16. doi: 10.1021/bi0477541.
Human erythrocyte hexose transfer is mediated by the glucose transport protein GLUT1 and is characterized by a complexity that is unexplained by available hypotheses for carrier-mediated sugar transport [Cloherty, E. K., Heard, K. S., and Carruthers, A. (1996) Biochemistry 35, 10411-10421]. The study presented here examines the possibility that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1), mutant GLUT1 (GLUT1(338)(-)(A3)), or carboxy-terminal hemagglutinin-polyHis-tagged GLUT1 (GLUT1-HA-H6). GLUT1 and GLUT1-HA-H6 are expressed at the yeast cell membrane and restore 2-deoxy-d-glucose, 3-O-methylglucose, and d-glucose transport capacity to RE700A. GLUT1-HA-H6 confers GLUT1-specific sugar transport characteristics to transfected RE700A, including inhibition by cytochalasin B and high-affinity transport of the nonmetabolized sugar 3-O-methylglucose. GLUT1(338)(-)(A3), a catalytically inactive GLUT1 mutant, is expressed but fails to restore RE700A sugar uptake capacity or growth on glucose. In contrast to transport in human red cells, K(m(app)) for 2-deoxy-d-glucose uptake equals K(i(app)) for 2-deoxy-d-glucose inhibition of 3-O-methylglucose uptake. Unlike transport in human red cells or transport in human embryonic kidney cells transfected with GLUT1-HA-H6, unidirectional sugar uptake in RE700A-GLUT1-HA-H6 is not inhibited by reductant and is not stimulated by intracellular sugar. Net uptake of subsaturating 3-O-methylglucose by RE700A-GLUT1-HA-H6 is a simple, first-order process. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific.
人类红细胞己糖转运由葡萄糖转运蛋白GLUT1介导，其特征在于具有一定复杂性，现有载体介导的糖转运假说无法解释这种复杂性[克洛赫蒂，E.K.，赫德，K.S.，和卡拉瑟斯，A.（1996年）《生物化学》35卷，10411 - 10421页]。此处呈现的研究探讨了GLUT1的操作特性是否由宿主细胞环境决定的可能性。用含有编码野生型人类葡萄糖转运蛋白（GLUT1）、突变型GLUT1（GLUT1(338)(-)(A3)）或羧基末端血凝素 - 多组氨酸标签化的GLUT1（GLUT1 - HA - H6）的DNA的p426 GPD酵母表达载体转染酿酒酵母的葡萄糖转运缺陷型菌株（RE700A）。GLUT1和GLUT1 - HA - H6在酵母细胞膜上表达，并将2 - 脱氧 - d - 葡萄糖、3 - O - 甲基葡萄糖和d - 葡萄糖的转运能力恢复到RE700A。GLUT1 - HA - H6赋予转染的RE700A GLUT1特异性的糖转运特性，包括细胞松弛素B的抑制作用以及非代谢性糖3 - O - 甲基葡萄糖的高亲和力转运。GLUT1(338)(-)(A3)是一种催化无活性的GLUT1突变体，虽有表达，但无法恢复RE700A的糖摄取能力或在葡萄糖上的生长能力。与人类红细胞中的转运情况不同，2 - 脱氧 - d - 葡萄糖摄取的K(m(app))等于2 - 脱氧 - d - 葡萄糖对3 - O - 甲基葡萄糖摄取抑制作用的K(i(app))。与人类红细胞中的转运或用GLUT1 - HA - H6转染的人类胚胎肾细胞中的转运不同，RE700A - GLUT1 - HA - H6中的单向糖摄取不受还原剂抑制，也不受细胞内糖的刺激。RE700A - GLUT1 - HA - H6对亚饱和3 - O - 甲基葡萄糖的净摄取是一个简单的一级过程。这些发现支持了红细胞糖转运复杂性具有宿主细胞特异性的假说。
