Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter
Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter
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DOI：
10.1042/bj20030044
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发表时间：
2003-05-15
影响因子：
4.1
通讯作者：
Kasahara, M
中科院分区：
文献类型：
--
作者：
Kasahara, T;Kasahara, M
Hxt 2是酿酒酵母的一种高亲和力的葡萄糖转运蛋白，属于主要的易化剂超家族。Hxt 1在其跨膜区段（TM）和TM间环中与Hxt 2共享约70%的氨基酸同一性，但以Hxt 2的亲和力的约十分之一转运D-葡萄糖。为了确定Hxt 2的哪些TM对于高亲和力葡萄糖转运是重要的，我们通过将Hxt 2的12个TM中的每一个用Hxt 1的相应片段随机替换来构建Hxt 2和Hxt 1的嵌合体，总共4096种不同的转运蛋白。在筛选的> 20 000个酵母转化体中，通过高亲和力葡萄糖转运活性的平板测定选择39个不同的克隆并测序。除了两个例外，所选嵌合体包含Hxt 2 TM 1、5、7和8。然后，我们构建了对应于Hxt 2 TM 1、5、7和8的所有16种可能组合的嵌合体。只有一个嵌合体，即含有所有四个Hxt 2 TM，表现出运输活动可比的Hxt 2。该嵌合体的D-葡萄糖转运的Km和V-max值以及底物特异性与Hxt 2几乎相同。这些结果表明，TM 1、5、7和8是显示Hxt 2的高亲和力葡萄糖转运活性所必需的。
Hxt2 is a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae and belongs to the major facilitator super-family. Hxt 1 shares approximate to 70 % amino acid identity with Hxt2 in its transmembrane segments (TMs) and inter-TM loops, but transports D-glucose with an affinity about one-tenth of that of Hxt2. To determine which TMs of Hxt2 are important for high-affinity glucose transport, we constructed chimaeras of Hxt2 and Hxt] by randomly replacing each of the 12 TMs of Hxt2 with the corresponding segment of Hxt1, for a total of 4096 different transporters. Among > 20 000 yeast transformants screened, 39 different clones were selected by plate assays of high-affinity glucose-transport activity and sequenced. With only two exceptions, the selected chimaeras contained Hxt2 TMs 1, 5, 7 and 8. We then constructed chimaeras corresponding to all 16 possible combinations of Hxt2 TMs 1, 5, 7 and 8. Only one chimaera, namely that containing all four Hxt2 TMs, exhibited transport activity comparable with that of Hxt2. The K-m and V-max values for D-glucose transport, and the substrate specificity of this chimaera were almost identical with those of Hxt2. These results indicate that TMs 1, 5, 7 and 8 are necessary for exhibiting high-affinity glucose-transport activity of Hxt2.