Functional expression of the human UDP-galactose transporters in the yeast Saccharomyces cerevisiae.
Functional expression of the human UDP-galactose transporters in the yeast Saccharomyces cerevisiae.
复制标题
人类 UDP-半乳糖转运蛋白在酿酒酵母中的功能表达。
DOI：
10.1093/oxfordjournals.jbchem.a022024
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发表时间：
1998
影响因子：
2.7
通讯作者：
M. Kawakita
中科院分区：
文献类型：
--
作者：
G. Sun;S. Yoshioka;N. Ishida;M. Kawakita
我们描述了假定的人类高尔基体udp -半乳糖转运蛋白（hUGT1和hUGT2）在酵母中的功能表达。hUGT1和hUGT2均在酵母组成型GAPDH启动子的控制下表达。虽然hUGT1与hUGT2的氨基酸序列除了在c端有5个氨基酸残基不同，但其表达水平似乎明显低于hUGT2。hUGT产物在高尔基体和其他细胞器室的膜中表达。由表达hUGT1-或hugt2的酵母细胞制备的膜泡表现出udp -半乳糖特异性转运活性。酵母表达的hUGT1和hUGT2对udp -半乳糖的表观Km值分别为1.2和2微米，与哺乳动物高尔基囊泡的表观Km值相当。在哺乳动物高尔基囊泡中观察到，这种转运依赖于温度和囊泡的完整性，并被UMP抑制。我们的研究结果表明，先前描述的hUGT1和hUGT2编码udp -半乳糖转运蛋白，而不是调节蛋白。开发一种方便的酵母表达系统将有助于分析udp -半乳糖转运体的结构-功能关系。
We describe the functional expression of the putative human Golgi UDP-galactose transporters (hUGT1 and hUGT2) in the yeast Saccharomyces cerevisiae. Both hUGT1 and hUGT2 were expressed under the control of the yeast constitutive GAPDH promoter. The expression level of hUGT1 seemed to be considerably lower than that of hUGT2, although hUGT1 has an amino acid sequence identical to that of hUGT2 except for 5 amino acid residues at the C-terminus. The hUGT product was expressed in the membranes of Golgi and other organellar compartments. The membrane vesicles prepared from the hUGT1- or the hUGT2-expressing yeast cells exhibited UDP-galactose specific transport activity. The apparent Km values of the yeast-expressed hUGT1 and hUGT2 for UDP-galactose were 1.2 and 2 microM, respectively, which were comparable with the Km obtained with mammalian Golgi vesicles. Transport was dependent on temperature and integrity of vesicles, and was inhibited by UMP, as observed with mammalian Golgi vesicles. Our results demonstrate that the previously described hUGT1 and hUGT2 encode the UDP-galactose transporters, rather than regulatory proteins. The development of a convenient yeast expression system should facilitate analysis of the structure-function relationships of the UDP-galactose transporters.