Membrane topology of the mammalian CMP-sialic acid transporter
Membrane topology of the mammalian CMP-sialic acid transporter
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DOI：
10.1074/jbc.274.13.8779
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发表时间：
1999-03-26
影响因子：
4.8
通讯作者：
Gerardy-Schahn, R
中科院分区：
文献类型：
--
作者：
Eckhardt, M;Gotza, B;Gerardy-Schahn, R
核苷酸糖转运蛋白是高尔基体和内质网的一个远亲膜蛋白家族。第一个转运蛋白序列是在最近两年内确定的。然而，关于这些分子的二级和三级结构的信息仅限于理论考虑。在本研究中，采用表位插入方法研究了cmp -唾液酸转运体的膜拓扑结构。免疫荧光研究分析了引入的表位在半透性细胞中的取向。发现一个氨基末端引入的FLAG序列和一个羧基末端的血凝素标签都指向细胞质。在潜在的膜间环结构中含有血凝素表位的CMP唾液酸转运体变体与10个跨膜区域的存在具有良好的相关性。这种构建概念似乎在其他哺乳动物和非哺乳动物的核苷酸糖转运体中也得到了保留。此外，对产生的突变体的功能分析表明，插入或非常接近膜跨越区域的插入使运输过程失活，而亲水环结构中的插入对活性没有可检测到的影响。这项研究为理解核苷酸糖转运体的结构-功能关系指明了方向。
Nucleotide sugar transporters form a family of distantly related membrane proteins of the Golgis apparatus and the endoplasmic reticulum. The first transporter sequences have been identified within the last 2 years. However, information about the secondary and tertiary structure for these molecules has been limited to theoretical considerations. In the present study, an epitope-insertion approach was used to investigate the membrane topology of the CMP-sialic acid transporter. Immunofluorescence studies were carried out to analyze the orientation of the introduced epitopes in semipermeabilized cells. Both an amino-terminally introduced FLAG sequence and a carboxyl-terminal hemagglutinin tag were found to be oriented toward the cytosol. Results obtained with CMP sialic acid transporter variants that contained the hemagglutinin epitope in potential intermembrane loop structures were in good correlation with the presence of 10 transmembrane regions. This building concept seems to be preserved also in other mammalian and nonmammalian nucleotide sugar transporters. Moreover, the functional analysis of the generated mutants demonstrated that insertions in or very close to membrane-spanning regions inactivate the transport process, whereas those in hydrophilic loop structures have no detectable effect on the activity. This study points the way toward understanding structure-function relationships of nucleotide sugar transporters.