Pajor A M, Sun N, Bai L, Markovich D, Sule P
Department of Physiology, University of Arizona, College of Medicine, Tucson, AZ 85724, USA.
Biochim Biophys Acta. 1998 Mar 6;1370(1):98-106. doi: 10.1016/s0005-2736(97)00249-6.
The Na+/dicarboxylate cotransporter, NaDC-1, and the Na+/sulfate cotransporter, NaSi-1, share 43% sequence identity, but they exhibit no overlap in substrate specificity. A functional chimera, SiDC-4, was prepared from NaDC-1 and NaSi-1 by homologous recombination and expressed in Xenopus oocytes. SiDC-4 contains putative transmembrane domains 1-4 of NaSi-1 (amino acids 1-139) and putative transmembrane domains 5-11 of NaDC-1 (amino acids 141-593). SiDC-4 retains the substrate specificity of NaDC-1, which suggests that the substrate recognition domain is found in the carboxy-terminal portion of the protein, past amino acid 141. However, residues that affect substrate affinity and inhibition by furosemide and flufenamate are found in the amino terminal third of the protein. The cation binding properties of SiDC-4, including a stimulation of transport by lithium, differed from both parental transporters, suggesting that cation binding is determined by interactions between the amino- and carboxy-terminal portions of the protein. We conclude that the substrate recognition site of NaDC-1 and NaSi-1 is found in the carboxy-terminal portion of the protein, past amino acid 141, but residues in the amino terminus can affect substrate affinity, inhibitor sensitivity, and cation selectivity.
钠离子/二羧酸共转运体NaDC-1和钠离子/硫酸盐共转运体NaSi-1的序列一致性为43%，但它们在底物特异性方面没有重叠。通过同源重组从NaDC-1和NaSi-1制备了一种功能性嵌合体SiDC-4，并在非洲爪蟾卵母细胞中表达。SiDC-4包含NaSi-1的推定跨膜结构域1-4（氨基酸1-139）和NaDC-1的推定跨膜结构域5-11（氨基酸141-593）。SiDC-4保留了NaDC-1的底物特异性，这表明底物识别结构域位于蛋白质的羧基末端部分，在氨基酸141之后。然而，影响底物亲和力以及呋塞米和氟苯那酸抑制作用的残基位于蛋白质的氨基末端三分之一处。SiDC-4的阳离子结合特性，包括锂对转运的刺激作用，与两个亲本转运体都不同，这表明阳离子结合是由蛋白质的氨基末端和羧基末端部分之间的相互作用决定的。我们得出结论，NaDC-1和NaSi-1的底物识别位点位于蛋白质的羧基末端部分，在氨基酸141之后，但氨基末端的残基可以影响底物亲和力、抑制剂敏感性和阳离子选择性。
