THE SUBSTRATE RECOGNITION DOMAIN IN THE NA+ DICARBOXYLATE AND NA+/SULFATE COTRANSPORTERS IS LOCATED IN THE CARBOXY-TERMINAL PORTION OF THE PROTEIN/

The Na+/dicarboxylate cotransporter, NaDC-1, and the Na+/sulfate cotra nsporter, NaSi-1, share 43% sequence identity, but they exhibit no ove rlap in substrate specificity. A functional chimera, SiDC-4, was prepa red from NaDC-1 and NaSi-1 by homologous recombination and expressed i n 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 specific ity 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 sti mulation of transport by lithium, differed from both parental transpor ters, suggesting that cation binding is determined by interactions bet ween the amino-and carboxy-terminal portions of the protein. We conclu de that the substrate recognition site of NaDC-1 and NaSi-1 is found i n the carboxy-terminal portion of the protein, past amino acid 141, bu t residues in the amino terminus can affect substrate affinity, inhibi tor sensitivity, and cation selectivity. (C) 1998 Elsevier Science B.V .