Role of conserved prolines in the structure and function of the Na+/dicarboxylate cotransporter 1, NaDC1

The Na+/dicarboxylate cotransporter 1 (NaDC1) is a low-affinity transporter for citric acid cycle intermediates such as succinate and citrate. The sequence of NaDC1 contains a number of conserved proline residues in predicted transmembrane helices (TMs) 7 and 10. These transmembrane domains are of particular importance because they may be involved in determining the substrate or cation-binding affinity in NaDC1. Four conserved proline residues in TMs 7 and 10 of rabbit NaDC1 were replaced with alanine to promote ideal alpha helix or glycine to promote free conformation, and the mutant transporters were expressed in the HRPE cell line. Mutations of prolines in TM 10 produced decreased protein expression and activity, whereas mutations of prolines in TM 7 completely abolished protein expression and activity. The chemical chaperone glycerol was found to improve the expression of the Pro-351 mutants in TM 7, suggesting that these mutants had defects in trafficking. The inactive mutant transporters at position 351 could also be rescued by the addition of a proline at a second site. For example, the P351A-F347P mutant had restored activity, although its substrate specificity was altered. We conclude that, in TM 7, Pro-327 may be of particular importance in the function of the transporter, whereas Pro-351 may affect protein targeting. The prolines in TM 10, at positions 523 and 524, may not be directly involved in the transporter function but may be necessary for maintaining structure.