Cysteine residues in the Na+/dicarboxylate co-transporter, NaDC-1

The role of cysteine residues in the Na+/dicarboxylate cotransporter (NaDC- 1) was tested using site-directed mutagenesis. The transport activity of Na DC-1 was not affected by mutagenesis of any of the 11 cysteine residues, in dicating that no individual cysteine residue is necessary for function. NaD C-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located i n transmembrane domain 9. Although cysteine residues are not required for f unction in NaDC-1, their presence appears to be important for protein stabi lity or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the t ransport activity and expression of NaDC-1. The results indicate that mutag enesis of multiple cysteine residues in NaDC-1 may alter the shape or confi guration of the protein, leading to alterations in protein trafficking or s tability.