Vulnerability of the cysteine-less proton-coupled folate transporter (PCFT-SLC46A1) to mutational stress associated with the substituted cysteine accessibility method

► The highly conserved Lys 422 is fully replaceable in wild-type PCFT. ► K422C and K422E mutants are inactive in the functional Cys-less PCFT. ► The K422C in Cys-less PCFT loses activity due to decreased protein expression. ► Additions of Cys residues cumulatively restore K422C PCFT expression and activity.