Rescuing Proper Trafficking of Cysteine Mutant Proteins

Abstract

Cysteine substitution has been widely used in structure and function studies, but sometimes mutant proteins get retained during trafficking and the cell is unable to deliver full-length membrane proteins to the cell surface. Similarly, in many inheritable genetic diseases cysteine mutant proteins encounter the same fate. In the visual system, for example, some cysteine mutants of cyclic nucleotide-gated channel (CNG channels) are retained in the ER, leading to achromatopsia (color blindness). We reasoned that it should be possible to modify the chemical structure of the mutation in order to mimic the side chain of the wild type amino acid and recover proper trafficking. As proof of principle, we have studied two naturally occurring cysteine mutants (Y181C and R277C in CNGA3). These mutations are responsible for hereditary cone photoreceptor disorders. We introduced both achromatopsia-related cysteines in a cysteine-less CNG channel, and used them as targets for specific chemical modification with hydroxybenzyl- (MTSHB) and aminoethyl-methanethiosulfonate (MTSEA). These reagents readily attach to the side chain of cysteines and mimic the chemistry of tyrosine and arginine, respectively. Cell surface expression was assayed in Xenopus oocytes using fluorescence microscopy and electrophysiology. We successfully restored trafficking and normal function to CNG mutant channels R→C and Y→C, as well as three more cysteine mutants within the S4 transmembrane segment that are known to be retained in the ER. This chemical method provides a unique opportunity to functionally characterize previously inaccessible proteins, as well as it can be readily implemented to assess the chemical nature of misfolding problems, conformational dynamics of folding processes or to study protein conformational changes at the site of the cysteine mutation.