title: Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method: evidence for glutathionylation.
abstract: The substituted cysteine accessibility method (SCAM) is widely used to study the structure and function of channels, receptors and transporters. In its usual application, a cysteine residue is introduced into a protein which lacks native cysteines following which the accessibility of the residue to the aqueous compartment is assessed. Implicit, and generally assumed, is that if the cysteine-substituted residue is not available to react with sulfhydryl reagents it is not exposed to the extracellular compartment or within the aqueous translocation pathway. We demonstrate here, in a Hela-derived cell line, that some cysteine-substituted residues of the proton-coupled folate transporter (PCFT, SLC46A1) that are inaccessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate are glutathionylated by biotinylated glutathione ethyl ester in the absence of an oxidizing agent. Intramolecular disulfide formation involving cysteine-substituted residues was also identified in some instances. These posttranslational modifications limit the accessibility of the cysteine residues to sulfhydryl-reactive reagents and can have a profound impact on the interpretation of SCAM but may not alter function. When a posttranslationally modified residue is used as a reference extracellular control, the high level of exposure required for detection on Western blot results in erroneous detection of otherwise inaccessible intracellular cysteine-substituted residues. The data indicate that in the application of SCAM, when a cysteine-substituted residue does not appear to be accessible to sulfhydryl-reactive reagents, the possibility of a posttranslational modification should be excluded. The data explain the discrepancies in the assessment, and confirm the localization, of the first intracellular loop of PCFT.
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; and.|Department of Medicine, Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Rongbao
authors.initials: R
authors.lastname: Zhao
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; and.
authors.forename: Mitra
authors.initials: M
authors.lastname: Najmi
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; and.
authors.forename: Srinivas
authors.initials: S
authors.lastname: Aluri
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; and i.david.goldman@einstein.yu.edu.|Department of Medicine, Albert Einstein College of Medicine, Bronx, New York.
authors.forename: I David
authors.initials: ID
authors.lastname: Goldman
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D013329:Structure-Activity Relationship
chemical_list: D005978:Glutathione; D003545:Cysteine
keywords: PCFT SLC46A1; SCAM; glutathionylation; posttranslational modification; proton-coupled folate transporter; substituted cysteine accessibility method