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.

Residues in the eighth transmembrane domain of the proton-coupled folate transporter (SLC46A1) play an important role in defining the aqueous translocation pathway and in folate substrate binding

“…In this construct, serine was substituted for Cys66 and Cys298 and an HA (hemagglutinin) tag was added to the C-terminus. This template eliminates the disulfide bond and reaction with a sulfhydryl-reacting reagent should the procedure require pretreatment with dithiothreitol to remove a modifying molecule that interferes with biotinylation and function (13,14). This results in only a negligible decrease in function (13,14).…”

“…The topology of the transporter has been defined as well as residues that play a role in proton-coupling, proton binding, the affinity of the carrier for its folate substrates and the rate of oscillation of the carrier among its conformational states (4,7–13). Recently, transmembrane helices were identified that participate in the formation of a gate at the external interface of the aqueous pathway (14).…”

Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

“…5,6 The topology of this transporter has been defined and is typical of the solute carrier (SLC) superfamily of transporters with 12 transmembrane helices, 6 on either side of a large internal loop, with N-and C-termini located within the cytoplasm. [7][8][9][10] The exofacial regions at the membrane-aqueous interface of transmembrane helices 1, 2, 7, and 11 were recently shown to come together to form a gate at the extracellular entrance to the aqueous translocation pathway, 11 as has been reported for other solute transporters. 12,13 The proximity of residues in these helices was established by crosslinkages between cysteine-substituted residues within this region of the gate.…”

“…18,19 Analysis of PCFT protein expression at the cell surface Cell surface expression was determined by biotinylation of accessible PCFT lysine residues with EZ-Link sulfo-NHS-LC, as has been previously described in detail. 10,11 Briefly, washed cells were treated with 1 mg/mL of EZ-Link sulfo-NHS-LC for 30 min at room temperature (RT). All subsequent steps were at 4°C.…”

“…Protein samples were resolved on 4% to 12% polyacrylamide gels (BioRad), electroblotted onto polyvinylidine difluoride membranes (Millipore), and followed by blocking with 10% nonfat milk in Tris-buffered saline, as has been previously described. 10,11 The blots were probed with anti-HA primary antibody (Sigma, St. Louis, MO) at RT, followed by treatment with HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA). Actin, the internal loading control, was probed with monoclonal anti-b-actin antibody (Sigma, St. Louis, MO) and HRP conjugated anti-mouse antibody (Cell Signaling Technology, Danvers, MA).…”