Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method

Evidence for glutathionylation

Rongbao Zhao, Mitra Najmi, Srinivas Aluri, I. David Goldman

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)C517-C526
JournalAmerican Journal of Physiology - Cell Physiology
Volume312
Issue number4
DOIs
StatePublished - Apr 7 2017

Fingerprint

Post Translational Protein Processing
Cysteine
Sulfhydryl Reagents
Proton-Coupled Folate Transporter
HeLa Cells
Oxidants
Disulfides
Western Blotting
Cell Line

Keywords

  • Glutathionylation
  • PCFT
  • Posttranslational modification
  • Proton-coupled folate transporter
  • SCAM
  • SLC46A1
  • Substituted cysteine accessibility method

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

Cite this

@article{1fb8d551d1be475ea2d947ad1ae2bcf4,
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.",
keywords = "Glutathionylation, PCFT, Posttranslational modification, Proton-coupled folate transporter, SCAM, SLC46A1, Substituted cysteine accessibility method",
author = "Rongbao Zhao and Mitra Najmi and Srinivas Aluri and Goldman, {I. David}",
year = "2017",
month = "4",
day = "7",
doi = "10.1152/ajpcell.00350.2016",
language = "English (US)",
volume = "312",
pages = "C517--C526",
journal = "American Journal of Physiology - Renal Fluid and Electrolyte Physiology",
issn = "1931-857X",
publisher = "American Physiological Society",
number = "4",

}

TY - JOUR

T1 - Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method

T2 - Evidence for glutathionylation

AU - Zhao, Rongbao

AU - Najmi, Mitra

AU - Aluri, Srinivas

AU - Goldman, I. David

PY - 2017/4/7

Y1 - 2017/4/7

N2 - 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.

AB - 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.

KW - Glutathionylation

KW - PCFT

KW - Posttranslational modification

KW - Proton-coupled folate transporter

KW - SCAM

KW - SLC46A1

KW - Substituted cysteine accessibility method

UR - http://www.scopus.com/inward/record.url?scp=85017292912&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85017292912&partnerID=8YFLogxK

U2 - 10.1152/ajpcell.00350.2016

DO - 10.1152/ajpcell.00350.2016

M3 - Article

VL - 312

SP - C517-C526

JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

SN - 1931-857X

IS - 4

ER -