Increased redox-sensitive green fluorescent protein reduction potential in the endoplasmic reticulum following glutathione-mediated dimerization

Deboleena Dipak Sarkar, Sarah K. Edwards, Justin A. Mauser, Allen M. Suarez, Maxwell A. Serowoky, Natalie L. Hudok, Phylicia L. Hudok, Martha Nuñez, Craig S. Weber, Ron Lynch, Osamu Miyashita, Tsu Shuen Tsao

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

As the endoplasmic reticulum (ER) is the compartment where disulfide bridges in secreted and cell surface proteins are formed, the disturbance of its redox state has profound consequences, yet regulation of ER redox potential remains poorly understood. To monitor the ER redox state in live cells, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. One particular green fluorescent protein (GFP)-based redox sensor, roGFP1-iL, could detect oxidizing changes in the ER despite having a reduction potential significantly lower than that previously reported for the ER. We have confirmed these observations and determined the mechanisms by which roGFP1-iL detects oxidizing changes. First, glutathione mediates the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitates dimerization of roGFP1-iL, which shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule's reduction potential compared with that of a dithiol redox buffer. We conclude that the glutathione redox couple in the ER significantly increased the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in the ER with a high degree of reliability in real time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggests a range of ER redox potentials consistent with those determined by earlier approaches that did not involve fluorescent sensors.

Original languageEnglish (US)
Pages (from-to)3332-3345
Number of pages14
JournalBiochemistry
Volume52
Issue number19
DOIs
StatePublished - May 14 2013

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Dimerization
Green Fluorescent Proteins
Endoplasmic Reticulum
Oxidation-Reduction
Glutathione
Sensors
Disulfides
Dimers
Buffers
Monomers
Fluorescence
Dynamic response
Membrane Proteins
Molecules
Monitoring

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sarkar, D. D., Edwards, S. K., Mauser, J. A., Suarez, A. M., Serowoky, M. A., Hudok, N. L., ... Tsao, T. S. (2013). Increased redox-sensitive green fluorescent protein reduction potential in the endoplasmic reticulum following glutathione-mediated dimerization. Biochemistry, 52(19), 3332-3345. https://doi.org/10.1021/bi400052u

Increased redox-sensitive green fluorescent protein reduction potential in the endoplasmic reticulum following glutathione-mediated dimerization. / Sarkar, Deboleena Dipak; Edwards, Sarah K.; Mauser, Justin A.; Suarez, Allen M.; Serowoky, Maxwell A.; Hudok, Natalie L.; Hudok, Phylicia L.; Nuñez, Martha; Weber, Craig S.; Lynch, Ron; Miyashita, Osamu; Tsao, Tsu Shuen.

In: Biochemistry, Vol. 52, No. 19, 14.05.2013, p. 3332-3345.

Research output: Contribution to journalArticle

Sarkar, DD, Edwards, SK, Mauser, JA, Suarez, AM, Serowoky, MA, Hudok, NL, Hudok, PL, Nuñez, M, Weber, CS, Lynch, R, Miyashita, O & Tsao, TS 2013, 'Increased redox-sensitive green fluorescent protein reduction potential in the endoplasmic reticulum following glutathione-mediated dimerization', Biochemistry, vol. 52, no. 19, pp. 3332-3345. https://doi.org/10.1021/bi400052u
Sarkar, Deboleena Dipak ; Edwards, Sarah K. ; Mauser, Justin A. ; Suarez, Allen M. ; Serowoky, Maxwell A. ; Hudok, Natalie L. ; Hudok, Phylicia L. ; Nuñez, Martha ; Weber, Craig S. ; Lynch, Ron ; Miyashita, Osamu ; Tsao, Tsu Shuen. / Increased redox-sensitive green fluorescent protein reduction potential in the endoplasmic reticulum following glutathione-mediated dimerization. In: Biochemistry. 2013 ; Vol. 52, No. 19. pp. 3332-3345.
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AU - Suarez, Allen M.

AU - Serowoky, Maxwell A.

AU - Hudok, Natalie L.

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AU - Lynch, Ron

AU - Miyashita, Osamu

AU - Tsao, Tsu Shuen

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