1 research outputs found
Increased Redox-Sensitive Green Fluorescent Protein Reduction Potential in the Endoplasmic Reticulum following Glutathione-Mediated Dimerization
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 <i>in vivo</i> 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 <i>in vitro</i> suggests a range of ER
redox potentials consistent with those determined by earlier approaches
that did not involve fluorescent sensors