The Mitochondria-to-Cytosol H2O2 Gradient Is Caused by Peroxiredoxin-Dependent Cytosolic Scavenging

Abstract

Fluorescent protein-based reporters used to measure intracellular H2O2 were developed to overcome the limitations of small permeable dyes. The two major families of genetically encoded redox reporters are the reduction-oxidation sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins and HyPer and derivatives. We have used the most sensitive probes of each family, roGFP2-Tpx1.C169S and HyPer7, to monitor steady-state and fluctuating levels of peroxides in fission yeast. While both are able to monitor the nanomolar fluctuations of intracellular H2O2, the former is two-five times more sensitive than HyPer7, and roGFP2-Tpx1.C169S is partially oxidized in the cytosol of wild-type cells while HyPer7 is fully reduced. We have successfully expressed HyPer7 in the mitochondrial matrix, and it is ~40% oxidized, suggesting higher steady-state levels of peroxides, in the low micromolar range, than in the cytosol. Cytosolic HyPer7 can detect negligible H2O2 in the cytosol from mitochondrial origin unless the main H2O2 scavenger, the cytosolic peroxiredoxin Tpx1, is absent, while mitochondrial HyPer7 is oxidized to the same extent in wild-type and ∆tpx1 cells. We conclude that there is a bidirectional flux of H2O2 across the matrix and the cytosol, but Tpx1 rapidly and efficiently scavenges mitochondrial-generated peroxides and stops their steady-state cytosolic levels rising.

Resistance to H2O2-induced oxidative stress in human cells of different phenotypes

“…We found that the cytoplasmic HyPer sensor is fully reduced in cells with normal metabolism suspended in a complete growth medium. These observations are consistent with the already published research [ 22 ] but differ from our previously obtained data related to K-562 cells suspended in PBS [ 12 ]. According to [ 12 ], HyPer is slightly oxidized (∼10%) under such conditions.…”

“…(2) ) that the sensor can be fully oxidized only under the condition of , i.e. at complete oxidation of thiol-disulfide exchange enzymes which are responsible for the HyPer redox-active domain reduction (presumably, thioredoxin system [ 10 , 22 , 23 ]). Correspondingly, we suggested that it is the functional status of the thioredoxin-depended enzymatic system that drives the dependence of the H 2 O 2 gradient on the oxidative load.…”

Antagonistic effects of mitochondrial matrix and intermembrane space proteases on yeast aging

“…To uncover the consequences of these alterations of the OXPHOS system, we measured H 2 O 2 abundance at the mitochondrial matrix by expressing the peroxide reporter HyPer7 at this subcellular compartment and determining its oxidation levels [ 27 , 28 ]. The percentage of oxidation of this reporter depends on the steady-state H 2 O 2 levels at the matrix, being around 40% (OxD 0 of 0.4) in wild-type cells and increasing up to 60% when the main H 2 O 2 scavenger, Tpx1, is missing [ 28 ]. As shown in Fig.…”

“…A detailed description of plasmid construction and the experimental procedure has been published [ 28 ]. Briefly, plasmid p730 carrying the reporter HyPer7 with the MTS (mitochondrial targeting sequence) of Aco1 was transformed in the mitochondrial protease mutants.…”

“…In two wells, final concentrations of 50 mM dithiothreitol (DTT) and 1 mM of H 2 O 2 were added as controls of fully reduced and oxidized reporter, respectively. A formula described in [ 28 ] was applied to determine the degree of oxidation of the H 2 O 2 sensor MTS-HyPer7. For each strain, we grew cultures of the wild-type background 972 and performed the same treatments on 96-well plates; after recording, we subtracted the fluorescence values of the wild-type strain to those of the strain expressing the reporter.…”