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.
Highlights
Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, C/ Dr Aiguader 88, Laboratory of Experimental Oncology, Pirogov Russian National Research Medical University, Department of Metabolism and Redox Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia
To quantitatively monitor the sensitivity of HyPer7 to H2 O2 fluctuations, we expressed it in the cytosol of S. pombe cells under the control of the constitutive sty1 promoter, as previously described for the original HyPer and for roGFP2-Tpx1.C169S
Using biochemical approaches [25], as well as the basal oxidation levels of cytosolic roGFP2-Tpx1.C169S as a readout of the steady–state levels of H2 O2 [26], we demonstrated that cells lacking Tpx1 display toxic concentrations of peroxides in the cytosol in the range of 0.2–0.3 μM
Summary
We have used the most sensitive probes of each family, roGFP2-Tpx.C169S and HyPer, to monitor steady-state and fluctuating levels of peroxides in fission yeast. While both are able to monitor the nanomolar fluctuations of intracellular. Generation and scavenging of H2 O2 have been a matter of study for many decades, as well as the fluxes of peroxides between different cellular compartments Both electron transport chain (ETC) components and other mitochondrial enzymes, such as 2-oxoglutarate dehydrogenase, pyruvate dehydrogenase, and the mitochondrial membrane forms of glycerol 3-phosphate dehydrogenase, contribute to the formation, directly or indirectly, of H2 O2 (for a review, see [5]). A wide range of published maps and institutional affiliations
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