Abstract
Oocytes are postulated to repress the proton pumps (e.g., complex IV) and ATP synthase to safeguard mitochondrial DNA homoplasmy by curtailing superoxide production. Whether the ATP synthase is inhibited is, however, unknown. Here we show that: oligomycin sensitive ATP synthase activity is significantly greater (~170 vs. 20 nmol/min−1/mg−1) in testes compared to oocytes in Xenopus laevis (X. laevis). Since ATP synthase activity is redox regulated, we explored a regulatory role for reversible thiol oxidation. If a protein thiol inhibits the ATP synthase, then constituent subunits must be reversibly oxidised. Catalyst-free trans-cyclooctene 6-methyltetrazine (TCO-Tz) immunocapture coupled to redox affinity blotting reveals several subunits in F1 (e.g., ATP-α-F1) and Fo (e.g., subunit c) are reversibly oxidised. Catalyst-free TCO-Tz Click PEGylation reveals significant (~60%) reversible ATP-α-F1 oxidation at two evolutionary conserved cysteine residues (C244 and C294) in oocytes. TCO-Tz Click PEGylation reveals ~20% of the total thiols in the ATP synthase are substantially oxidised. Chemically reversing thiol oxidation significantly increased oligomycin sensitive ATP synthase activity from ~12 to 100 nmol/min−1/mg−1 in oocytes. We conclude that reversible thiol oxidation inhibits the mitochondrial ATP synthase in X. laevis oocytes.
Highlights
Human sperm rely on oxidative phosphorylation (OXPHOS) to swim 103 times their own length to fertilise an oocyte [1,2]
We conclude that reversible thiol oxidation inhibits the mitochondrial ATP synthase in X. laevis oocytes
F1 -Fo ATP synthase activity should be greater in the soma compared to the female germline
Summary
Human sperm rely on oxidative phosphorylation (OXPHOS) to swim 103 times their own length to fertilise an oocyte [1,2]. Paternal mitochondrial DNA (mtDNA) is purged and/or heavily diluted after fertilisation to ensure maternal inheritance dominates in the embryo [3,4]. Maternal inheritance avoids deleterious mtDNA heteroplasmy because OXPHOS sensitises sperm to oxidative DNA damage [5]. Oxidative DNA damage can occur when thermodynamically and kinetically competent reduced electron donors (e.g., prosthetic semiquinone radicals) catalyse the univalent reduction of ground state molecular dioxygen (O2 ) to superoxide [6,7]. If OXPHOS imperils mtDNA homoplasmy, oocyte mitochondria may repress it to curtail superoxide production
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