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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.
Figure 1. Oligomycin sensitive F1-Fo ATP synthase activity is significantly greater in testes compared to oocytes. (A) The F1-Fo ATP synthase hydrolysis ATP to ADP. Pyruvate kinase regenerates ATP by using phosphoenolpyruvate (PEP) to phosphorylate ADP to ATP. Lactate dehydrogenase reduces pyruvate to lactate using NADH derived electrons. F1-Fo ATP synthase activity is followed by monitoring the loss of NADH absorbance at 340 nm. (B). Oligomycin sensitive F1-Fo ATP synthase activity is higher significantly (p ≤ 0.0001) in testes (n = 6) compared to oocytes (n = 6) in X. laevis. Statistical significance is indicated by an asterix as assessed by an independent Student’s t-test. (C). Native ATP-α-F1 blot image showing the F1-Fo ATP synthase is fully assembled in X. laevis oocytes (n = 4). A minor fraction is present as an F1 subcomplex. Each n is the weighted mean of 10 oocytes.
Figure 2. Cysteine residues in bovine Fo-F1 ATP synthase in catalytic state 3A. Numbered cysteine residues are highlighted in yellow. The key lists the cysteine residue by amino acid number for the bovine enzyme with the equivalent X. laevis residue in brackets in bold. A coloured asterix denotes the predicted position of additional cysteine residues in X. laevis. Additional cysteine residues are highlighted in bold in the key if they are likely to be solvent exposed. No structural data is available for subunit g and C3.
Figure 3. Several F1-Fo ATP synthase subunits are reversibly oxidised. (A). Catalyst-free trans-cyclooctene-6methyltetrazine (TCO-Tz) immunocapture coupled to redox affinity blotting workflow. From left to right: Primary amines in the ATP-α-F1 antibody are labelled with a heterobifunctional NHS-PEG4-TCO linker. After excess NHS is quenched with Tris (not shown), the labelled antibody is incubated with Biotin functionalised maleimide labelled reversibly oxidised thiols in mitochondrial membranes to capture the F1-Fo ATP synthase. Agarose beads substituted with 6-methyltetrazine are then used to selectively capture the antibody-synthase complex. After washing away contaminants with a spin cup, samples are boiled, denatured, and reduced to elute subunits for streptavidin blotting. Streptavidin, conjugated Alexa Fluor™ 647 positive bands denote reversibly oxidised subunits. (B). A predicted reversibly oxidised subunit profile based on Table 1. (C). Representative image of an experimentally observed reversibly oxidised subunit profile alongside a molecular weight (MW) ladder. Arrows indicate the predicted identity of the observed bands. The image shows several F1-Fo ATP synthase subunits are reversibly oxidised. An unpredicted band at 100 kDa was observed (see main text). Clickable TCO-Tz immunocapture coupled to redox affinity blotting was performed on five pools of 10 X. laevis oocytes. Each lane represents the weighted mean of 10 oocytes.
Figure 4. Reversible ATP-α-F1 oxidation is significant in oocytes. (A). Catalyst-free trans-cyclooctene-6methyltetrazine (TCO-Tz) Click PEGylation scheme for reversibly oxidised thiols. Left to right: Reduced thiols are alkylated with NEM. Reversibly oxidised thiols are reduced with TCEP before being alkylated with TCO-PEG3-NEM (TPN). TPN labelled thiols are incubated with Tz-PEG5 to initiate the catalyst-free IEDDA Click reaction. Reversibly oxidised thiols are then mass shifted when assessed by Western Blot owing to a PEG induced electrophoretic mobility shift. (B). Western blot image showing reversibly oxidised (i.e., mass shifted 5 and 10 kDa bands) relative to reduced ATP-α-F1 (i.e., lower band) in X. laevis oocytes (n = 5). MW = molecular weight. (C). Percent reversibly oxidised (i.e., mass shifted) compared to reduced (unshifted) ATP-α-F1 quantified. Percent reversibly oxidised ATP-α-F1 is significantly (p = 0.0007) greater than the amount of reduced ATP-α-F1. An asterix denotes statistical significance as assessed by a paired Student’s t-test. (D). Quantified percentage contribution of the 5 and 10 kDa bands to the total mass shifted (i.e., reversibly oxidised) signal. No significant difference (p = 0.09611) in the contribution of the 5 and 10 kDa band signal was observed as assessed by a paired Student’s t-test. Each n is the weighted mean of 10 oocytes.
Figure 5. Reversible thiol oxidation inhibits the F1-Fo ATP synthase. (A). Chemically reversing thiol oxidation using TCEP substantially increases F1-Fo ATP synthase activity. Specifically, oligomycin sensitive F1-Fo ATP synthase activity is significantly greater (p = 0.0007) in TCEP (n = 6) compared to control oocytes (n = 6) in X. laevis. (B). Inverted Hr-CNPAGE image of F1-Fo ATP synthase mediated in-gel ATP hydrolysis. No signal is observed in oligomycin treated controls and a decreased signal in the TCEP condition. (C). Densitometry based quantification reveals a significant (p = 0.0067) increased F1-Fo ATP synthase mediated ATP hydrolysis in TCEP (n = 3) compared to control oocytes (n = 3) in X. laevis. Statistical significance is indicated by an asterix as assed by an independent Studentâs t-test. Each n is the weighted mean of 10 oocytes.
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