Fig. 2: A substantial fraction of mitochondrial dGTP is bound to protein.
From: Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit
a Comparison between different dNTP extraction methods. dNTP levels (pmol dNTP/mg of protein) in liver mitochondria extracts prepared with acid (0.6 M PCA or 0.5 M TCA), 60% methanol treatment, followed by centrifugation and then boiling the supernatant (MeOH + C + B), or 60% methanol treatment followed by boiling before centrifugation (MeOH + B + C). dGTP values obtained after MeOH + C + B extraction were significantly different from values obtained by any other tested method. Results are mean values of N independent experiments (symbols): PCA = 3–5; TCA = 4–7; MeOH + C + B = 4–7; MeOH + B + C = 3–5. Error bars represent standard deviation. b Ratio of protein bound to free dNTP fractions in mouse liver (top plot) and brain (bottom plot) mitochondria. Fractions were obtained after treatment of mitochondria with 60% methanol, centrifugation, and complete denaturation of resulting pellets (protein-bound fraction) and supernatants (free fraction) with TCA. N = 5–7 (top plot) and 5–6 (bottom plot) of independent experiments c Electrophoretic mobility shift assay (EMSA) of radiolabeled [α-32P]-dGTP (0.06 pmol) after incubation with 10 µg of native protein lysates from fresh mouse liver mitochondria. Specificity of shifted bands was tested against a 10- to 1,000-fold molar excess of cold dGTP, a 100-fold molar excess of the other cold dNTPs (dATP, dTTP, and dCTP) or different guanine-containing nucleotides (dGDP, dGMP, and GTP). Competitors were incubated with the protein lysate for 30 min prior to the addition of [α-32P]-dGTP (last lane resolves an assay where radiolabeled dGTP was incubated before cold dGTP addition). Black arrows indicate specific dGTP-protein complexes. The white arrow tags unspecific [α-32P]-dGTP binding. d Autoradiography of [α-32P]-dGTP-protein complexes resolved by BN-PAGE. 30 µg of native protein lysates from fresh mouse liver mitochondria were incubated with 1 µM [α-32P]-dGTP (300 Ci/mmol) and resolved by blue native-polyacrylamide gel electrophoresis (BN-PAGE). Competition assays were performed by pre-incubating the lysates with an excess of 10- to 1,000-fold of cold dGTP or 1,000-fold of dATP, dTTP, and dCTP. A white arrow indicates a signal from dNTP-protein complexes (not dGTP-specific). Black arrows indicate specific dGTP-protein complexes as revealed by subsequent autoradiography of dried BN-PAGE gels. Migration of a 669 kDa MW marker is indicated. Image shows representative data of at least 2 independent experiments e Analysis of retardation in electrophoretic mobility of radiolabeled [α-32P]-dGTP (300 Ci/mmol) after incubation with mouse liver mitochondria lysed under native conditions and resolved by BN-PAGE gel. Several competition assays were performed by pre-incubating the lysate with a 100-fold molar excess of different guanine-containing nucleotides (dGTP, dGDP, dGMP, and GTP), dNTPs, rNTPs, and dGuo. Lanes 1–9 were discontinuously loaded in one same gel while lanes 10–13 were loaded in a second gel. Both gels were run simultaneously with the same sample treated with the different competitors. Two-tailed Mann-Whitney U test, *p < 0.05, **p < 0.01, ***p < 0.005.
