Fig. 3: Retrotranslocation of MAD substrates is ATP-dependent and membrane potential-independent. | Communications Biology

Fig. 3: Retrotranslocation of MAD substrates is ATP-dependent and membrane potential-independent.

From: Tom40 functions as a channel for protein retrotranslocation in the mitochondria-associated degradation (MAD) pathway

Fig. 3: Retrotranslocation of MAD substrates is ATP-dependent and membrane potential-independent.The alt text for this image may have been generated using AI.

A, D Representative western blots of mitochondrial pellet after releasing (Pellet) and released proteins (Released) from isolated mitochondria from WT (+) and doa1Δ (−) cells in the SUB592 background in the absence or presence of 2 mM ATP and NADH, or 5 µg/ml valinomycin. Release was assessed after incubation of isolated mitochondria for 30 min at 30 °C. The blot was probed with antibodies against GFP for detection of Kgd1p (A) or Pim1p (D), and antibodies against Tom40p and Cyb2p. Total protein load was assessed using TCE. B, E Quantification of Kgd1p (B) and Pim1p (E) in mitochondria in (A) and (D), respectively. C, F Quantification of Kgd1p (C) and Pim1p (F) released from isolated mitochondria in (A) and (D), respectively (n = 4 in (B and C) and n = 3 in (E and F), 1-way ANOVA with Sidak’s multiple comparison test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns no significance). All signals were first normalized to pellet Cyb2p signals and then normalized to WT (+).

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