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Fig. 3. | BMC Biology

Fig. 3.

From: The C-terminal region of the oxidoreductase MIA40 stabilizes its cytosolic precursor during mitochondrial import

Fig. 3.

The C-terminal region of MIA40 slows mitochondrial import and concomitantly protects MIA40 from proteasomal degradation. a Steps in the biogenesis of MIA40 involve synthesis of the precursor by cytosolic ribosomes, translocation over the OMM, interaction with AIFM1 for translocation, and oxidation by the disulfide relay. b Interaction between MIA40 variants and AIFM1. HEK293 cells stably expressing different variants of MIA40-HA (24 h doxycycline) were subjected to native immunoprecipitation (IP) against the HA-tag. Precipitates were analyzed by SDS-PAGE and immunoblotting against the indicated proteins. AIFM1 co-precipitates with both, MIA40WT and MIA40Δ108, but not with MIA40Δ40. Blue arrowhead, signal of MIA40Δ108. Quantification using Image lab. Data from 5 experiments were combined and standard deviations are presented. c In organello import assay of MIA40 variants. In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 cells. Non-imported proteins were removed by treatment with Proteinase K. Imported proteins were analyzed by reducing SDS-PAGE and autoradiography. Signals were quantified and the amount of imported protein was plotted. MIA40Δ108 is imported more rapidly than MIA40WT. Quantification using ImageQuantTL. Data from 4 experiments were combined and standard deviations are presented. d In cellulo translocation assay. Synthesis of MIA40 variants was induced 1.5 h before the experiment using 1 μg ml−1 doxycycline. Cells were pulse-labeled for 5 min with 35S-methionine and chased with cold methionine for different times. After the chase, cells were fractionated into a cytosolic and mitochondrial fraction. Both MIA40Δ108 and MIA40WT are imported into mitochondria, but less of MIA40Δ108. Cytosolic MIA40WT is stable in contrast to cytosolic MIA40Δ108. Quantification using ImageQuantTL. Data from 2 experiments were combined and standard deviations are presented. e, f In cellulo oxidation kinetics assay to follow oxidative folding of MIA40 variants and COX19 in intact cells. Synthesis of MIA40 variants was induced 1.5 h before the experiment using 1 μg/ml doxycycline. Cells were pulse-labeled for 5 min with 35S-methionine and chased with cold methionine for different times. The chase was stopped by trichloroacetic acid (TCA) precipitation, and then the lysate was treated with mmPEG12 to determine protein redox states, followed by IP against the HA tag. Eluates were analyzed by Tris-Tricine-PAGE and autoradiography. Reduced proteins were modified with mmPEG12, whereas oxidized proteins remained unmodified. MIA40 becomes oxidized very slow compared to COX19 with half oxidation times in the range of 90 min. Yet, the reduced cytosolic form of MIA40WT is stable over the course of the experiment. Conversely, reduced MIA40Δ108 disappears rapidly. Quantification using ImageQuantTL. Data from 2 to 3 experiments were combined and standard deviations are presented if n > 2. Red., reduced; ox., oxidized; un., unmodified. g, h In cellulo oxidation kinetics assay to follow oxidative folding of MIA40Δ108 in the presence or absence of proteasomal inhibition. As e, except that oxidation kinetics were performed in the presence or absence of MG132. Reduced MIA40Δ108 is strongly stabilized in an import-competent form by MG132. Quantification using ImageQuantTL. Data from 3 experiments were combined (MIA40WT, Additional file 3: Figure S3B) and standard deviations are presented. Red., reduced; ox., oxidized; un., unmodified

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