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Fig. 4. | BMC Biology

Fig. 4.

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

Fig. 4.

Bypassing the AIFM1- and disulfide relay-dependent import pathway stabilizes MIA40Δ108. a Steady-state levels of MIA40Δ108 and MIA40WT upon rerouting the MIA40 import pathway. Expression of MIA40 variants with and without the N-terminal bipartite mitochondrial targeting sequence of AIFM1 (MTSAIFM1) was induced in HEK293 cells using 1 μg ml−1 doxycycline for 24 h. MIA40Δ108 is stabilized by MTSAIFM1. N = 2 biological replicates. Black arrowhead, endogenous MIA40; blue arrowhead, signal of MIA40Δ108. b Like a, except that concomitantly to expression (16 h doxycyclin), cells were incubated with 1 μM of the proteasome inhibitor MG132. Then, cells were lysed and analyzed by immunoblotting. MIA40Δ108 cannot be further stabilized by MG132 if it is targeted to mitochondria with the MTSAIFM1. Quantification using Image lab. Data from 4 experiments were combined and standard deviations are presented. Black arrowhead, endogenous MIA40; blue arrowhead, signal of MIA40Δ108. c Steady-state levels of COX19 variants extended by the C-terminal stretch of MIA40. Stable HEK293 cells were generated that either expressed COX19WT-HA or a COX19 lacking its four cysteines (COX194CS-HA), or fusion proteins of these variants that were extended by either the last 35 amino acids of MIA40 (C-MIA) or the stretch of amino acids in which charged residues were mutated to neutral residues (C-neutral). Expression of COX19 variants was induced using 1 μg ml−1 doxycycline for 24 h. Then, cells were lysed and analyzed by immunoblotting. Elongation of COX19 variants increased steady-state levels. The fusion to the endogenous C-terminal stretch of MIA40 thereby had the strongest stabilizing effect. Quantification using Image lab. Data from 5 experiments were combined and standard deviations are presented. Light red arrowhead, COX19WT; dark red arrowhead, COX194CS. d Model. Compared to other disulfide relay substrates, MIA40 follows an AIFM1- and disulfide-relay dependent import pathway. As a consequence, import and oxidation of MIA40 is a comparatively slow process. Thus, the MIA40 precursor needs to be stabilized in the cytosol. The negatively charged C-terminus of MIA40 contributes to this cytosolic stabilization but also appears to delay MIA40 import

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