Fig. 2.
FAM136A becomes oxidized by MIA40 during its maturation
A. Direct redox shift assay to assess redox state of FAM136A. Cells were lysed and treated with the maleimide mmPEG12 that modifies free thiols (reduced, red.) but not thiols in disulfide bonds (oxidized, ox.). Modification of proteins with mmPEG12 results in a slower migration of the protein on SDS-PAGE.
B. Redox state analysis of FAM136A as described in (A.). To test for the redox state of FAM136A, cells were lysed and either treated with the strong reductant TCEP (lanes 2 and 4) or left untreated (lane 1 and 3). Then lysates were left untreated (lanes 1 and 2) or incubated with mmPEG12 (lanes 3 and 4). Lysates were analysed by SDS-PAGE and immunoblotting. Signals were quantified using Image Lab, and the amount of reduced and oxidized protein was plotted. FAM136A-HA is mainly present in the oxidized state. Virtually all of the protein is oxidized at steady state. Black and white circles represent the indicated treatment and not‐treated samples. N = 3 replicates.
C.-E. Assessment of the MIA40-FAM136A interaction. FAM136A-HA (C.,D.) or MIA40-Step (E) were precipitated (immunoprecipitation with HA-beads [IP] or affinity precipitation using streptactin beads [AP]) after stopping thiol-disulfide exchange reactions by NEM incubation from HEK293 cells stably and inducibly expressing these proteins. Precipitates were tested for MIA40 (C.,D.) or FAM136A (E.). Indicated amounts of the total lysate were loaded as input control. FAM136A coprecipitates MIA40 and vice versa. For the FAM136A-HA IP, this coprecipitation was also present when cells were lysed under denaturing conditions (D.) indicating a covalent interaction between both proteins. In (C.-E.), CPOX served as negative control, while in (E.), AIFM1 served as positive control. Black and white circles represent the presence of the indicated protein.
F. Duration of the FAM136A–MIA40 interaction. HEK293 cells stably expressing FAM136A‐HA were treated for 4 h with the translation inhibitor cycloheximide (CHX). Then, cells were lysed under native conditions, and FAM136A‐HA was precipitated. Precipitates were analysed by reducing SDS–PAGE and immunoblotting. MIA40 and FAM136A interact transiently with each other comparable to other MIA40 substrates. CPOX served as negative control.
G. Scheme of assay to assess MIA40-FAM136A interaction in vitro. GST‐tagged variants of MIA40 (wild‐type, WT, redox‐inactive C4, 53, 55S (SPS) and chaperone‐inactive F68E (FE) and GST alone were purified and bound to beads. The beads were incubated with in vitro translated radioactive FAM136A. Subsequently, beads were washed and analysed for bound FAM136A by SDS‐PAGE and autoradiography. Incubation with GST-only bound beads served as control.
H. In vitro assessment of MIA40-FAM136A interaction. WT MIA40 (lane 2) interacts with FAM136A. By comparison, the interaction between FAM136A and the MIA40C4, 53, 53S (lane 3) or the MIA40 F68E variants (lane 4) were significantly decreased (one-way ANOVA with post hoc Tukey HSD test). The control (GST-only bound beads) revealed only minor background binding (lane 1). 5 % of the total lysate was loaded as input control (lane 5). TCE staining served as loading control. For quantification, the co‐precipitated FAM136A levels were normalized to the levels precipitated by WT MIA40 and in addition normalized to the according MIA40 signal in the TCE staining. Black and white circles represent the indicated treatment and not‐treated samples. N = 3
I. In vitro oxidation kinetics assay of FAM136A. Recombinant expressed and purified human MIA40 WT was added to 35S-labelled FAM136A to allow disulphide exchange reactions. The reaction was stopped by rapid acidification via addition of trichloroacetic acid (TCA). Lysates were treated with mmPEG12 to determine protein redox states, followed by non-reducing SDS-PAGE and autoradiography.
J. Assessment of FAM136A oxidation by purified MIA40 as described in (I). Reduced FAM136A is modified with nine mmPEG12, whereas oxidized FAM136A was modified by only one mmPEG24. In the control experiment, FAM136A did not become oxidized in the absence of MIA40 during the indicated times. Addition of MIA40 to FAM136A resulted in an oxidation of the protein over time. Reduced and oxidized forms as well as semi-oxidized intermediates of FAM136A, and the FAM136A-MIA40 mixed disulphide complex are indicated. N = 3 replicates.