The zinc-binding protein Hot13 promotes oxidation of the mitochondrial import receptor Mia40

Abstract

A disulphide relay system mediates the import of cysteine-containing proteins into the intermembrane space of mitochondria. This system consists of two essential proteins, Mia40 and Erv1, which bind to newly imported proteins by disulphide transfer. A third component, Hot13, was proposed to be important in the biogenesis of cysteine-rich proteins of the intermembrane space, but the molecular function of Hot13 remained unclear. Here, we show that Hot13, a conserved zinc-binding protein, interacts functionally and physically with the import receptor Mia40. It improves the Erv1-dependent oxidation of Mia40 both in vivo and in vitro. As a consequence, in mutants lacking Hot13, the import of substrates of Mia40 is impaired, particularly in the presence of zinc ions. In mitochondria as well as in vitro, Hot13 can be functionally replaced by zinc-binding chelators. We propose that Hot13 maintains Mia40 in a zinc-free state, thereby facilitating its efficient oxidation by Erv1.

Figure 1

Hot13 proteins form a ubiquitous protein family conserved among fungi, plants and animals. (A) Alignment of the conserved domain of Hot13 proteins. Cysteine and histidine residues are highlighted in black and conserved residues are shown in grey. The consensus line shows residues that are invariant among all Hot13 proteins. The proteins used here are: Saccharomyces cerevisiae (Hot13, AAS56591), Candida albicans (EAL02591), Schizosaccharomyces pombe (NP_596663), Neurospora crassa (EAA28094), Aspergillus nidulans (XP_001400209), Dictyostelium discoideum (EAL72440), Paramecium tetraurelia (XP_001445542), Danio rerio (AAH78283), Rattus norvegicus (AAH83739), Homo sapiens (AAH47393), Arabidopsis thaliana (BAE98773), Oryza sativum (NP_001050105) and Staphylococcus haemolyticus (YP_254069). The last protein sequence belongs to a Gram-positive bacterium and homologues of Hot13 are widely distributed in prokaryotes. (B) Model of zinc-ion coordination by conserved cysteine and histidine residues within Hot13 (pfam05495; Marchler-Bauer et al, 2007). (C) Zinc content in recombinant MBP-Hot13 and MBP as measured by inductively coupled plasma atomic emission spectroscopy. (D) Radiolabelled Hot13 was synthesized in reticulocyte lysate and pretreated in the absence or presence of 5 mM EDTA, 1 mM o-phenanthroline or 10 μM zinc acetate. Trypsin was added at the indicated concentrations for 30 min on ice. Hot13 was visualized by autoradiography. MBP, maltose-binding protein.

Figure 2

Hot13 modulates the redox state of Mia40. (A) Proteins in 100 μg of wild-type (wt) and Δhot13 mitochondria were resolved on a non-reducing SDS gel. Steady-state levels of the matrix protein fumarase, of the inner membrane protein Mia40, and the intermembrane space (IMS) proteins Erv1, Sod1, Cox17 and Tim13 were detected by western blotting. (B) Mitochondria were incubated with various concentrations of glutathione (GSH) for 10 min before free thiols were trapped with N-ethylmaleimide. The redox state of Mia40 was analysed by non-reducing SDS–PAGE. (C) Wild-type, Δhot13 and GAL-ERV1 cells were grown to log phase. Equal amounts of cells were spread onto glucose-containing plates. Filter discs were placed onto the cell lawn that was soaked with 10 μl of 3 M DTT, 500 mM diamide or 10 M hydrogen peroxide, respectively. The plates were grown at 30°C for 2 days. (D) Oxidized cytochrome c (40 μM) was incubated with DTT (2 mM) and recombinant Erv1 (10 μM) in a cuvette in the presence or absence of 10 μM Hot13. The reduction of cytochrome c was monitored over time by absorbance spectroscopy at 550 nm. DTT, dithiothreitol; SDS–PAGE, SDS–polyacrylamide gel electrophoresis.

Figure 3

Hot13 promotes oxidation of Mia40 in a direct and metal-dependent manner. (A) Mitochondria (500 μg) expressing untagged or octahistidinyl-tagged variants of Hot13 and of Erv1 were lysed with 1% Triton X-100, 300 mM NaCl, 10 mM imidazol, 1 mM phenylmethanesulphonyl fluoride and 50 mM sodium phosphate, pH 8.0. For the samples shown in the right panel, 10 mM β-mercaptoethanol was added to the extract to reduce disulphide bonds. The extract was cleared by centrifugation and applied to nickel affinity chromatography. After extensive washing, bound proteins were eluted and analysed by western blotting for the presence of Mia40. Western blots of the intermembrane space (IMS) protein cytochrome c peroxidase (CCPO) and of the matrix protein Mba1 acted as controls. Ten per cent of the extract used for the His₈-Hot13 pulldown was loaded for control (T, total). The asterisk indicates mixed disulphides of Mia40 and Erv1 due to incomplete reduction by the SDS sample buffer. (B) Radiolabelled Hot13 was incubated with mitoplasts from a GAL-MIA40 strain grown either in the presence of galactose (Mia40↑) or glucose (Mia40↓). The mitoplasts were washed, and bound Hot13 protein was detected by autoradiography. (C) Wild-type and Δhot13 mitoplasts were incubated with 67 μM recombinant MBP-Hot13 or 25 mM EDTA and 10 mM o-phenanthroline (o-Phe) in the presence or absence of 100 μM zinc acetate (ZnAc) for 10 min at 25°C. Free thiols were trapped and the redox state of Mia40 was determined. (D) The influence of glutathione (GSH) on the redox state of Mia40 was assessed as described in Fig 2B with or without 25 mM EDTA and 10 mM o-Phe. (E) The graph shows the mean values and standard deviations of four independent glutathione titration experiments. MBP, maltose-binding protein.

Figure 4

Hot13 suppresses the inhibitory effect of zinc ions on the Erv1-mediated oxidation of Mia40. (A) The carboxy-terminal domain of Mia40 (Mia40C) was purified in its oxidized (Oxid.) form. The protein was reduced by incubation with 10 mM DTT and further incubated without (−Zn) or with (+Zn) 50 μM zinc acetate for 20 min at 25°C. DTT and free zinc ions were removed by gel filtration. Samples shown in lanes 5–12 were further incubated with purified Erv1 at 25°C for the time points indicated. In all samples, free thiol groups were derivatized with 15 mM 4-acetamido-4′-maleimidylstilbene-2,2′-disulphonic acid (AMS). Proteins were stained with Coomassie blue. (B) Mia40C was reduced and incubated with zinc as in (A) and re-isolated by gel filtration. It was further incubated in the presence or absence of 1 mM EDTA or purified maltose-binding protein (MBP)-Hot13. Erv1 was added at the indicated time points. All samples were treated with AMS and analysed as in (A). The concentrations of Mia40C, Hot13 and Erv1 were 50, 50 and 7 μM, respectively. DTT, dithiothreitol.

Figure 5

The import of purified Cox17 shifts Mia40 into its reduced conformation. (A) Recombinant Cox17 was expressed in the presence of ³⁵S-sulphate and purified. Cox17 (12.5 pmol) was incubated with isolated wild-type or Δhot13 mitochondria in the presence of 14 mM glutathione and the indicated zinc concentrations at 25°C. After 10 min, the mitochondria were treated with proteinase K to remove non-imported protein, re-isolated, washed and subjected to SDS–PAGE. For a control, 10% of the Cox17 used per import reaction was loaded in lane 1. (B) The experiment shown in (A) was repeated four times. The radioactive signals were quantified and expressed in relation to the total Cox17 protein added per reaction. The mean values are shown. (C) Recombinant Cox17 was imported into mitochondria in the presence of 0.5 mM zinc and increasing concentrations of glutathione. After autoradiography, the imported protein was quantified and expressed in comparison with the amount of protein imported in the absence of glutathione. (D) Recombinant Cox17 was expressed in the presence of 200 μM zinc acetate and purified. The protein was incubated with mitochondria or mitoplasts from wild-type or Erv1-depleted yeast strains for 10 min. For a control, the samples were incubated with elution buffer. Samples were analysed as in Fig 2B. (E) Model for the influence of metal ions on protein import into the IMS. Imported proteins can be associated with zinc ions. These are passed through Mia40 to Hot13 and further to an unknown acceptor. Hot13-mediated demetalation of Mia40 presumably improves the reoxidation of the import receptor by Erv1. IMS, intermembrane space; SDS–PAGE, SDS–polyacrylamide gel electrophoresis.