Role of twin Cys-Xaa9-Cys motif cysteines in mitochondrial import of the cytochrome C oxidase biogenesis factor Cmc1

Abstract

The Mia40 import pathway facilitates the import and oxidative folding of cysteine-rich protein substrates into the mitochondrial intermembrane space. Here we describe the in vitro and in organello oxidative folding of Cmc1, a twin CX(9)C-containing substrate, which contains an unpaired cysteine. In vitro, Cmc1 can be oxidized by the import receptor Mia40 alone when in excess or at a lower rate by only the sulfhydryl oxidase Erv1. However, physiological and efficient Cmc1 oxidation requires Erv1 and Mia40. Cmc1 forms a stable intermediate with Mia40 and is released from this interaction in the presence of Erv1. The three proteins are shown to form a ternary complex in mitochondria. Our results suggest that this mechanism facilitates efficient formation of multiple disulfides and prevents the formation of non-native disulfide bonds.

FIGURE 1.

Cmc1 is an Mia40-Erv1 pathway import substrate. A, in organello mitochondrial protein import of in vitro synthesized wild-type ³⁵S-labeled precursor Cmc1 into mitochondria isolated from wild-type and permissive temperature grown erv1-101 and mia40-3 strains. The import of Su9-DHFR was followed as Erv1-Mia40-independent import control. p, precursor; m, mature. In the bar graphs, the bands in the right panel were quantified using the histogram function of the Adobe Photoshop program and expressed as percentages of WT values. B and C, reconstitution of Cmc1 oxidative folding following 4 h of incubation and subsequent analysis of AMS-trapped thiols (B) or H₂O₂ production (C, fluorometric Amplex Red assay). D, formation of intermolecular disulfides involving Cmc1, Mia40, and Erv1.

FIGURE 2.

Cmc1 can be fully oxidized in vitro by Mia40 alone. Reconstitution time course with reduced Cmc1 alone or incubated with either 5- (A) or 20-fold (B) excess of Mia40 followed by analysis of AMS-trapped thiols by nonreducing SDS-PAGE.

FIGURE 3.

Cmc1 is an in vitro substrate of Erv1. A, time course of oxidation of reduced Cmc1 by Erv1. B, effect of Mia40 in Cmc1 partially oxidized by Erv1. Reduced Cmc1 was mixed with Erv1 for 4 h. Next, Erv1 was removed by His tag affinity cobalt beads. The Erv1-free sample was then incubated with or without Mia40 for 2 h. C, titration of Erv1 with Cmc1 held constant at 1 μm. In A–C, the samples were treated as in Fig. 1.

FIGURE 4.

Cmc1 interacts with the second cysteine of the CPC active site of Mia40, and release from Mia40 requires a functional Erv1 protein. A, reconstitution of Cmc1 oxidative folding following 4 h of incubation and analysis of AMS-trapped thiols using wild-type, C30S, and C133S Erv1 purified protein in the presence and absence of Mia40. The first line includes Cmc1 alone without AMS; all of the other samples contain AMS. B–D, reconstitution of Cmc1 oxidative folding and analysis of AMS-trapped thiols using wild-type, SX₉C, SPC, and CPS purified Mia40 protein in the presence and absence of Erv1 in the indicated stoichiometries. E, fluorometric Amplex Red assay performed as in Fig. 1C with mutant forms of Erv1 and Mia40.

FIGURE 5.

Identification of disulfide bonds by mass spectrometry. A, amino acid sequence of Cmc1. Tryptic peptides 1–4, which contain cysteines, are highlighted. B, Coomassie-stained gel of varying states of Cmc1 oxidation. The bands were excised from the gel and sent for mass spectrometry analysis. The excised bands include: band 1 (B1), fully reduced and unmodified Cmc1; band 2 (B2), fully reduced and AMS-modified Cmc1; band 3 (B3), partially oxidized and AMS-modified Cmc1; band 4 (B4), fully oxidized and AMS-modified Cmc1; and band 5 (B5), Mia40-Cmc1 intermediate. Below the gel are indications of the presence or absence of modified cysteine-containing peptides as indicated in A. Note that for excised band 1, the unmodified versions of the peptides were detected. C, model presenting the cysteine residues involved in the Mia40-Cmc1 interaction in band 5 in B.

FIGURE 6.

Folding requirements and oxidized kinetics are altered in Cmc1 cysteine mutant variants. A, schematic diagram of Cmc1 showing the cysteine residues in a hypothetical folding based on the structure of other CX₉C proteins. B, reconstitution of oxidative folding and analysis of AMS-trapped thiols using Cmc1 cysteine mutants. C, reconstitution of wild-type and mutant variants of Cmc1 oxidative folding with Mia40 and Erv1 (molar ratio, 5:1:1) following time course incubations up to 4 h as indicated and subsequent analysis of AMS-trapped thiols. D, the bands for the reconstitution assays in C corresponding to fully reduced (4/5 AMS), partially oxidized (2/3 AMS), and fully oxidized (0/1 AMS) Cmc1 were quantified using the histogram function of the Adobe Photoshop program and expressed as percentages of total signal at each time point for each protein (WT or mutant Cmc1).

FIGURE 7.

Importance of the CX₉C cysteines for Cmc1 import by Mia40 and Erv1. A, in vivo yeast complementation of a Δcmc1 strain with overexpressed cysteine to alanine Cmc1 variants. Serial dilutions were spotted on solid synthetic media containing dextrose (WO-D) or ethanol-glycerol (WO-EG). Pictures were taken after 2 and 4 days of incubation at 30 °C. B, steady state levels of Cmc1 in mitochondria isolated from a Δcmc1 mutant strain overexpressing each Cmc1 variant. Porin was used as a loading control. The bands in the upper panel were quantified using the histogram function of the Adobe Photoshop program and expressed as percentages of WT values. C, in organello import of ³⁵S-labeled WT, C42A, C52A, C64A, and C74A Cmc1 into wild-type mitochondria. The proteins were separated by nonreducing SDS-PAGE. The signals were quantified as in B and expressed as percentages of WT values. D, same as in C, but the proteins were separated by both reducing and nonreducing SDS-PAGE. E, in organello import of ³⁵S-labeled wild-type Cmc1 into wild-type and mia40-3 mitochondria. The proteins were separated by nonreducing PAGE except the sample labeled as Red, which was reducing. In C–E, the symbols + and − indicate treatment or not with PK. F, in organello import of ³⁵S-labeled wild-type and C104A Cmc1 into wild-type mitochondria. The proteins were separated by nonreducing SDS-PAGE. G, in organello import of Cmc1 C64A using wild-type and erv1-101 mitochondria. The mitochondria were incubated for 15 min at either 30 or 37 °C prior to proceeding with the import reaction. Following import, the mitochondria were reisolated and left proteinase-untreated. In C–F, the lower panel is a longer exposition and more contrasted version of the higher molecular mass portion of the upper panel. H, following import of ³⁵S-labeled Cmc1 into WT, mia40-3 or mia40-4 mitoplasts, extracts were subjected to immunoprecipitation with the indicated antibodies.

FIGURE 8.

Model of Cmc1 oxidative folding in the mitochondrial IMS. See explanation under “Discussion.” For simplification, a possible role of Erv1 in substrate oxidation in steps 2 and 3 is not depicted.