Recombinant Saccharomyces cerevisiae Mitochondrial intermembrane space import and assembly protein 40 (MIA40)

What are the key structural features of Saccharomyces cerevisiae Mia40?

S. cerevisiae Mia40 contains two essential functional elements:

• An N-terminal redox-active cysteine-proline-cysteine (CPC) motif critical for substrate oxidation

• A C-terminal hydrophobic substrate-binding pocket for interaction with incoming proteins

The protein contains six conserved cysteine residues forming three disulfide bonds: the redox-active C1-C2 pair within the CPC motif, and two structural disulfides (C3-C6 and C4-C5) that stabilize the hydrophobic substrate-binding domain . Full-length yeast Mia40 also possesses an N-terminal bipartite presequence that targets it to mitochondria, although this element is absent in metazoan and plant homologs .

What is the primary function of Mia40 in mitochondria?

Mia40 serves dual functions as both a receptor and an oxidase in the mitochondrial IMS:

• As a trans-site receptor, it binds incoming precursor proteins via hydrophobic interactions, driving their translocation across the outer membrane through a "holding trap" mechanism

• As an oxidase, it introduces disulfide bonds into substrate proteins, promoting their oxidative folding and stable retention in the IMS

Genetic studies using Mia40 variants with mutations in either the CPC motif or the substrate-binding domain have demonstrated that the receptor function (substrate binding) is both necessary and sufficient for protein import, whereas the oxidase activity is essential for subsequent protein folding and stability .

What expression systems are recommended for producing recombinant S. cerevisiae Mia40?

For recombinant expression of S. cerevisiae Mia40, the following approaches have proven successful:

E. coli expression system:

• Expression constructs typically use His-tagged versions of Mia40, particularly Mia40core (lacking the N-terminal 70-225 residues)

• Expression in E. coli can be performed using pN-His10-Mia40Δ1-225 vectors with standard IPTG induction

Yeast expression system:

• Endogenous promoter-driven expression using pFL39-derived plasmids

• Galactose-inducible overexpression using GAL promoters, which allows for controlled high-level expression

Purification typically involves:

• Affinity chromatography using Ni-NTA resins for His-tagged versions

• Size exclusion chromatography to achieve high purity

• Maintaining reducing conditions during initial purification steps if the functional redox-active form is desired

How can I assess the oxidation state of purified recombinant Mia40?

The oxidation state of Mia40 can be determined using the following methodology:

• Alkylation assay with maleimide reagents:

  • Precipitate proteins with trichloroacetic acid (TCA) to preserve redox state

  • Treat with alkylating agents like mmPEG24 or mmPEG12

  • Analyze by SDS-PAGE to detect mass shifts (approximately 1.2 kDa per alkylated thiol for mmPEG24 and 0.7 kDa for mmPEG12)

• Mobility shift analysis:

  • Compare migration patterns under reducing versus non-reducing SDS-PAGE

  • Fully oxidized Mia40 migrates faster under non-reducing conditions

  • Different redox states can be identified based on distinct band patterns

The oxidation state analysis typically reveals:

• Two structural disulfides of the substrate-binding domain (C3-C6 and C4-C5)

• Variable oxidation states of the CPC motif (C1-C2), which cycles between oxidized and reduced forms during its functional cycle

What are the key assays to evaluate Mia40's import function in isolated mitochondria?

Several complementary approaches can be used to assess Mia40's functionality:

In vitro import assays with radiolabeled substrates:

• Generate 35S-labeled precursor proteins (Tim9, Tim10, Cox17, Cox19)

• Incubate with isolated mitochondria (50-75 μg) at 30°C

• Remove non-imported material by proteinase K treatment (50 μg/ml)

• Analyze by SDS-PAGE under reducing and non-reducing conditions

Tracking covalent intermediates:

• After import, solubilize mitochondria in detergent buffer with 50 mM iodoacetamide

• Analyze mixed disulfide intermediates by blue native electrophoresis or SDS-PAGE

• Verify Mia40 involvement through antibody shift assays using anti-Mia40 antibodies

Reconstitution with purified components:

• Purify recombinant Mia40core with His10 tag

• Reduce with 50 mM DTT and denature in 8 M urea

• Import into isolated mitochondria (50-75 μg protein with 1.25-2.5 μg Mia40core)

• Assess import efficiency by immunodecoration and Ni-NTA affinity purification

How can I monitor the oxidative folding pathway mediated by Mia40?

To track the oxidative folding pathway:

• Pulse-chase oxidation kinetics:

  • Radiolabel translation products in yeast cells for 3 minutes

  • Stop labeling by washing cells and adding excess non-radioactive methionine

  • Take samples at different time points and treat with alkylating agent (mmPEG24)

  • Immunoprecipitate substrate proteins and analyze by SDS-PAGE

  • This reveals temporal progression of disulfide bond formation

• Substrate intermediate trapping:

  • Import radiolabeled substrates in the presence of oxidizing agent CuCl2

  • Analyze by BN-PAGE to detect Mia40-substrate intermediates

  • Confirm identity using antibody-shift BN-PAGE with anti-Mia40 antibodies

In wild-type cells, substrates like Cox19 show complete oxidation within 2 minutes, while temperature-sensitive mia40 mutants exhibit significantly slower oxidation kinetics (4-8 minutes for half-oxidation) .

What are the most informative Mia40 mutants for dissecting its dual functionality?

Several key mutants have been instrumental in understanding Mia40's functions:

For dissecting the receptor function from oxidase activity, the Mia40-SPS mutant is particularly valuable as it retains only the substrate-binding capability, demonstrating that the binding function alone can partially support protein import .

How can genetic complementation approaches be used to study Mia40 function?

Genetic complementation strategies:

• Plasmid shuffling method:

  • Start with a strain harboring chromosomal deletion of MIA40 rescued by a URA3-containing plasmid expressing wild-type Mia40

  • Transform with test plasmids expressing Mia40 variants

  • Select on 5-FOA medium to counter-select against the URA3 plasmid

  • Assess growth to determine if the variant complements essential Mia40 function

• Heterologous complementation:

  • Human MIA40 can complement yeast mia40 deletion despite lacking the presequence

  • Chimeric constructs combining cytochrome b2 presequence with Mia40core demonstrate importance of targeting signals

• Temperature-sensitive complementation:

  • Use conditional mia40 mutants (mia40-3, mia40-4) grown at non-permissive temperature

  • Express Mia40 variants and assess rescue capacity

  • Measure mitochondrial protein levels to determine functionality

The complementation studies revealed that the C-terminal domain contains the essential functional elements of Mia40, and that the protein contains dual targeting information that can direct it to either the presequence pathway or the MIA pathway .

How does Mia40 cooperate with Erv1 to ensure complete oxidation of substrate proteins?

Mia40 and Erv1 cooperation involves a complex mechanism:

• Ternary complex formation:

  • Mia40 initially engages with incoming substrates via its CPC motif

  • Erv1 subsequently binds to the Mia40-substrate conjugate at a site distinct from the C2 region

  • This forms a ternary complex (Mia40-substrate-Erv1) essential for complete oxidation

• Electron transfer cascade:

  • A total of four electrons from substrate dithiols are accepted in consecutive steps

  • The redox-active CPC motif of Mia40 accepts electrons from the substrate

  • Erv1 reoxidizes the CPC motif, transferring electrons to cytochrome c or O2

Experimental approach to study the ternary complex:

• Import saturating amounts of substrates into mitochondria

• Perform affinity purification from detergent-solubilized mitochondria

• Analyze binding partners by Western blotting or mass spectrometry

• Compare wild-type and erv1 mutant (erv1-2, erv1-5) backgrounds

Experiments with temperature-sensitive erv1-2 mutants demonstrated that under restrictive conditions, Mia40 adopts a partially reduced form and fails to bind substrates, confirming the essential role of Erv1 in maintaining functional Mia40 .

What is the mechanism by which Mia40 achieves substrate specificity?

Mia40 achieves substrate specificity through a two-step "sliding-and-docking" mechanism:

• Initial recognition via ITS/MISS signals:

  • Substrates contain a 9-amino acid internal peptide sequence called IMS-targeting signal (ITS) or mitochondrial IMS sorting signal (MISS)

  • The ITS forms an amphipathic helix with crucial hydrophobic residues positioned one or two turns away from the docking cysteine

• Precise positioning of substrate cysteines:

  • Hydrophobic interactions between the ITS/MISS motif and Mia40's substrate-binding cleft orient the substrate noncovalently

  • This positions specific substrate cysteines to interact with Mia40's redox-active CPC motif

The specificity mechanism can be studied by:

• Mutational analysis of the ITS/MISS regions in substrate proteins

• NMR structure determination of Mia40-substrate complexes

• Measuring binding affinities of Mia40 with wild-type and mutant substrates using surface plasmon resonance or isothermal titration calorimetry

This mechanism explains how Mia40 can selectively target different cysteines in different substrates (e.g., N-terminal cysteines in small Tims versus inner disulfide cysteines in Cox17) .

How does Mia40 overexpression affect cellular proteostasis beyond mitochondrial protein import?

Recent research has uncovered unexpected roles for Mia40 in cellular proteostasis:

• Suppression of cytosolic protein aggregation:

  • Overexpression of Mia40 strongly protects cells against toxic protein aggregates like polyQ-GFP

  • This protective effect extends to both yeast and human cells

  • Mia40 prevents the formation of SDS-resistant aggregates and promotes homogeneous distribution of aggregation-prone proteins

• Competition mechanism for cellular quality control resources:

  • Mitochondrial precursor protein import competes with aggregation-prone cytosolic proteins for chaperones and proteasome capacity

  • Mia40 regulates this competition as it has a rate-limiting role in mitochondrial protein import

  • Temperature-sensitive mia40 mutants show hypersensitivity to polyQ expression even at permissive conditions

• Experimental approaches to study this phenomenon:

  • Co-expression of Mia40 with aggregation-prone reporter proteins

  • Fluorescence microscopy to track aggregate formation

  • Biochemical fractionation to measure soluble versus insoluble protein distribution

  • Cell growth assays to quantify toxicity suppression

The data reveal that endogenous Mia40 levels are rate-limiting under physiological conditions, as overexpression leads to significantly higher levels of many Mia40 substrates .

What are common problems encountered when expressing and purifying functional recombinant Mia40?

When purifying Mia40 for functional studies, it's critical to verify the oxidation state of the protein using alkylation assays with mmPEG reagents to confirm proper formation of both the structural disulfides and the redox-active CPC motif .

How can I distinguish between import and oxidation defects when characterizing Mia40 variants?

Distinguishing between these functions requires multiple complementary approaches:

• Analysis with oxidase-deficient mutants:

  • Compare Mia40-SPS (lacks oxidase activity) with wild-type Mia40

  • Measure substrate accumulation in mitochondria

  • If Mia40-SPS supports import but not oxidation, defects in oxidation can be isolated

• Chemical complementation approach:

  • Supplement with chemical oxidants like diamide

  • If defects are due to oxidation function, diamide should partially rescue the phenotype

• Substrate-specific analysis:

  • Track early binding intermediates via BN-PAGE

  • Monitor formation of mature oxidized forms under non-reducing conditions

  • Compare kinetics of various steps in the pathway

• Pulse-chase experiments:

  • Radiolabel substrates and follow oxidation over time

  • Compare wild-type and mutant conditions

  • Slower oxidation with normal initial binding indicates specific oxidation defects

A key finding from such analyses is that protein import driven by Mia40's receptor function occurs independently of its oxidase activity, as demonstrated by the partial restoration of IMS import in the Mia40-SPS mutant .

How has the Mia40 system evolved across different eukaryotic lineages?

The Mia40 system shows notable evolutionary variation:

• Structural differences across lineages:

  • Fungal Mia40: Contains large N-terminal region with presequence

  • Metazoan and plant Mia40: Consists only of conserved C-terminal domain

  • The C-terminal domain with twin CX9C motifs is universally conserved

• Targeting pathway evolution:

  • Fungal Mia40: Typically uses presequence pathway for import

  • Human MIA40: Lacks presequence, imported via the MIA pathway itself

  • Yeast Mia40core: Can use the MIA pathway, demonstrating evolutionary flexibility

• Mechanistic variation in plants:

  • In Arabidopsis, Mia40 is not essential

  • Plant Erv1 can directly interact with and oxidatively fold substrate proteins

  • This indicates stepwise evolution of the IMS disulfide relay system

This evolutionary perspective suggests that the mitochondrial disulfide relay system evolved from a simple Erv1-only system to the more complex Mia40-Erv1 system seen in fungi and animals, with plants maintaining an intermediate state .

What is known about the substrate specificity of Mia40 across different species?

Substrate specificity features across species:

• Conserved recognition elements:

  • The MISS/ITS motif (mitochondrial intermembrane space sorting signal/intermembrane space targeting signal) is broadly conserved

  • Hydrophobic substrate-binding cleft of Mia40 is structurally preserved across species

• Species-specific differences:

  • Human Mia40 (CHCHD4) shows preference for oxidizing the inner disulfide of Cox17

  • Yeast Mia40 preferentially oxidizes the outer disulfide of Cox17

  • These differences correlate with evolutionary changes in substrate sequences

• Methodological approaches for comparative studies:

  • Heterologous complementation (e.g., human MIA40 in yeast)

  • In vitro import assays with substrates and Mia40 from different species

  • Structural studies of Mia40-substrate interactions across species

The conserved mechanism involves recognition of hydrophobic patches in substrate proteins that position specific cysteines for interaction with Mia40's redox-active CPC motif, though the exact cysteine residues targeted may differ between species .