Mitochondrial Intermembrane Space Assembly Pathway Research Articles

Aim32 is a dual-localized 2Fe-2S mitochondrial protein that functions in redox quality control

Yeast is a facultative anaerobe and uses diverse electron acceptors to maintain redox-regulated import of cysteine-rich precursors via the mitochondrial intermembrane space assembly (MIA) pathway. With the growing diversity of substrates utilizing the MIA pathway, understanding the capacity of the intermembrane space (IMS) to handle different types of stress is crucial. We used MS to identify additional proteins that interacted with the sulfhydryl oxidase Erv1 of the MIA pathway. Altered inheritance of mitochondria 32 (Aim32), a thioredoxin-like [2Fe-2S] ferredoxin protein, was identified as an Erv1-binding protein. Detailed localization studies showed that Aim32 resided in both the mitochondrial matrix and IMS. Aim32 interacted with additional proteins including redox protein Osm1 and protein import components Tim17, Tim23, and Tim22. Deletion of Aim32 or mutation of conserved cysteine residues that coordinate the Fe-S center in Aim32 resulted in an increased accumulation of proteins with aberrant disulfide linkages. In addition, the steady-state level of assembled TIM22, TIM23, and Oxa1 protein import complexes was decreased. Aim32 also bound to several mitochondrial proteins under nonreducing conditions, suggesting a function in maintaining the redox status of proteins by potentially targeting cysteine residues that may be sensitive to oxidation. Finally, Aim32 was essential for growth in conditions of stress such as elevated temperature and hydroxyurea, and under anaerobic conditions. These studies suggest that the Fe-S protein Aim32 has a potential role in general redox homeostasis in the matrix and IMS. Thus, Aim32 may be poised as a sensor or regulator in quality control for a broad range of mitochondrial proteins.

Aim32, a dual‐localized 2Fe‐2S mitochondrial protein that functions in redox quality control

Maintaining redox homeostasis under normal and stress conditions in the mitochondrion is complex. Yeast being a facultative anaerobe, likely uses diverse electron acceptors to sustain the redox-dependent mitochondrial intermembrane space assembly (MIA) pathway under different environmental conditions. We used mass spectrometry in an effort to identify additional proteins that interacted with the sulfhydryl oxidase Erv1 of the MIA pathway. Aim32, a thioredoxin-like [2Fe-2S] ferredoxin protein, was identified as an Erv1 binding protein. Detailed and complementary localization studies showed that Aim32 resided in both the mitochondrial matrix and intermembrane space, placing it in a rare protein class that is dual-localized within mitochondria. Aim32 interacted with additional proteins including redox protein Osm1 as well as protein import components Tim17, Tim23, and Tim22. Deletion of Aim32 or conserved cysteine residues that coordinate the Fe-S center resulted in an increased accumulation of proteins with aberrant disulfide linkages. In addition, the steady-state level of assembled TIM22, TIM23, and Oxa1 protein import complexes was decreased and a subset of the import complexes showed misassembly, among other phenotypes. Aim32 also bound to several mitochondrial proteins under non-reducing conditions, suggesting a function in maintaining the redox status of oxidized proteins by potentially targeting cysteine residues that may be sensitive to oxidation. Finally, Aim32 was essential for growth in conditions of stress, and upon challenge with hydroxyurea (HU). This is the first evidence of the Fe-S protein, Aim32 being uniquely localized to both the mitochondrial intermembrane space and matrix with a role in redox regulation.

Bisphenol-A inhibits mitochondrial biogenesis via impairment of GFER mediated mitochondrial protein import in the rat brain hippocampus

Mitochondrial biogenesis relies on different protein import machinery, as mitochondrial proteins are imported from the cytosol. The mitochondrial intermembrane space assembly (MIA) pathway consists of GFER/ALR and CHCHD4/Mia40, responsible for importing proteins and their oxidative folding inside the mitochondria. The MIA pathway plays an essential role in complex IV (COX IV) biogenesis via importing copper chaperone COX17, associated with the respiratory chain. BPA, an environmental toxicant, found in consumable plastics, causes neurotoxicity via impairment in mitochondrial dynamics, neurogenesis, and cognitive functions. We studied the levels of key regulatory proteins of mitochondrial import pathways and mitochondrial biogenesis after BPA exposure in the rat hippocampus. BPA caused a significant reduction in the levels of mitochondrial biogenesis proteins (PGC1α, and TFAM) and mitochondrial import protein (GFER). Immunohistochemical analysis showed reduced co-localization of NeuN with GFER, PGC-1α, and TFAM suggesting impaired mitochondrial biogenesis and protein import. BPA exposure resulted in damaged mitochondria with distorted cristae in neurons and caused a significant reduction in GFER localization inside IMS as depicted by immunogold electron microscopy. The reduced levels of GFER resulted in defective COX17 import. The translocation of cytochrome c into the cytosol and increased cleaved caspase-3 levels triggered apoptosis due to BPA toxicity. Overall, our study implicates GFER as a potential target for impaired mitochondrial protein machinery, biogenesis, and apoptosis against BPA neurotoxicity in the rat hippocampus.

Inhibiting the Mitochondrial Sulfhydryl Oxidase Alr Reduces Cox17 and Alters Mitochondrial Cristae Structure Leading to the Differentiation of AML and Stem Cells

The vast majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and then imported into the mitochondria. A subset of these imported proteins are folded into their mature and functional forms in the mitochondrial inter-membrane space (IMS) by the Mitochondrial IMS Assembly (MIA) pathway. We found that genes encoding substrates of the MIA pathway are over-expressed in leukemic stem cells compared to bulk AML cells. Therefore, we assessed the effects of inhibiting the MIA pathway in AML. We knocked down the mitochondrial sulfhydryl oxidase ALR, a key regulator of the MIA pathway. Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, knockdown of ALR reduced the engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6, mimicked the effects of ALR knockdown and killed AML cells with an IC50 of 5-10 μM. MitoBloCK-6 preferentially reduced the clonogenic growth of primary AML cells (n=4/5) over normal hematopoietic cells (n=4). However, only 3/10 bulk AML cells were sensitive to MitoBloCK-6 induced cell death by Annexin V/PI staining.Next, we evaluated the efficacy and toxicity of ALR inhibition in vivo . We injected primary AML cells or normal cord blood into the femurs of mice and then treated mice with MitoBloCK-6 (80 mg/kg i.p. 5 of 7 days x 2 weeks). MitoBloCK-6 strongly reduced the engraftment of primary AML samples but did not affect engraftment of cord blood. In secondary transplants, MitoBloCK-6 also targeted leukemic stem cells. No change in mouse body weight, serum chemistries, or organ histology was seen.As expression levels of ALR substrates are increased in AML stem cells, we assessed the effects of ALR inhibition on differentiation in AML. Genetic or chemical inhibition of ALR induced the differentiation of AML cells as evidenced by increased CD surface marker expression and increased non-specific esterase. In addition, ALR inhibition was preferentially cytotoxic towards undifferentiated cells and stem cells over differentiated bulk AML cells.Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Inhibition of ALR selectively reduced levels of Cox17 protein and altered mitochondrial cristae structure. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation, induced differentiation of AML cells, and altered mitochondrial cristae structure, without changing respiratory chain activity or oxygen consumption. Of note, cristae remodelling independent of respiratory chain function has been recently implicated in cellular differentiation and in yeast, Cox17 regulates the cristae organizing machinery.Thus, we have identified novel mechanisms by which mitochondrial pathways regulate the fate and differentiation of AML cells and stem cells Moreover, inhibition of ALR may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells. DisclosuresSchimmer:Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.

Differential proteomic and oxidative profiles unveil dysfunctional protein import to adipocyte mitochondria in obesity-associated aging and diabetes

Human age-related diseases, including obesity and type 2 diabetes (T2DM), have long been associated to mitochondrial dysfunction; however, the role for adipose tissue mitochondria in these conditions remains unknown. We have tackled the impact of aging and T2DM on adipocyte mitochondria from obese patients by quantitating not only the corresponding abundance changes of proteins, but also the redox alterations undergone by Cys residues thereof. For that, we have resorted to a high-throughput proteomic approach based on isobaric labeling, liquid chromatography and mass spectrometry. The alterations undergone by the mitochondrial proteome revealed aging- and T2DM-specific hallmarks. Thus, while a global decrease of oxidative phosphorylation (OXPHOS) subunits was found in aging, the diabetic patients exhibited a reduction of specific OXPHOS complexes as well as an up-regulation of the anti-oxidant response. Under both conditions, evidence is shown for the first time of a link between increased thiol protein oxidation and decreased protein abundance in adipose tissue mitochondria. This association was stronger in T2DM, where OXPHOS mitochondrial- vs. nuclear-encoded protein modules were found altered, suggesting impaired mitochondrial protein translocation and complex assembly. The marked down-regulation of OXPHOS oxidized proteins and the alteration of oxidized Cys residues related to protein import through the redox-active MIA (Mitochondrial Intermembrane space Assembly) pathway support that defects in protein translocation to the mitochondria may be an important underlying mechanism for mitochondrial dysfunction in T2DM and physiological aging. The present draft of redox targets together with the quantification of protein and oxidative changes may help to better understand the role of oxidative stress in both a physiological process like aging and a pathological condition like T2DM.

Biogenesis of yeast Mia40 - uncoupling folding from import and atypical recognition features

The discovery of the mitochondrial intermembrane space assembly (MIA) pathway was followed by studies that focused mainly on the typical small substrates of this disulfide relay system and the interactions between its two central partners: the oxidoreductase Mia40 and the FAD-protein Erv1. Recent studies have revealed that more complex proteins utilize this pathway, including Mia40 itself. In the present study, we dissect the Mia40 biogenesis in distinct stages, supporting a kinetically coordinated sequence of events, starting with (a) import and insertion through the Tim23 translocon, followed by (b) folding of the core of imported Mia40 assisted by the endogenous Mia40 and (c) final interaction with Erv1. The interaction with endogenous Mia40 and the subsequent interaction with Erv1 represent kinetically distinguishable steps that rely on completely different determinants. Interaction with Mia40 proceeds very early (within 30 s) and is characterized by no Cys-specificity, an increased tolerance to mutations of the hydrophobic substrate-binding cleft and no apparent dependence on glutathione as a proofreading mechanism. All of these features illustrate a very atypical behaviour for the Mia40 precursor compared to other substrates of the MIA pathway. By contrast, interaction with Erv1 occurs after 5 min of import and relies on a more stringent specificity.

Dual Role of Mitofilin in Mitochondrial Membrane Organization and Protein Biogenesis

The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central componentof MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.

Identification of the Signal Directing Tim9 and Tim10 into the Intermembrane Space of Mitochondria

The intermembrane space of mitochondria contains the specific mitochondrial intermembrane space assembly (MIA) machinery that operates in the biogenesis pathway of precursor proteins destined to this compartment. The Mia40 component of the MIA pathway functions as a receptor and binds incoming precursors, forming an essential early intermediate in the biogenesis of intermembrane space proteins. The elements that are crucial for the association of the intermembrane space precursors with Mia40 have not been determined. In this study, we found that a region within the Tim9 and Tim10 precursors, consisting of only nine amino acid residues, functions as a signal for the engagement of substrate proteins with the Mia40 receptor. Furthermore, the signal contains sufficient information to facilitate the transfer of proteins across the outer membrane to the intermembrane space. Thus, here we have identified the mitochondrial intermembrane space sorting signal required for delivery of proteins to the mitochondrial intermembrane space.