Redox-Mediated Regulation of Mitochondrial Biogenesis, Dynamics, and Respiratory Chain Assembly in Yeast and Human Cells

Mitochondria are double-membrane organelles that contain their own genome, the mitochondrial DNA (mtDNA), and reminiscent of its endosymbiotic origin. Mitochondria are responsible for cellular respiration via the function of the electron oxidative phosphorylation system (OXPHOS), located in the mitochondrial inner membrane and composed of the four electron transport chain (ETC) enzymes (complexes I-IV), and the ATP synthase (complex V). Even though the mtDNA encodes essential OXPHOS components, the large majority of the structural subunits and additional biogenetical factors (more than seventy proteins) are encoded in the nucleus and translated in the cytoplasm. To incorporate these proteins and the rest of the mitochondrial proteome, mitochondria have evolved varied, and sophisticated import machineries that specifically target proteins to the different compartments defined by the two membranes. The intermembrane space (IMS) contains a high number of cysteine-rich proteins, which are mostly imported via the MIA40 oxidative folding system, dependent on the reduction, and oxidation of key Cys residues. Several of these proteins are structural components or assembly factors necessary for the correct maturation and function of the ETC complexes. Interestingly, many of these proteins are involved in the metalation of the active redox centers of complex IV, the terminal oxidase of the mitochondrial ETC. Due to their function in oxygen reduction, mitochondria are the main generators of reactive oxygen species (ROS), on both sides of the inner membrane, i.e., in the matrix and the IMS. ROS generation is important due to their role as signaling molecules, but an excessive production is detrimental due to unwanted oxidation reactions that impact on the function of different types of biomolecules contained in mitochondria. Therefore, the maintenance of the redox balance in the IMS is essential for mitochondrial function. In this review, we will discuss the role that redox regulation plays in the maintenance of IMS homeostasis as well as how mitochondrial ROS generation may be a key regulatory factor for ETC biogenesis, especially for complex IV.

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The mitochondrial intermembrane space: the most constricted mitochondrial sub-compartment with the largest variety of protein import pathways

Open Biology ◽

10.1098/rsob.210002 ◽

2021 ◽

Vol 11 (3) ◽

Author(s):

Ruairidh Edwards ◽

Ross Eaglesfield ◽

Kostas Tokatlidis

Keyword(s):

Protein Import ◽

Atp Hydrolysis ◽

Current Knowledge ◽

Intermembrane Space ◽

Inner Membrane ◽

Mitochondrial Proteome ◽

Normal Site ◽

The Matrix ◽

Human Disorders ◽

Mitochondrial Intermembrane Space

The mitochondrial intermembrane space (IMS) is the most constricted sub-mitochondrial compartment, housing only about 5% of the mitochondrial proteome, and yet is endowed with the largest variability of protein import mechanisms. In this review, we summarize our current knowledge of the major IMS import pathway based on the oxidative protein folding pathway and discuss the stunning variability of other IMS protein import pathways. As IMS-localized proteins only have to cross the outer mitochondrial membrane, they do not require energy sources like ATP hydrolysis in the mitochondrial matrix or the inner membrane electrochemical potential which are critical for import into the matrix or insertion into the inner membrane. We also explore several atypical IMS import pathways that are still not very well understood and are guided by poorly defined or completely unknown targeting peptides. Importantly, many of the IMS proteins are linked to several human diseases, and it is therefore crucial to understand how they reach their normal site of function in the IMS. In the final part of this review, we discuss current understanding of how such IMS protein underpin a large spectrum of human disorders.

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Mutant huntingtin disrupts mitochondrial proteostasis by interacting with TIM23

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904101116 ◽

2019 ◽

Vol 116 (33) ◽

pp. 16593-16602 ◽

Cited By ~ 12

Author(s):

Svitlana Yablonska ◽

Vinitha Ganesan ◽

Lisa M. Ferrando ◽

JinHo Kim ◽

Anna Pyzel ◽

...

Keyword(s):

Cell Lines ◽

Protein Import ◽

Mitochondrial Protein ◽

Biological Significance ◽

Intermembrane Space ◽

Mutant Huntingtin ◽

Mitochondrial Protein Import ◽

Inner Membrane ◽

Mitochondrial Proteome ◽

Brain Tissues

Mutant huntingtin (mHTT), the causative protein in Huntington’s disease (HD), associates with the translocase of mitochondrial inner membrane 23 (TIM23) complex, resulting in inhibition of synaptic mitochondrial protein import first detected in presymptomatic HD mice. The early timing of this event suggests that it is a relevant and direct pathophysiologic consequence of mHTT expression. We show that, of the 4 TIM23 complex proteins, mHTT specifically binds to the TIM23 subunit and that full-length wild-type huntingtin (wtHTT) and mHTT reside in the mitochondrial intermembrane space. We investigated differences in mitochondrial proteome between wtHTT and mHTT cells and found numerous proteomic disparities between mHTT and wtHTT mitochondria. We validated these data by quantitative immunoblotting in striatal cell lines and human HD brain tissue. The level of soluble matrix mitochondrial proteins imported through the TIM23 complex is lower in mHTT-expressing cell lines and brain tissues of HD patients compared with controls. In mHTT-expressing cell lines, membrane-bound TIM23-imported proteins have lower intramitochondrial levels, whereas inner membrane multispan proteins that are imported via the TIM22 pathway and proteins integrated into the outer membrane generally remain unchanged. In summary, we show that, in mitochondria, huntingtin is located in the intermembrane space, that mHTT binds with high-affinity to TIM23, and that mitochondria from mHTT-expressing cells and brain tissues of HD patients have reduced levels of nuclearly encoded proteins imported through TIM23. These data demonstrate the mechanism and biological significance of mHTT-mediated inhibition of mitochondrial protein import, a mechanism likely broadly relevant to other neurodegenerative diseases.

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Tim23–Tim50 pair coordinates functions of translocators and motor proteins in mitochondrial protein import

The Journal of Cell Biology ◽

10.1083/jcb.200808068 ◽

2009 ◽

Vol 184 (1) ◽

pp. 129-141 ◽

Cited By ~ 99

Author(s):

Yasushi Tamura ◽

Yoshihiro Harada ◽

Takuya Shiota ◽

Koji Yamano ◽

Kazuaki Watanabe ◽

...

Keyword(s):

Motor Proteins ◽

Protein Import ◽

Protein Translocation ◽

Mitochondrial Protein ◽

Intermembrane Space ◽

Mitochondrial Protein Import ◽

Inner Membrane ◽

The Matrix ◽

General Protein ◽

Mitochondrial Hsp70

Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23–Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23–Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.

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Cox18p Is Required for Export of the Mitochondrially EncodedSaccharomyces cerevisiaeCox2p C-Tail and Interacts with Pnt1p and Mss2p in the Inner Membrane

Molecular Biology of the Cell ◽

10.1091/mbc.01-12-0580 ◽

2002 ◽

Vol 13 (4) ◽

pp. 1122-1131 ◽

Cited By ~ 80

Author(s):

Scott A. Saracco ◽

Thomas D. Fox

Keyword(s):

Intermembrane Space ◽

Biosynthetic Enzyme ◽

Epitope Tag ◽

Inner Membrane ◽

Multiple Alleles ◽

Functional Interactions ◽

C Terminus ◽

The Matrix ◽

Inner Membrane Protein ◽

Carboxy Terminal

The amino- and carboxy-terminal domains of mitochondrially encoded cytochrome c oxidase subunit II (Cox2p) are translocated out of the matrix to the intermembrane space. We have carried out a genetic screen to identify components required to export the biosynthetic enzyme Arg8p, tethered to the Cox2p C terminus by a translational gene fusion inserted into mtDNA. We obtained multiple alleles of COX18, PNT1, and MSS2, as well as mutations in CBP1 and PET309. Focusing on Cox18p, we found that its activity is required to export the C-tail of Cox2p bearing a short C-terminal epitope tag. This is not a consequence of reduced membrane potential due to loss of cytochrome oxidase activity because Cox2p C-tail export was not blocked in mitochondria lacking Cox4p. Cox18p is not required to export the Cox2p N-tail, indicating that these two domains of Cox2p are translocated by genetically distinct mechanisms. Cox18p is a mitochondrial integral inner membrane protein. The inner membrane proteins Mss2p and Pnt1p both coimmunoprecipitate with Cox18p, suggesting that they work together in translocation of Cox2p domains, an inference supported by functional interactions among the three genes.

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Discrimination of distinct proteinases at the four structural levels of rat liver mitochondria

Biochemical Journal ◽

10.1042/bj2330283 ◽

1986 ◽

Vol 233 (1) ◽

pp. 283-286 ◽

Cited By ~ 3

Author(s):

M C Duque-Magalhães ◽

P Régnier

Keyword(s):

Outer Membrane ◽

Rat Liver ◽

Degradation Products ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Intermembrane Space ◽

Peptidase Activity ◽

Inner Membrane ◽

Mitochondrial Fractions ◽

The Matrix

Rat liver mitochondrial fractions corresponding to four morphological structures (matrix, inner membrane, intermembrane space and outer membrane) contain proteinases that cleave casein components at different rates. Proteinases of the intermembrane space preferentially cleave kappa-casein, whereas the proteinases of the outer membrane, inner membrane and matrix fractions degrade alpha S1-casein more rapidly. Electrophoretic separation of the degradation products of alpha S1-casein and kappa-casein in polyacrylamide gels shows that different polypeptides are produced when the substrate is degraded by the matrix, by both membranes and by the intermembrane-space fraction. Some of the degradation products resulting from incubation of the caseins with the mitochondrial fractions are probably the result of digestion by contaminating lysosomal proteinase(s). The matrix has a high peptidase activity, since glucagon, a small peptide, is very rapidly degraded by this fraction. These observations strongly suggest that distinct proteinases, with different specificities, are associated respectively with the intermembrane space and with both membrane fractions.

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The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments

BMC Biology ◽

10.1186/s12915-019-0733-6 ◽

2020 ◽

Vol 18 (1) ◽

Cited By ~ 10

Author(s):

Heike Rampelt ◽

Iva Sucec ◽

Beate Bersch ◽

Patrick Horten ◽

Inge Perschil ◽

...

Keyword(s):

Intermembrane Space ◽

Inner Mitochondrial Membrane ◽

Inner Membrane ◽

Mitochondrial Carrier ◽

Transmembrane Segments ◽

Mitochondrial Inner Membrane ◽

N Terminus ◽

The Matrix ◽

Mitochondrial Carrier Family ◽

Mitochondrial Intermembrane Space

Abstract Background The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. Results Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. Conclusions The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.

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Import of Carrier Proteins into Mitochondria

Biological Chemistry ◽

10.1515/bc.1999.146 ◽

1999 ◽

Vol 380 (10) ◽

pp. 1151-1156 ◽

Cited By ~ 18

Author(s):

Kaye N. Truscott ◽

Nikolaus Pfanner

Keyword(s):

Protein Import ◽

Carrier Protein ◽

Intermembrane Space ◽

Carrier Proteins ◽

Inner Membrane ◽

The Matrix

AbstractCarrier proteins located in the inner membrane of mitochondria are responsible for the exchange of metabolites between the intermembrane space and the matrix of this organelle. All members of this family are nuclear-encoded and depend on translocation machineries for their import into mitochondria. Recently many new translocation components responsible for the import of carrier proteins were identified. It is now possible to describe a detailed import pathway for this class of proteins. This review highlights the contribution made by translocation components to the process of carrier protein import into mitochondria.

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Membrane translocation of mitochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p.

Molecular Biology of the Cell ◽

10.1091/mbc.8.8.1449 ◽

1997 ◽

Vol 8 (8) ◽

pp. 1449-1460 ◽

Cited By ~ 136

Author(s):

S He ◽

T D Fox

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Gene ◽

Leader Peptide ◽

Intermembrane Space ◽

Inner Membrane ◽

C Terminus ◽

The Matrix ◽

Inner Membrane Protein ◽

Export System

To study in vivo the export of mitochondrially synthesized protein from the matrix to the intermembrane space, we have fused a synthetic mitochondrial gene, ARG8m, to the Saccharomyces cerevisiae COX2 gene in mitochondrial DNA. The Arg8mp moiety was translocated through the inner membrane when fused to the Cox2p C terminus by a mechanism dependent on topogenic information at least partially contained within the exported Cox2p C-terminal tail. The pre-Cox2p leader peptide did not signal translocation. Export of the Cox2p C-terminal tail, but not the N-terminal tail, was dependent on the inner membrane potential. The mitochondrial export system does not closely resemble the bacterial Sec translocase. However, normal translocation of both exported domains of Cox2p was defective in cells lacking the widely conserved inner membrane protein Oxa1p.

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Mitochondrial inner-membrane protease Yme1 degrades outer-membrane proteins Tom22 and Om45

The Journal of Cell Biology ◽

10.1083/jcb.201702125 ◽

2017 ◽

Vol 217 (1) ◽

pp. 139-149 ◽

Cited By ~ 16

Author(s):

Xi Wu ◽

Lanlan Li ◽

Hui Jiang

Keyword(s):

Outer Membrane ◽

Outer Membrane Proteins ◽

Ubiquitin Proteasome System ◽

Mitochondrial Outer Membrane ◽

Intermembrane Space ◽

Inner Membrane ◽

Mitochondrial Proteome ◽

Mitochondrial Inner Membrane ◽

Ubiquitin Proteasome

Mitochondria are double-membraned organelles playing essential metabolic and signaling functions. The mitochondrial proteome is under surveillance by two proteolysis systems: the ubiquitin–proteasome system degrades mitochondrial outer-membrane (MOM) proteins, and the AAA proteases maintain the proteostasis of intramitochondrial compartments. We previously identified a Doa1–Cdc48-Ufd1-Npl4 complex that retrogradely translocates ubiquitinated MOM proteins to the cytoplasm for degradation. In this study, we report the unexpected identification of MOM proteins whose degradation requires the Yme1-Mgr1-Mgr3 i-AAA protease complex in mitochondrial inner membrane. Through immunoprecipitation and in vivo site-specific photo–cross-linking experiments, we show that both Yme1 adapters Mgr1 and Mgr3 recognize the intermembrane space (IMS) domains of the MOM substrates and facilitate their recruitment to Yme1 for proteolysis. We also provide evidence that the cytoplasmic domain of substrate can be dislocated into IMS by the ATPase activity of Yme1. Our findings indicate a proteolysis pathway monitoring MOM proteins from the IMS side and suggest that the MOM proteome is surveilled by mitochondrial and cytoplasmic quality control machineries in parallel.

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Abstract 995: Blockade Of Electron Transport Preserves The Contents Of Bcl-2 And Cytochrome c In Subsarcolemmal Mitochondria During Ischemia

Circulation ◽

10.1161/circ.116.suppl_16.ii_197-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Qun Chen ◽

Edward J Lesnefsky

Keyword(s):

Electron Transport ◽

Outer Membrane ◽

Rabbit Heart ◽

Intermembrane Space ◽

Inner Mitochondrial Membrane ◽

Complex Iv ◽

Inner Membrane ◽

Transport Chain ◽

Subsarcolemmal Mitochondria

Cardiac ischemia decreases the rate of oxidative phosphorylation (OXPHOS) and the contents of cardiolipin and cytochrome c (CYTc) in subsarcolemmal mitochondria (SSM) in the isolated rabbit heart. CYTc release first requires damage to the inner mitochondrial membrane to delocalize CYTc to the intermembrane space, followed by breach of the outer membrane. The decrease in cardiolipin content allows CYTc detachment from the inner membrane. It is still unclear how CYTc passes the outer membrane for release into cytosol. We propose that ischemia increases outer-membrane leakage by depletion of bcl-2 content, and that oxidants generated by the electron transport chain (ETC) during ischemia favor bcl-2 depletion. We used blockade of the proximal ETC at complex I during ischemia with amobarbital (AMO) to test the role of ETC during ischemia. Langendorff perfused rabbit hearts were treated with AMO (2.5 mM for 1 min) or vehicle immediately before 30 min global ischemia (37°C). Time controls were perfused for 45 min. SSM were isolated at the end of ischemia. CYTc content (reduced minus oxidized spectra), OXPHOS and bcl-2 (western blotting) were measured. Ischemia decreased OXPHOS with TMPD-ascorbate as substrate (electron donor to complex IV via CYTc) and the contents of CYTc and bcl-2. In contrast, AMO preserves OXPHOS, CYTc and bcl-2. Thus, blockade of electron transport preserves bcl-2 content during ischemia with enhanced CYTc retention by SSM. The ETC contributes to mitochondrial damage during ischemia, depleting cardiolipin in the inner membrane and bcl-2 in the outer membrane favoring the two steps required for release of CYTc from mitochondria during ischemia and reperfusion.

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