scholarly journals Structural organization of mitochondrial human complex I: role of the ND4 and ND5 mitochondria-encoded subunits and interaction with prohibitin

2004 ◽  
Vol 383 (3) ◽  
pp. 491-499 ◽  
Author(s):  
Ingrid BOURGES ◽  
Claire RAMUS ◽  
Bénédicte MOUSSON de CAMARET ◽  
Réjane BEUGNOT ◽  
Claire REMACLE ◽  
...  
Keyword(s):  
Complex I ◽  
Nadh Dehydrogenase ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Membrane Association ◽  
Oxidoreductase Activity ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Complex I Assembly ◽  
Human Complex

Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of these subunits have significant pathogenic consequences in humans. However, the way these events affect complex I assembly is poorly documented. To understand the effects of particular mutations in ND subunits on complex I assembly, we studied four human cell lines: ND4 non-expressing cells, ND5 non-expressing cells, and rho° cells that do not express any ND subunits, in comparison with normal complex I control cells. In control cells, all the seven analysed nuclear-encoded complex I subunits were found to be attached to the mitochondrial inner membrane, except for the 24 kDa subunit, which was nearly equally partitioned between the membranes and the matrix. Absence of a single ND subunit, or even all the seven ND subunits, caused no major changes in the nuclear-encoded complex I subunit content of mitochondria. However, in cells lacking ND4 or ND5, very low amounts of 24 kDa subunit were found associated with the membranes, whereas most of the other nuclear-encoded subunits remained attached. In contrast, membrane association of most of the nuclear subunits was significantly reduced in the absence of all seven ND proteins. Immunopurification detected several subcomplexes. One of these, containing the 23, 30 and 49 kDa subunits, also contained prohibitin. This is the first description of prohibitin interaction with complex I subunits and suggests that this protein might play a role in the assembly or degradation of mitochondrial complex I.

External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency in Yarrowia lipolytica

2001 ◽  
Vol 114 (21) ◽  
pp. 3915-3921 ◽  
Author(s):  
Stefan J. Kerscher ◽  
Andrea Eschemann ◽  
Pamela M. Okun ◽  
Ulrich Brandt
Keyword(s):  
Yarrowia Lipolytica ◽  
Complex I ◽  
Functional Expression ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Cytosolic Side ◽  
Respiratory Membrane

Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.

A single external enzyme confers alternative NADH:ubiquinone oxidoreductase activity in Yarrowia lipolytica

1999 ◽  
Vol 112 (14) ◽  
pp. 2347-2354 ◽  
Author(s):  
S.J. Kerscher ◽  
J.G. Okun ◽  
U. Brandt
Keyword(s):  
Yarrowia Lipolytica ◽  
Complex I ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Inner Mitochondrial Membrane ◽  
Gene Encoding ◽  
Oxidoreductase Activity ◽  
Mitochondrial Inner Membrane ◽  
Energy Conserving ◽  
And Function ◽  
Complex I Activity

NADH:ubiquinone oxidoreductases catalyse the first step within the diverse pathways of mitochondrial NADH oxidation. In addition to the energy-conserving form commonly called complex I, fungi and plants contain much simpler alternative NADH:ubiquinone oxido-reductases that catalyze the same reaction but do not translocate protons across the inner mitochondrial membrane. Little is known about the distribution and function of these enzymes. We have identified YLNDH2 as the only gene encoding an alternative NADH:ubiquinone oxidoreductase (NDH2) in the obligate aerobic yeast Yarrowia lipolytica. Cells carrying a deletion of YLNDH2 were fully viable; full inhibition by piericidin A indicated that complex I activity was the sole NADH:ubiquinone oxidoreductase activity left in the deletion strains. Studies with intact mitochondria revealed that NDH2 in Y. lipolytica is oriented towards the external face of the mitochondrial inner membrane. This is in contrast to the situation seen in Saccharomyces cerevisiae, Neurospora crassa and in green plants, where internal alternative NADH:ubiquinone oxidoreductases have been reported. Phylogenetic analysis of known NADH:ubiquinone oxidoreductases suggests that during evolution conversion of an ancestral external alternative NADH:ubiquinone oxidoreductase to an internal enzyme may have paved the way for the loss of complex I in fermenting yeasts like S. cerevisiae.

scholarly journals Analysis of the Assembly Profiles for Mitochondrial- and Nuclear-DNA-Encoded Subunits into Complex I

2007 ◽  
Vol 27 (12) ◽  
pp. 4228-4237 ◽  
Author(s):  
Michael Lazarou ◽  
Matthew McKenzie ◽  
Akira Ohtake ◽  
David R. Thorburn ◽  
Michael T. Ryan
Keyword(s):  
Complex I ◽  
Nuclear Dna ◽  
De Novo ◽  
Nuclear Gene ◽  
Subunit Exchange ◽  
Mitochondrial Inner Membrane ◽  
Large Size ◽  
Membrane Defects ◽  
Complex I Assembly ◽  
Human Complex

ABSTRACT Complex I of the respiratory chain is composed of at least 45 subunits that assemble together at the mitochondrial inner membrane. Defects in human complex I result in energy generation disorders and are also implicated in Parkinson's disease and altered apoptotic signaling. The assembly of this complex is poorly understood and is complicated by its large size and its regulation by two genomes, with seven subunits encoded by mitochondrial DNA (mtDNA) and the remainder encoded by nuclear genes. Here we analyzed the assembly of a number of mtDNA- and nuclear-gene-encoded subunits into complex I. We found that mtDNA-encoded subunits first assemble into intermediate complexes and require significant chase times for their integration into the holoenzyme. In contrast, a set of newly imported nuclear-gene-encoded subunits integrate with preexisting complex I subunits to form intermediates and/or the fully assembly holoenzyme. One of the intermediate complexes represents a subassembly associated with the chaperone B17.2L. By using isolated patient mitochondria, we show that this subassembly is a productive intermediate in complex I assembly since import of the missing subunit restores complex I assembly. Our studies point to a mechanism of complex I biogenesis involving two complementary processes, (i) synthesis of mtDNA-encoded subunits to seed de novo assembly and (ii) exchange of preexisting subunits with newly imported ones to maintain complex I homeostasis. Subunit exchange may also act as an efficient mechanism to prevent the accumulation of oxidatively damaged subunits that would otherwise be detrimental to mitochondrial oxidative phosphorylation and have the potential to cause disease.

scholarly journals The mitochondrial AAA protease FTSH3 regulates Complex I abundance by promoting its disassembly

2021 ◽  
Author(s):  
Aneta Ivanova ◽  
Abi S Ghifari ◽  
Oliver Berkowitz ◽  
James Whelan ◽  
Monika W Murcha
Keyword(s):  
Complex I ◽  
Nadh Dehydrogenase ◽  
Slow Growth ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Forward Genetic Screen ◽  
Slow Growth Rate ◽  
Partial Restoration ◽  
Screen Approach

Abstract ATP is generated in mitochondria by oxidative phosphorylation. Complex I (NADH:ubiquinone oxidoreductase or NADH dehydrogenase) is the first multisubunit protein complex of this pathway, oxidising NADH and transferring electrons to the ubiquinone pool. Typically Complex I mutants display a slow growth rate compared to wild-type plants. Here, using a forward genetic screen approach for restored growth of a Complex I mutant, we have identified the mitochondrial ATP dependent metalloprotease, Filamentous Temperature Sensitive H 3 (FTSH3), as a factor that is required for the disassembly of Complex I. An ethyl methanesulfonate-induced mutation in FTSH3, named rmb1 (restoration of mitochondrial biogenesis 1), restored Complex I abundance and plant growth. Complementation could be achieved with FTSH3 lacking proteolytic activity, suggesting the unfoldase function of FTSH3 has a role in Complex I disassembly. The introduction of the rmb1 to an additional, independent, and extensively characterised Complex I mutant, ndufs4, resulted in similar increases to Complex I abundance and a partial restoration of growth. These results show that disassembly or degradation of Complex I plays a role in determining its steady-state abundance and thus turnover may vary under different conditions.

Leishmania type II dehydrogenase is essential for parasite viability irrespective of the presence of an active complex I

2021 ◽  
Vol 118 (42) ◽  
pp. e2103803118
Author(s):  
Margarida Duarte ◽  
Cleide Ferreira ◽  
Gurleen Kaur Khandpur ◽  
Tamara Flohr ◽  
Jannik Zimmermann ◽  
...  
Keyword(s):  
Complex I ◽  
Nadh Dehydrogenase ◽  
Leishmania Major ◽  
Proton Translocation ◽  
Protozoan Parasite ◽  
Type I ◽  
Type Ii ◽  
Active Complex ◽  
Mitochondrial Inner Membrane ◽  
Candidate Target

Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I–expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I–overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2—an enzyme without a counterpart in mammals—as a candidate target for leishmanicidal drugs.

Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated human complex I deficiency

2005 ◽  
Vol 289 (4) ◽  
pp. C881-C890 ◽  
Author(s):  
Werner J. H. Koopman ◽  
Henk-Jan Visch ◽  
Sjoerd Verkaart ◽  
Lambertus W. P. J. van den Heuvel ◽  
Jan A. M. Smeitink ◽  
...  
Keyword(s):  
Form Factor ◽  
Aspect Ratio ◽  
Complex I ◽  
Mitochondrial Morphology ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Mitochondrial Network ◽  
Mitochondrial Structure ◽  
Complex I Deficiency ◽  
Complex I Activity ◽  
Human Complex

Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency.

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+removal: aberrations in human complex I deficiency

2006 ◽  
Vol 291 (2) ◽  
pp. C308-C316 ◽  
Author(s):  
Henk-Jan Visch ◽  
Werner J. H. Koopman ◽  
Dimphy Zeegers ◽  
Sjenet E. van Emst-de Vries ◽  
Frank J. M. van Kuppeveld ◽  
...  
Keyword(s):  
Digital Imaging ◽  
Complex I ◽  
Removal Rate ◽  
Mitochondrial Matrix ◽  
Atp Production ◽  
Complex I Deficiency ◽  
Deficient Patient ◽  
Atp Supply ◽  
Human Complex

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]M) and the rate of cytosolic Ca2+removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial Ca2+indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial Ca2+concentration ([Ca2+]M) and, consequently, the Ca2+-stimulated increase in [ATP]M, thus allowing studies of how the increase in [ATP]Mand the cytosolic Ca2+removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose dependently decreased the Bk-induced increase in [Ca2+]Mand [ATP]Mby maximally 80 and 90%, respectively. Digital imaging microscopy of cells coloaded with the cytosolic Ca2+indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic Ca2+removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic Ca2+removal during stimulation with a Ca2+-mobilizing agonist. In contrast, complex I-deficient patient fibroblasts displayed a cytosolic Ca2+removal rate that was already decreased by 40% compared with healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic Ca2+pumps. This work reveals the usefulness of rhodamine-based Ca2+indicators in examining the role of intramitochondrial Ca2+in mitochondrial (patho) physiology.

scholarly journals Structure of inhibitor-bound mammalian complex I

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hannah R. Bridges ◽  
Justin G. Fedor ◽  
James N. Blaza ◽  
Andrea Di Luca ◽  
Alexander Jussupow ◽  
...  
Keyword(s):  
Complex I ◽  
Energy Coupling ◽  
Competitive Inhibitor ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Mouse Heart ◽  
Coupling Mechanism ◽  
Substrate Reduction ◽  
Mitochondrial Inner Membrane ◽  
Respiratory Complex I ◽  
Ubiquinone Binding

Abstract Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.

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