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The inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration. The ETC relies on mobile carriers; therefore, it has long been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology. However, it is unclear if cells temporally modulate IMM fluidity upon metabolic or other stimulation. Using a photostable, red-shifted, cell-permeable molecular-rotor, Mitorotor-1, we present a multiplexed approach for quantitatively mapping IMM fluidity in living cells. This reveals IMM fluidity to be linked to cellular-respiration and responsive to stimuli. Multiple approaches combining in vitro experiments and live-cell fluorescence (FLIM) lifetime imaging microscopy (FLIM) show Mitorotor-1 to robustly report IMM 'microviscosity'/fluidity through changes in molecular free volume. Interestingly, external osmotic stimuli cause controlled swelling/compaction of mitochondria, thereby revealing a graded Mitorotor-1 response to IMM microviscosity. Lateral diffusion measurements of IMM correlate with microviscosity reported via Mitorotor-1 FLIM-lifetime, showing convergence of independent approaches for measuring IMM local-order. Mitorotor-1 FLIM reveals mitochondrial heterogeneity in IMM fluidity; between-and-within cells and across single mitochondrion. Multiplexed FLIM lifetime imaging of Mitorotor-1 and NADH autofluorescence reveals that IMM fluidity positively correlates with respiration, across individual cells. Remarkably, we find that stimulating respiration, through nutrient deprivation or chemically, also leads to increase in IMM fluidity. These data suggest that modulating IMM fluidity supports enhanced respiratory flux. Our study presents a robust method for measuring IMM fluidity and suggests a dynamic regulatory paradigm of modulating IMM local order on changing metabolic demand.

The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body (BB) of the flagellum. Here, we studied the architecture of the exclusion zone filament (EZF) of the TAC, the only known component of which is p197, that connects the BB with the mitochondrial outer membrane (OM). We show that p197 has three domains that are all essential for mitochondrial DNA inheritance. The C terminus of p197 interacts with the mature and probasal body (pro-BB), whereas its N terminus binds to the peripheral OM protein TAC65. The large central region of p197 has a high α-helical content and likely acts as a flexible spacer. Ultrastructure expansion microscopy (U-ExM) of cell lines exclusively expressing p197 versions of different lengths that contain both N- and C-terminal epitope tags demonstrates that full-length p197 alone can bridge the ∼270-nm distance between the BB and the cytosolic face of the OM. Thus U-ExM allows the localization of distinct domains within the same molecules and suggests that p197 is the TAC subunit most proximal to the BB. In addition, U-ExM revealed that p197 acts as a spacer molecule, as two shorter versions of p197, with the repeat domain either removed or replaced by the central domain of the Trypanosoma cruzi p197 ortholog reduced the distance between the BB and the OM in proportion to their predicted molecular weight.

Cellular respiration is powered by membrane-bound redox enzymes that convert chemical energy into an electrochemical proton gradient and drive the energy metabolism. By combining large-scale classical and quantum mechanical simulations with cryo-electron microscopy data, we resolve here molecular details of conformational changes linked to proton pumping in the mammalian complex I. Our data suggest that complex I deactivation blocks water-mediated proton transfer between a membrane-bound quinone site and proton-pumping modules, decoupling the energy-transduction machinery. We identify a putative gating region at the interface between membrane domain subunits ND1 and ND3/ND4L/ND6 that modulates the proton transfer by conformational changes in transmembrane helices and bulky residues. The region is perturbed by mutations linked to human mitochondrial disorders and is suggested to also undergo conformational changes during catalysis of simpler complex I variants that lack the "active"-to-"deactive" transition. Our findings suggest that conformational changes in transmembrane helices modulate the proton transfer dynamics by wetting/dewetting transitions and provide important functional insight into the mammalian respiratory complex I., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The structure of the dimeric ATP synthase from bovine mitochondria determined in three rotational states by electron cryo-microscopy provides evidence that the proton uptake from the mitochondrial matrix via the proton inlet half channel proceeds via a Grotthus mechanism, and a similar mechanism may operate in the exit half channel. The structure has given information about the architecture and mechanical constitution and properties of the peripheral stalk, part of the membrane extrinsic region of the stator, and how the action of the peripheral stalk damps the side-to-side rocking motions that occur in the enzyme complex during the catalytic cycle. It also describes wedge structures in the membrane domains of each monomer, where the skeleton of each wedge is provided by three α-helices in the membrane domains of the b-subunit to which the supernumerary subunits e, f, and g and the membrane domain of subunit A6L are bound. Protein voids in the wedge are filled by three specifically bound cardiolipin molecules and two other phospholipids. The external surfaces of the wedges link the monomeric complexes together into the dimeric structures and provide a pivot to allow the monomer-monomer interfaces to change during catalysis and to accommodate other changes not related directly to catalysis in the monomer-monomer interface that occur in mitochondrial cristae. The structure of the bovine dimer also demonstrates that the structures of dimeric ATP synthases in a tetrameric porcine enzyme have been seriously misinterpreted in the membrane domains., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Mitochondria have a characteristic ultrastructure with invaginations of the inner membrane called cristae that contain the protein complexes of the oxidative phosphorylation system. How this particular morphology of the respiratory membrane impacts energy conversion is currently unknown. One proposed role of cristae formation is to facilitate the establishment of local proton gradients to fuel ATP synthesis. Here, we determined the local pH values at defined sublocations within mitochondria of respiring yeast cells by fusing a pH-sensitive GFP to proteins residing in different mitochondrial subcompartments. Only a small proton gradient was detected over the inner membrane in wild type or cristae-lacking cells. Conversely, the obtained pH values did barely permit ATP synthesis in a reconstituted system containing purified yeast F 1 F 0 ATP synthase, although, thermodynamically, a sufficiently high driving force was applied. At higher driving forces, where robust ATP synthesis was observed, a P -side pH value of 6 increased the ATP synthesis rate 3-fold compared to pH 7. In contrast, when ATP synthase was coreconstituted with an active proton-translocating cytochrome oxidase, ATP synthesis readily occurred at the measured, physiological pH values. Our study thus reveals that the morphology of the inner membrane does not influence the subcompartmental pH values and is not necessary for robust oxidative phosphorylation in mitochondria. Instead, it is likely that the dense packing of the oxidative phosphorylation complexes in the cristae membranes assists kinetic coupling between proton pumping and ATP synthesis., Competing Interests: The authors declare no competing interest.

Mitochondrial ATP synthases form dimers, which assemble into long ribbons at the rims of the inner membrane cristae. We reconstituted detergent-purified mitochondrial ATP synthase dimers from the green algae Polytomella sp. and the yeast Yarrowia lipolytica into liposomes and examined them by electron cryotomography. Tomographic volumes revealed that ATP synthase dimers from both species self-assemble into rows and bend the lipid bilayer locally. The dimer rows and the induced degree of membrane curvature closely resemble those in the inner membrane cristae. Monomers of mitochondrial ATP synthase reconstituted into liposomes do not bend membrane visibly and do not form rows. No specific lipids or proteins other than ATP synthase dimers are required for row formation and membrane remodelling. Long rows of ATP synthase dimers are a conserved feature of mitochondrial inner membranes. They are required for cristae formation and a main factor in mitochondrial morphogenesis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

In this Inaugural Article, I trace some key steps that have enabled the development of mass spectrometry for the study of intact protein complexes from a variety of cellular environments. Beginning with the preservation of the first soluble complexes from plasma, I describe our early experiments that capitalize on the heterogeneity of subunit composition during assembly and exchange reactions. During these investigations, we observed many assemblies and intermediates with different subunit stoichiometries, and were keen to ascertain whether or not their overall topology was preserved in the mass spectrometer. Adapting ion mobility and soft-landing methodologies, we showed how ring-shaped complexes could survive the phase transition. The next logical progression from soluble complexes was to membrane protein assemblies but this was not straightforward. We encountered many pitfalls along the way, largely due to the use of detergent micelles to protect and stabilize complexes. Further obstacles presented when we attempted to distinguish lipids that copurify from those that are important for function. Developing new experimental protocols, we have subsequently defined lipids that change protein conformation, mediate oligomeric states, and facilitate downstream coupling of G protein-coupled receptors. Very recently, using a radical method-ejecting protein complexes directly from native membranes into mass spectrometers-we provided insights into associations within membranes and mitochondria. Together, these developments suggest the beginnings of mass spectrometry meeting with cell biology., Competing Interests: The author declares no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Current strategies used to quantitatively describe the biological diversity of lipids by mass spectrometry are often limited in assessing the exact structural variability of individual molecular species in detail. A major challenge is represented by the extensive isobaric overlap present among lipids, hampering their accurate identification. This is especially true for cardiolipins, a mitochondria-specific class of phospholipids, which are functionally involved in many cellular functions, including energy metabolism, cristae structure, and apoptosis. Substituted with four fatty acyl side chains, cardiolipins offer a particularly high potential to achieve complex mixtures of molecular species. Here, we demonstrate how systematically generated high-performance liquid chromatography-mass spectral data can be utilized in a mathematical structural modeling approach, to comprehensively analyze and characterize the molecular diversity of mitochondrial cardiolipin compositions in cell culture and disease models, cardiolipin modulation experiments, and a broad variety of frequently studied model organisms., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Male sterility is an essential trait in hybrid seed production for monoclinous crops, including rice and wheat. However, compared with the high percentage of hybrid rice planted in the world, little commercial hybrid wheat is planted globally as a result of the lack of a suitable system for male sterility. Therefore, understanding the molecular nature of male fertility in wheat is critical for commercially viable hybrid wheat. Here, we report the cloning and characterization of Male Sterility 1 ( Ms1 ) in bread wheat by using a combination of advanced genomic approaches. MS1 is a newly evolved gene in the Poaceae that is specifically expressed in microsporocytes, and is essential for microgametogenesis. Orthologs of Ms1 are expressed in diploid and allotetraploid ancestral species. Orthologs of Ms1 are epigenetically silenced in the A and D subgenomes of allohexaploid wheat; only Ms1 from the B subgenome is expressed. The encoded protein, Ms1, is localized to plastid and mitochondrial membranes, where it exhibits phospholipid-binding activity. These findings provide a foundation for the development of commercially viable hybrid wheat., Competing Interests: The authors declare no conflict of interest.

Mitochondria constantly divide and fuse. Homotypic fusion of the outer mitochondrial membranes requires the mitofusin (MFN) proteins, a family of dynamin-like GTPases. MFNs are anchored in the membrane by transmembrane (TM) segments, exposing both the N-terminal GTPase domain and the C-terminal tail (CT) to the cytosol. This arrangement is very similar to that of the atlastin (ATL) GTPases, which mediate fusion of endoplasmic reticulum (ER) membranes. We engineered various MFN-ATL chimeras to gain mechanistic insight into MFN-mediated fusion. When MFN1 is localized to the ER by TM swapping with ATL1, it functions in the maintenance of ER morphology and fusion. In addition, an amphipathic helix in the CT of MFN1 is exchangeable with that of ATL1 and critical for mitochondrial localization of MFN1. Furthermore, hydrophobic residues N-terminal to the TM segments of MFN1 play a role in membrane targeting but not fusion. Our findings provide important insight into MFN-mediated membrane fusion., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques-surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations-suggest that α-tubulin's amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic "mitochondrial" membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents., Competing Interests: The authors declare no conflict of interest.

The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.

The voltage-dependent anion channel (VDAC), located in the outer mitochondrial membrane, acts as a gatekeeper for the entry and exit of mitochondrial metabolites. Here we reveal functional dynamics of isoform one of VDAC (VDAC1) by a combination of solution NMR spectroscopy, Gaussian network model analysis, and molecular dynamics simulation. Micro- to millisecond dynamics are significantly increased for the N-terminal six β-strands of VDAC1 in micellar solution, in agreement with increased B-factors observed in the same region in the bicellar crystal structure of VDAC1. Molecular dynamics simulations reveal that a charge on the membrane-facing glutamic acid 73 (E73) accounts for the elevation of N-terminal protein dynamics as well as a thinning of the nearby membrane. Mutation or chemical modification of E73 strongly reduces the micro- to millisecond dynamics in solution. Because E73 is necessary for hexokinase-I-induced VDAC channel closure and inhibition of apoptosis, our results imply that micro- to millisecond dynamics in the N-terminal part of the barrel are essential for VDAC interaction and gating.

Humans and most other animals use 2 different genetic codes to translate their hereditary information: the standard code for nuclear-encoded proteins and a modern variant of this code in mitochondria. Despite the pivotal role of the genetic code for cell biology, the functional significance of the deviant mitochondrial code has remained enigmatic since its first description in 1979. Here, we show that profound and functionally beneficial alterations on the encoded protein level were causative for the AUA codon reassignment from isoleucine to methionine observed in most mitochondrial lineages. We demonstrate that this codon reassignment leads to a massive accumulation of the easily oxidized amino acid methionine in the highly oxidative inner mitochondrial membrane. This apparently paradoxical outcome can yet be smoothly settled if the antioxidant surface chemistry of methionine is taken into account, and we present direct experimental evidence that intramembrane accumulation of methionine exhibits antioxidant and cytoprotective properties in living cells. Our results unveil that methionine is an evolutionarily selected antioxidant building block of respiratory chain complexes. Collective protein alterations can thus constitute the selective advantage behind codon reassignments, which authenticates the "ambiguous decoding" hypothesis of genetic code evolution. Oxidative stress has shaped the mitochondrial genetic code.

Exchange of ATP and ADP across mitochondrial membrane replenishes the cytoplasm with newly synthesized ATP and provides the mitochondria with the substrate ADP for oxidative phosphorylation. The sole means of this exchange is the mitochondrial ADP/ATP carrier (AAC), a membrane protein that is suggested to cycle between two conformationally distinct states, cytosolic-open (c-state) and matrix-open (m-state), thereby shuttling nucleotides across the inner mitochondrial membrane. However, the c-state is the only structurally resolved state, and the binding site of ADP remains elusive. Here, we present approximately 0.3 mus of all-atom MD simulations of the c-state revealing rapid, spontaneous binding of ADP to deeply positioned binding sites within the AAC lumen. To our knowledge, a complete ligand-binding event has heretofore not been described in full atomic detail in unbiased simulations. The identified ADP-bound state and additional simulations shed light on key structural elements and the initial steps involved in conversion to the m-state. Electrostatic analysis of trajectories reveals the presence of an unusually strong positive electrostatic potential in the lumen of AAC that appears to be the main driving force for the observed spontaneous binding of ADP. We provide evidence that the positive electrostatic potential is likely a common attribute among the entire family of mitochondrial carriers. In addition to playing a key role in substrate recruitment and translocation, the electropositivity of mitochondrial carriers might also be critical for their binding to the negatively charged environment of the inner mitochondrial membrane.

MitoNEET is a protein of unknown function present in the mitochondrial membrane that was recently shown to bind specifically the antidiabetic drug pioglizatone. Here, we report the crystal structure of the soluble domain (residues 32-108) of human mitoNEET at 1.8-A resolution. The structure reveals an intertwined homodimer, and each subunit was observed to bind a [2Fe-2S] cluster. The [2Fe-2S] ligation pattern of three cysteines and one histidine differs from the known pattern of four cysteines in most cases or two cysteines and two histidines as observed in Rieske proteins. The [2Fe-2S] cluster is packed in a modular structure formed by 17 consecutive residues. The cluster-binding motif is conserved in at least seven distinct groups of proteins from bacteria, archaea, and eukaryotes, which show a consensus sequence of (hb)-C-X(1)-C-X(2)-(S/T)-X(3)-P-(hb)-C-D-X(2)-H, where hb represents a hydrophobic residue; we term this a CCCH-type [2Fe-2S] binding motif. The nine conserved residues in the motif contribute to iron ligation and structure stabilization. UV-visible absorption spectra indicated that mitoNEET can exist in oxidized and reduced states. Our study suggests an electron transfer function for mitoNEET and for other proteins containing the CCCH motif.