PMF-seq: a highly scalable screening strategy for linking genetics to mitochondrial bioenergetics.

Our current understanding of mitochondrial organelle physiology has benefited from two broad approaches: classically, cuvette-based measurements with suspensions of isolated mitochondria, in which bioenergetic parameters are monitored acutely in response to respiratory chain substrates and inhibitors1-4, and more recently, highly scalable genetic screens for fitness phenotypes associated with coarse-grained properties of the mitochondrial state5-10. Here we introduce permeabilized-cell mitochondrial function sequencing (PMF-seq) to combine strengths of these two approaches to connect genes to detailed bioenergetic phenotypes. In PMF-seq, the plasma membranes within a pool of CRISPR mutagenized cells are gently permeabilized under conditions that preserve mitochondrial physiology, where detailed bioenergetics can be probed in the same way as with isolated organelles. Cells with desired bioenergetic parameters are selected optically using flow cytometry and subjected to next-generation sequencing. Using PMF-seq, we recover genes differentially required for mitochondrial respiratory chain branching and reversibility. We demonstrate that human D-lactate dehydrogenase specifically conveys electrons from D-lactate into cytochrome c to support mitochondrial membrane polarization. Finally, we screen for genetic modifiers of tBID, a pro-apoptotic protein that acts directly and acutely on mitochondria. We find the loss of the complex V assembly factor ATPAF2 acts as a genetic sensitizer of tBID's acute action. We anticipate that PMF-seq will be valuable for defining genes critical to the physiology of mitochondria and other organelles.

METTL17 is an Fe-S cluster checkpoint for mitochondrial translation.

Friedreich's ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.

Nuclear genetic control of mtDNA copy number and heteroplasmy in humans.

Mitochondrial DNA (mtDNA) is a maternally inherited, high-copy-number genome required for oxidative phosphorylation1. Heteroplasmy refers to the presence of a mixture of mtDNA alleles in an individual and has been associated with disease and ageing. Mechanisms underlying common variation in human heteroplasmy, and the influence of the nuclear genome on this variation, remain insufficiently explored. Here we quantify mtDNA copy number (mtCN) and heteroplasmy using blood-derived whole-genome sequences from 274,832 individuals and perform genome-wide association studies to identify associated nuclear loci. Following blood cell composition correction, we find that mtCN declines linearly with age and is associated with variants at 92 nuclear loci. We observe that nearly everyone harbours heteroplasmic mtDNA variants obeying two principles: (1) heteroplasmic single nucleotide variants tend to arise somatically and accumulate sharply after the age of 70 years, whereas (2) heteroplasmic indels are maternally inherited as mixtures with relative levels associated with 42 nuclear loci involved in mtDNA replication, maintenance and novel pathways. These loci may act by conferring a replicative advantage to certain mtDNA alleles. As an illustrative example, we identify a length variant carried by more than 50% of humans at position chrM:302 within a G-quadruplex previously proposed to mediate mtDNA transcription/replication switching2,3. We find that this variant exerts cis-acting genetic control over mtDNA abundance and is itself associated in-trans with nuclear loci encoding machinery for this regulatory switch. Our study suggests that common variation in the nuclear genome can shape variation in mtCN and heteroplasmy dynamics across the human population.

The mitochondrial calcium uniporter transports Ca 2+ via a ligand-relay mechanism.

The mitochondrial calcium uniporter (mtCU) is a multicomponent Ca 2+ -specific channel that imparts mitochondria with the capacity to sense the cytosolic calcium signals. The metazoan mtCU comprises the pore-forming subunit MCU and the essential regulator EMRE, arranged in a tetrameric channel complex, and the Ca 2+ sensing peripheral proteins MICU1-3. The mechanism of mitochondrial Ca 2+ uptake by mtCU and its regulation is poorly understood. Our analysis of MCU structure and sequence conservation, combined with molecular dynamics simulations, mutagenesis, and functional studies, led us to conclude that the Ca 2+ conductance of MCU is driven by a ligand-relay mechanism, which depends on stochastic structural fluctuations in the conserved DxxE sequence. In the tetrameric structure of MCU, the four glutamate side chains of DxxE (the E-ring) chelate Ca 2+ directly in a high-affinity complex (site 1), which blocks the channel. The four glutamates can also switch to a hydrogen bond-mediated interaction with an incoming hydrated Ca 2+ transiently sequestered within the D-ring of DxxE (site 2), thus releasing the Ca 2+ bound at site 1. This process depends critically on the structural flexibility of DxxE imparted by the adjacent invariant Pro residue. Our results suggest that the activity of the uniporter can be regulated through the modulation of local structural dynamics. A preliminary account of this work was presented at the 67 th Annual Meeting of the Biophysical Society in San Diego, CA, February 18-22, 2023.

Lipoylation is dependent on the ferredoxin FDX1 and dispensable under hypoxia in human cells.

Iron-sulfur clusters (ISC) are essential cofactors that participate in electron transfer, environmental sensing, and catalysis. Amongst the most ancient ISC-containing proteins are the ferredoxin (FDX) family of electron carriers. Humans have two FDXs- FDX1 and FDX2, both of which are localized to mitochondria, and the latter of which is itself important for ISC synthesis. We have previously shown that hypoxia can eliminate the requirement for some components of the ISC biosynthetic pathway, but FDXs were not included in that study. Here, we report that FDX1, but not FDX2, is dispensable under 1% O2 in cultured human cells. We find that FDX1 is essential for production of the lipoic acid cofactor, which is synthesized by the ISC-containing enzyme lipoyl synthase. While hypoxia can rescue the growth phenotype of either FDX1 or lipoyl synthase KO cells, lipoylation in these same cells is not rescued, arguing against an alternative biosynthetic route or salvage pathway for lipoate in hypoxia. Our work reveals the divergent roles of FDX1 and FDX2 in mitochondria, identifies a role for FDX1 in lipoate synthesis, and suggests that loss of lipoic acid can be tolerated under low oxygen tensions in cell culture.

Nuclear genetic control of mtDNA copy number and heteroplasmy in humans.

Human mitochondria contain a high copy number, maternally transmitted genome (mtDNA) that encodes 13 proteins required for oxidative phosphorylation. Heteroplasmy arises when multiple mtDNA variants co-exist in an individual and can exhibit complex dynamics in disease and in aging. As all proteins involved in mtDNA replication and maintenance are nuclear-encoded, heteroplasmy levels can, in principle, be under nuclear genetic control, however this has never been shown in humans. Here, we develop algorithms to quantify mtDNA copy number (mtCN) and heteroplasmy levels using blood-derived whole genome sequences from 274,832 individuals of diverse ancestry and perform GWAS to identify nuclear loci controlling these traits. After careful correction for blood cell composition, we observe that mtCN declines linearly with age and is associated with 92 independent nuclear genetic loci. We find that nearly every individual carries heteroplasmic variants that obey two key patterns: (1) heteroplasmic single nucleotide variants are somatic mutations that accumulate sharply after age 70, while (2) heteroplasmic indels are maternally transmitted as mtDNA mixtures with resulting levels influenced by 42 independent nuclear loci involved in mtDNA replication, maintenance, and novel pathways. These nuclear loci do not appear to act by mtDNA mutagenesis, but rather, likely act by conferring a replicative advantage to specific mtDNA molecules. As an illustrative example, the most common heteroplasmy we identify is a length variant carried by >50% of humans at position m.302 within a G-quadruplex known to serve as a replication switch. We find that this heteroplasmic variant exerts cis -acting genetic control over mtDNA abundance and is itself under trans -acting genetic control of nuclear loci encoding protein components of this regulatory switch. Our study showcases how nuclear haplotype can privilege the replication of specific mtDNA molecules to shape mtCN and heteroplasmy dynamics in the human population.

A genetically encoded system for oxygen generation in living cells.

Oxygen plays a key role in supporting life on our planet. It is particularly important in higher eukaryotes where it boosts bioenergetics as a thermodynamically favorable terminal electron acceptor and has important roles in cell signaling and development. Many human diseases stem from either insufficient or excessive oxygen. Despite its fundamental importance, we lack methods with which to manipulate the supply of oxygen with high spatiotemporal resolution in cells and in organisms. Here, we introduce a genetic system, SupplemeNtal Oxygen Released from ChLorite (SNORCL), for on-demand local generation of molecular oxygen in living cells, by harnessing prokaryotic chlorite O2-lyase (Cld) enzymes that convert chlorite (ClO2-) into molecular oxygen (O2) and chloride (Cl-). We show that active Cld enzymes can be targeted to either the cytosol or mitochondria of human cells, and that coexpressing a chlorite transporter results in molecular oxygen production inside cells in response to externally added chlorite. This first-generation system allows fine temporal and spatial control of oxygen production, with immediate research applications. In the future, we anticipate that technologies based on SNORCL will have additional widespread applications in research, biotechnology, and medicine.

Characterization of tolloid-mediated cleavage of the GDF8 procomplex.

Growth differentiation factor 8 (GDF8), a.k.a. myostatin, is a member of the larger TGFß superfamily of signaling ligands. GDF8 has been well characterized as a negative regulator of muscle mass. After synthesis, GDF8 is held latent by a noncovalent complex between the N-terminal prodomain and the signaling ligand. Activation of latent GDF8 requires proteolytic cleavage of the prodomain at residue D99 by a member of the tolloid family of metalloproteases. While tolloid proteases cleave multiple substrates, they lack a conserved consensus sequence. Here, we investigate the tolloid cleavage site of the GDF8 prodomain to determine what residues contribute to tolloid recognition and subsequent proteolysis. Using sequential alanine mutations, we identified several residues adjacent to the scissile bond, including Y94, that when mutated, abolish tolloid-mediated activation of latent GDF8. Using the astacin domain of Tll1 (Tolloid Like 1) we determined that prodomain mutants were more resistant to proteolysis. Purified latent complexes harboring the prodomain mutations, D92A and Y94A, impeded activation by tolloid but could be fully activated under acidic conditions. Finally, we show that co-expression of GDF8 WT with prodomain mutants that were tolloid resistant, suppressed GDF8 activity. Taken together our data demonstrate that residues towards the N-terminus of the scissile bond are important for tolloid-mediated activation of GDF8 and that the tolloid-resistant version of the GDF8 prodomain can function dominant negative to WT GDF8.

MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations.

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

Structural perspective of BMP ligands and signaling.

The Bone Morphogenetic Proteins (BMPs) are the largest class signaling molecules within the greater Transforming Growth Factor Beta (TGFß) family, and are responsible for a wide array of biological functions, including dorsal-ventral patterning, skeletal development and maintenance, as well as cell homeostasis. As such, dysregulation of BMPs results in a number of diseases, including fibrodysplasia ossificans progressiva (FOP) and pulmonary arterial hypertension (PAH). Therefore, understanding BMP signaling and regulation at the molecular level is essential for targeted therapeutic intervention. This review discusses the recent advances in the structural and biochemical characterization of BMPs, from canonical ligand-receptor interactions to co-receptors and antagonists. This work aims to highlight how BMPs differ from other members of the TGFß family, and how that information can be used to further advance the field. Lastly, this review discusses several gaps in the current understanding of BMP structures, with the aim that discussion of these gaps will lead to advancements in the field.

Mutations in GDF11 and the extracellular antagonist, Follistatin, as a likely cause of Mendelian forms of orofacial clefting in humans.

Cleft lip with or without cleft palate (CL/P) is generally viewed as a complex trait with multiple genetic and environmental contributions. In 70% of cases, CL/P presents as an isolated feature and/or deemed nonsyndromic. In the remaining 30%, CL/P is associated with multisystem phenotypes or clinically recognizable syndromes, many with a monogenic basis. Here we report the identification, via exome sequencing, of likely pathogenic variants in two genes that encode interacting proteins previously only linked to orofacial clefting in mouse models. A variant in GDF11 (encoding growth differentiation factor 11), predicting a p.(Arg298Gln) substitution at the Furin protease cleavage site, was identified in one family that segregated with CL/P and both rib and vertebral hypersegmentation, mirroring that seen in Gdf11 knockout mice. In the second family in which CL/P was the only phenotype, a mutation in FST (encoding the GDF11 antagonist, Follistatin) was identified that is predicted to result in a p.(Cys56Tyr) substitution in the region that binds GDF11. Functional assays demonstrated a significant impact of the specific mutated amino acids on FST and GDF11 function and, together with embryonic expression data, provide strong evidence for the importance of GDF11 and Follistatin in the regulation of human orofacial development.

Crystal structure of the WFIKKN2 follistatin domain reveals insight into how it inhibits growth differentiation factor 8 (GDF8) and GDF11.

Growth differentiation factor 8 (GDF8; also known as myostatin) and GDF11 are closely related members of the transforming growth factor ß (TGF-ß) family. GDF8 strongly and negatively regulates skeletal muscle growth, and GDF11 has been implicated in various age-related pathologies such as cardiac hypertrophy. GDF8 and GDF11 signaling activities are controlled by the extracellular protein antagonists follistatin; follistatin-like 3 (FSTL3); and WAP, follistatin/kazal, immunoglobulin, Kunitz, and netrin domain-containing (WFIKKN). All of these proteins contain a follistatin domain (FSD) important for ligand binding and antagonism. Here, we investigated the structure and function of the FSD from murine WFIKKN2 and compared it with the FSDs of follistatin and FSTL3. Using native gel shift and surface plasmon resonance analyses, we determined that the WFIKKN2 FSD can interact with both GDF8 and GDF11 and block their interactions with the type II receptor activin A receptor type 2B (ActRIIB). Further, we solved the crystal structure of the WFIKKN2 FSD to 1.39 Å resolution and identified surface-exposed residues that, when substituted with alanine, reduce antagonism of GDF8 in full-length WFIKKN2. Comparison of the WFIKKN2 FSD with those of follistatin and FSTL3 revealed differences in both the FSD structure and position of residues within the domain that are important for ligand antagonism. Taken together, our results indicate that both WFIKKN and follistatin utilize their FSDs to block the type II receptor but do so via different binding interactions.

Structure of an EIIC sugar transporter trapped in an inward-facing conformation.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria and phosphorylates the sugar for metabolic consumption. The PTS is important for the survival of bacteria and thus a potential target for antibiotics, but its mechanism of sugar uptake and phosphorylation remains unclear. The PTS is composed of multiple proteins, and the membrane-embedded Enzyme IIC (EIIC) component transports sugars across the membrane. Crystal structures of two members of the glucose superfamily of EIICs, bcChbC and bcMalT, were solved in the inward-facing and outward-facing conformations, and the structures suggest that sugar translocation could be achieved by movement of a structured domain that contains the sugar-binding site. However, different conformations have not been captured on the same transporter to allow precise description of the conformational changes. Here we present a crystal structure of bcMalT trapped in an inward-facing conformation by a mercury ion that bridges two strategically placed cysteine residues. The structure allows direct comparison of the outward- and inward-facing conformations and reveals a large rigid-body motion of the sugar-binding domain and other conformational changes that accompany the rigid-body motion. All-atom molecular dynamics simulations show that the inward-facing structure is stable with or without the cross-linking. The conformational changes were further validated by single-molecule Föster resonance energy transfer (smFRET). Combined, these results establish the elevator-type mechanism of transport in the glucose superfamily of EIIC transporters.

Structure of the human myostatin precursor and determinants of growth factor latency.

Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have determined the structure of unprocessed pro-myostatin and analysed the properties of the protein in its different forms. Crystal structures and SAXS analyses show that pro-myostatin adopts an open, V-shaped structure with a domain-swapped arrangement. The pro-mature complex, after cleavage of the furin site, has significantly reduced activity compared with the mature growth factor and persists as a stable complex that is resistant to the natural antagonist follistatin. The latency appears to be conferred by a number of distinct features that collectively stabilise the interaction of the pro-domains with the mature growth factor, enabling a regulated stepwise activation process, distinct from the prototypical pro-TGF-ß1. These results provide a basis for understanding the effect of missense mutations in pro-myostatin and pave the way for the design of novel myostatin inhibitors.

Molecular characterization of latent GDF8 reveals mechanisms of activation.

Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-ß. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or "triggered" state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is "spring-loaded." Structural analysis shows that the prodomain:GDF8 complex adopts an "open" configuration, distinct from the latency state of TGF-ß and more similar to the open state of Activin A and BMP9 (nonlatent complexes). We determined that GDF8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared with WT GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.

Structural basis for potency differences between GDF8 and GDF11.

BACKGROUND: Growth/differentiation factor 8 (GDF8) and GDF11 are two highly similar members of the transforming growth factor ß (TGFß) family. While GDF8 has been recognized as a negative regulator of muscle growth and differentiation, there are conflicting studies on the function of GDF11 and whether GDF11 has beneficial effects on age-related dysfunction. To address whether GDF8 and GDF11 are functionally identical, we compared their signaling and structural properties. RESULTS: Here we show that, despite their high similarity, GDF11 is a more potent activator of SMAD2/3 and signals more effectively through the type I activin-like receptor kinase receptors ALK4/5/7 than GDF8. Resolution of the GDF11:FS288 complex, apo-GDF8, and apo-GDF11 crystal structures reveals unique properties of both ligands, specifically in the type I receptor binding site. Lastly, substitution of GDF11 residues into GDF8 confers enhanced activity to GDF8. CONCLUSIONS: These studies identify distinctive structural features of GDF11 that enhance its potency, relative to GDF8; however, the biological consequences of these differences remain to be determined.

Baculovirus FP25K Localization: Role of the Coiled-Coil Domain.

Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins. IMPORTANCE: Baculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha- and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane before occlusion, this function is not needed, and the domain was lost or never acquired.

The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport.

The phosphoenolpyruvate:carbohydrate phosphotransferase systems are found in bacteria, where they play central roles in sugar uptake and regulation of cellular uptake processes. Little is known about how the membrane-embedded components (EIICs) selectively mediate the passage of carbohydrates across the membrane. Here we report the functional characterization and 2.55-Å resolution structure of a maltose transporter, bcMalT, belonging to the glucose superfamily of EIIC transporters. bcMalT crystallized in an outward-facing occluded conformation, in contrast to the structure of another glucose superfamily EIIC, bcChbC, which crystallized in an inward-facing occluded conformation. The structures differ in the position of a structurally conserved substrate-binding domain that is suggested to play a central role in sugar transport. In addition, molecular dynamics simulations suggest a potential pathway for substrate entry from the periplasm into the bcMalT substrate-binding site. These results provide a mechanistic framework for understanding substrate recognition and translocation for the glucose superfamily EIIC transporters.