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Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and 2) are major facilitator superfamily transporters from the solute carrier family 49. Dysregulation of these ubiquitous transporters has been linked to various haematological and neurological disorders. While both FLVCRs were initially proposed to hold a physiological function in heme transport, subsequent studies questioned this notion. Here, we used structural, computational and biochemical methods and conclude that these two FLVCRs function as human choline transporters. We present cryo-electron microscopy structures of FLVCRs in different inward- and outward-facing conformations, captured in the apo state or in complex with choline in their translocation pathways. Our findings provide insights into the molecular framework of choline coordination and transport, largely mediated by conserved cation-π interactions, and further illuminate the conformational dynamics of the transport cycle. Moreover, we identified a heme binding site on the protein surface of the FLVCR2 N-domain, and observed that heme actively drives the conformational transitions of the protein. This auxiliary binding site might indicate a potential regulatory role of heme in the FLVCR2 transport mechanisms. Our work resolves the contested substrate specificity of the FLVCRs, and sheds light on the process of maintaining cellular choline homeostasis at the molecular level.
ER remodeling via ER-phagy
(2022)
The endoplasmic reticulum (ER) is a hotspot for many essential cellular functions. The ER membrane is highly dynamic, which affects many cellular processes that take place within the ER. One such process is ER-phagy, a selective degradation of ER fragments (including membranes and luminal content), which serves to preserve the size of ER while adapting its morphology under basal and stress conditions. In order to be degraded, the ER undergoes selective fragmentation facilitated by specialized ER-shaping proteins that also act as ER-phagy receptors. Their ability to sense and induce membrane curvature, as well as to bridge the ER with autophagy machinery, allows for a successful ER fragmentation and delivery of these fragments to the lysosome for degradation and recycling. In this review, we provide insights into ER-phagy from the perspective of membrane remodeling. We highlight the importance of ER membrane dynamics during ER-phagy and emphasize how its dysregulation reflects on human physiology and pathology.
Some quantitative data about the carbon-metabolism in Saccharomyces-cells of different ploidy were determined. The amount of carbon, necessary for the formation of a cell, proved to be proportional to the degree of ploidy of the cells. For the duplication of a diploid cell 6,7·10-11g glucose were used. In comparison with respiratory deficient cells the simultaneous utilization of fermentation and respiration metabolism in respiration sufficient cells leads to a decrease of the cell cycle duration, however, the energy needed for the formation of a cell is not decreased. The rate of cell multiplication has a maximum at about 30 °C for all classes of ploidy. Certain assumptions about the utilization of the carbon source were confirmed by experiments with 14C marked glucose.
Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential applications in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of the wild-type CbFDH at cryogenic and ambient temperatures as well as Val120Thr mutant at cryogenic temperature determined at the Turkish Light Source "Turkish DeLight". The structures reveal new hydrogen bonds between Thr120 and water molecules in the mutant CbFDH's active site, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, our findings provide invaluable insights into future protein engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.
Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential applications in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of the wild-type CbFDH at cryogenic and ambient temperatures as well as Val120Thr mutant at cryogenic temperature determined at the Turkish Light Source "Turkish DeLight". The structures reveal new hydrogen bonds between Thr120 and water molecules in the mutant CbFDH's active site, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, our findings provide invaluable insights into future protein engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.
Cryo-electron tomography (CryoET) resolves individual macromolecules inside living cells. However, the complex composition and high density of cells challenge the faithful identification of features in tomograms. Here, we capitalize on recent advances in electron tomography and demonstrate that 3D template matching (TM) localizes a wide range of structures inside crowded eukaryotic cells with confidence 10 to 100-fold above the noise level. We establish a TM pipeline with systematically tuned parameters for automated, objective and comprehensive feature identification. High-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, lipid membranes and microtubules, and individual subunits, demonstrate that TM is generic. We resolve ~100-kDa proteins, connect the functional states of complexes to their cellular localization, and capture vaults carrying ribosomal cargo in situ. By capturing individual molecular events inside living cells with defined statistical confidence, high-confidence TM greatly speeds up the CryoET workflow and sets the stage for visual proteomics.
Multiple resistance and pH adaptation (Mrp) cation/proton antiporters are essential for growth of a variety of halophilic and alkaliphilic bacteria under stress conditions. Mrp-type antiporters are closely related to the membrane domain of respiratory complex I. We determined the structure of the Mrp antiporter from Bacillus pseudofirmus by electron cryo-microscopy at 2.2 Å resolution. The structure resolves more than 99% of the sidechains of the seven membrane subunits MrpA to MrpG plus 360 water molecules, including ~70 in putative ion translocation pathways. Molecular dynamics simulations based on the high-resolution structure revealed details of the antiport mechanism. We find that switching the position of a histidine residue between three hydrated pathways in the MrpA subunit is critical for proton transfer that drives gated trans-membrane sodium translocation. Several lines of evidence indicate that the same histidine-switch mechanism operates in respiratory complex I.
Multiple resistance and pH adaptation (Mrp) cation/proton antiporters are essential for growth of a variety of halophilic and alkaliphilic bacteria under stress conditions. Mrp-type antiporters are closely related to the membrane domain of respiratory complex I. We determined the structure of the Mrp antiporter from Bacillus pseudofirmus by electron cryo-microscopy at 2.2 Å resolution. The structure resolves more than 99% of the sidechains of the seven membrane subunits MrpA to MrpG plus 360 water molecules, including ∼70 in putative ion translocation pathways. Molecular dynamics simulations based on the high-resolution structure revealed details of the antiport mechanism. We find that switching the position of a histidine residue between three hydrated pathways in the MrpA subunit is critical for proton transfer that drives gated transmembrane sodium translocation. Several lines of evidence indicate that the same histidine-switch mechanism operates in respiratory complex I.
Proton-powered c-ring rotation in mitochondrial ATP synthase is crucial to convert the transmembrane protonmotive force into torque to drive the synthesis of ATP. Capitalizing on recent cryo-EM structures, we aim at a structural and energetic understanding of how functional directional rotation is achieved. We performed multi-microsecond atomistic simulations to determine the free energy profiles along the c-ring rotation angle before and after the arrival of a new proton. Our results reveal that rotation proceeds by dynamic sliding of the ring over the a-subunit surface, during which interactions with conserved polar residues stabilize distinct intermediates. Ordered water chains line up for a Grotthuss-type proton transfer in one of these intermediates. After proton transfer, a high barrier prevents backward rotation and an overall drop in free energy favors forward rotation, ensuring the directionality of c-ring rotation required for the thermodynamically disfavored ATP synthesis. The essential arginine of the a-subunit stabilizes the rotated configuration through a salt-bridge with the c-ring. Overall, we describe a complete mechanism for the rotation step of the ATP synthase rotor, thereby illuminating a process critical to all life at atomic resolution.
Proton-powered c-ring rotation in mitochondrial ATP synthase is crucial to convert the transmembrane protonmotive force into torque to drive the synthesis of ATP. Capitalizing on recent cryo-EM structures, we aim at a structural and energetic understanding of how functional directional rotation is achieved. We performed multi-microsecond atomistic simulations to determine the free energy profiles along the c-ring rotation angle before and after the arrival of a new proton. Our results reveal that rotation proceeds by dynamic sliding of the ring over the a-subunit surface, during which interactions with conserved polar residues stabilize distinct intermediates. Ordered water chains line up for a Grotthuss-type proton transfer in one of these intermediates. After proton transfer, a high barrier prevents backward rotation and an overall drop in free energy favors forward rotation, ensuring the directionality of c-ring rotation required for the thermodynamically disfavored ATP synthesis. The essential arginine of the a-subunit stabilizes the rotated configuration through a salt-bridge with the c-ring. Overall, we describe a complete mechanism for the rotation step of the ATP synthase rotor, thereby illuminating a process critical to all life at atomic resolution.
Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.
Hemoproteinoids related to contemporary porphyrin-dependent peroxidases were synthesized under simple conditions. The peroxidative activity of hematin increased by a factor of 50 if the hematin was bound to proteinoids whereas the catalatic activity of hematin decreased rather under the same conditions. The peroxidative activity of hemoproteinoids particularly increased with their lysine content whereas the catalatic activity especially decreased in proteinoids with high phenylalanine content. The isoelectric points of the lysine-rich peroxidic hemoproteinoids were about 8. Their relatively broad pH-activity optimum was about pH 7.0. The molecular weights were a little below 20 000. Hematin content and amino acid composition of the synthetic materials were varied greatly. The substrate specificity appeared as broad as that of biogenous peroxidases, e. g., horseradish peroxidase. Among the many substrates was NADH. The possible importance of the peroxidative oxidation of NADH-type coenzymes by primitive heterotrophic organisms or prebiological systems in an anaerobic environment is discussed.
Physikalische und thermische Kontrastierung führt bei Fixierung in Glutaraldehyd und Einbettung in Vestopal bei Parenchymzellen der Leber zu weitgehend ähnlichen Kontrastunterschieden auch bei Mitochondrien und den Membranen des Retikulums. Beide Verfahren wirken also weitgehend unspezifisch. Von den chemischen Verfahren liefert Uranylacetat im Cytoplasma ähnliche Kontrastverhältnisse wie die beiden genannten Verfahren. Das spezifische Verhalten des Uranylacetats kann z. B. an der Kontrastierung des Chromatins demonstriert werden. Sie bleibt aus, wenn die färbbare Substanz auf der Wasseroberfläche des Messertroges herausgewaschen wurde. Bleicitrat-Kontrastierung hat hier im Gegensatz zu Uranylacetat eine spezifische Wirkung nur auf RNS-haltige Zellbestandteile.
Nucleic acid and histone modifications critically depend on the tricarboxylic acid (TCA) cycle for substrates and cofactors. Although a few TCA cycle enzymes have been reported in the nucleus, the corresponding pathways are considered to operate in mitochondria. Here, we show that a part of the TCA cycle is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Nuclear localization of the latter enzyme, which produces succinyl-CoA, changed from pluripotency to a differentiated state with accompanying changes in the nuclear protein succinylation. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus, warranting a revision of the canonical view on metabolic compartmentalization.
Nucleic acid and histone modifications critically depend on central metabolism for substrates and co-factors. Although a few enzymes related to the formation of these required metabolites have been reported in the nucleus, the corresponding metabolic pathways are considered to function elsewhere in the cell. Here we show that a substantial part of the mitochondrial tricarboxylic acid (TCA) cycle, the biosynthetic hub of epigenetic modification factors, is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass-spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins, supporting their nuclear location. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus warranting a revision of the canonical view on metabolic compartmentalization and gene expression regulation.
Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. The in situ ribosome structures upon tetracycline treatment show a virtually identical drug binding-site in both strains, yet the distribution of ribosomal complexes clearly differs. While K-12 retains ribosomes in a translation competent state, tRNAs are lost in the vast majority of ED1a ribosomes. A differential response is also reflected in proteome-wide abundance and thermal stability assessment. Our study underlines the need to include molecular analyses and to consider gut bacteria when addressing antibiotic mode of action.
Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.
Das aufgefundene Ribonucleoprotein besitzt die Eigenschaft eines Vollantigens. Die durchgeführten Immunisierungsversuche ergaben eine Artspezifität des Antigens. Eine Organspezifität war nicht nachweisbar. Erste Lokalisierungsversuche mit Hilfe der Immunfluoreszenz-Methode ergaben in der Niere eine besondere Anreicherung des Nucleoproteins in den Glomerulokapseln.