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Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability became compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and caused a reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogues of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. Importantly, binding and membrane aggregation of natamycin was paralleled by a decrease of the dipole potential in the PM, and this effect was enhanced in the presence of myriocin. We conclude that ergosterol promotes binding and aggregation of natamycin in the yeast PM, which can be synergistically enhanced by inhibitors of sphingolipid synthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Cellular homeostasis depends on the supply of metabolic energy in the form of ATP and electrochemical ion gradients. The construction of synthetic cells requires a constant supply of energy to drive membrane transport and metabolism. Here, we provide synthetic cells with long-lasting metabolic energy in the form of an electrochemical proton gradient. Leveraging the L-malate decarboxylation pathway we generate a stable proton gradient and electrical potential in lipid vesicles by electrogenic L-malate/L-lactate exchange coupled to L-malate decarboxylation. By co-reconstitution with the transporters GltP and LacY, the synthetic cells maintain accumulation of L-glutamate and lactose over periods of hours, mimicking nutrient feeding in living cells. We couple the accumulation of lactose to a metabolic network for the generation of intermediates of the glycolytic and pentose phosphate pathways. This study underscores the potential of harnessing a proton motive force via a simple metabolic network, paving the way for the development of more complex synthetic systems., (© 2024. The Author(s).)

The ability to adapt to osmotically diverse and fluctuating environments is critical to the survival and resilience of bacteria that colonize the human gut and urinary tract. Environmental stress often provides cross-protection against other challenges and increases antibiotic tolerance of bacteria. Thus, it is critical to understand how E. coli and other microbes survive and adapt to stress conditions. The osmotically inducible protein Y (OsmY) is significantly upregulated in response to hypertonicity. Yet its function remains unknown for decades. We determined the solution structure and dynamics of OsmY by nuclear magnetic resonance spectroscopy, which revealed that the two Bacterial OsmY and Nodulation (BON) domains of the protein are flexibly linked under low- and high-salinity conditions. In-cell solid-state NMR further indicates that there are no gross structural changes in OsmY as a function of osmotic stress. Using cryo-electron and super-resolution fluorescence microscopy, we show that OsmY attenuates plasmolysis-induced structural changes in E. coli and improves the time to growth resumption after osmotic upshift. Structure-guided mutational and functional studies demonstrate that exposed hydrophobic residues in the BON1 domain are critical for the function of OsmY. We find no evidence for membrane interaction of the BON domains of OsmY, contrary to current assumptions. Instead, at high ionic strength, we observe an interaction with the water channel, AqpZ. Thus, OsmY does not play a simple structural role in E. coli but may influence a cascade of osmoregulatory functions of the cell., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The adenosine triphosphate (ATP)-binding cassette (ABC) importer GlnPQ from Lactococcus lactis has two sequential covalently linked substrate-binding domains (SBDs), which capture the substrates and deliver them to the translocon. The two SBDs differ in their ligand specificities, binding affinities and the distance to the transmembrane domain; interestingly, both SBDs can bind their ligands simultaneously without affecting each other. In this work, we studied the binding of ligands to both SBDs using X-ray crystallography and molecular dynamics simulations. We report three high-resolution structures of SBD1, namely, the wild-type SBD1 with bound asparagine or arginine, and E184D SBD1 with glutamine bound. Molecular dynamics (MD) simulations provide a detailed insight into the dynamics associated with open-closed transitions of the SBDs.

SUMMARYNucleotide-derived second messengers are present in all domains of life. In prokaryotes, most of their functionality is associated with general lifestyle and metabolic adaptations, often in response to environmental fluctuations of physical parameters. In the last two decades, cyclic di-AMP has emerged as an important signaling nucleotide in many prokaryotic lineages, including Firmicutes, Actinobacteria, and Cyanobacteria. Its importance is highlighted by the fact that both the lack and overproduction of cyclic di-AMP affect viability of prokaryotes that utilize cyclic di-AMP, and that it generates a strong innate immune response in eukaryotes. In bacteria that produce the second messenger, most molecular targets of cyclic di-AMP are associated with cell volume control. Besides, other evidence links the second messenger to cell wall remodeling, DNA damage repair, sporulation, central metabolism, and the regulation of glycogen turnover. In this review, we take a biochemical, quantitative approach to address the main cellular processes that are directly regulated by cyclic di-AMP and show that these processes are very connected and require regulation of a similar set of proteins to which cyclic di-AMP binds. Altogether, we argue that cyclic di-AMP is a master regulator of cell volume and that other cellular processes can be connected with cyclic di-AMP through this core function. We further highlight important directions in which the cyclic di-AMP field has to develop to gain a full understanding of the cyclic di-AMP signaling network and why some processes are regulated, while others are not., Competing Interests: The authors declare no conflict of interest.

The emergence of new protein functions is crucial for the evolution of organisms. This process has been extensively researched for soluble enzymes, but it is largely unexplored for membrane transporters, even though the ability to acquire new nutrients from a changing environment requires evolvability of transport functions. Here, we demonstrate the importance of environmental pressure in obtaining a new activity or altering a promiscuous activity in members of the amino acid-polyamine-organocation (APC)-type yeast amino acid transporters family. We identify APC members that have broader substrate spectra than previously described. Using in vivo experimental evolution, we evolve two of these transporter genes, AGP1 and PUT4 , toward new substrate specificities. Single mutations on these transporters are found to be sufficient for expanding the substrate range of the proteins, while retaining the capacity to transport all original substrates. Nonetheless, each adaptive mutation comes with a distinct effect on the fitness for each of the original substrates, illustrating a trade-off between the ancestral and evolved functions. Collectively, our findings reveal how substrate-adaptive mutations in membrane transporters contribute to fitness and provide insights into how organisms can use transporter evolution to explore new ecological niches., Competing Interests: FK, ÚA, DS, BP, SO No competing interests declared, (© 2024, Karapanagioti et al.)

ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism., Competing Interests: Mv, PD, CP, BP No competing interests declared, (© 2023, van den Noort et al.)

Protein diffusion is a critical factor governing the functioning and organization of a cell's cytoplasm. In this study, we investigate the influence of (poly)ribosome distribution, cell aging, protein aggregation, and biomolecular condensate formation on protein mobility within the E. coli cytoplasm. We employ nanoscale single-molecule displacement mapping (SMdM) to determine the spatial distribution of the proteins and to meticulously track their diffusion. We show that the distribution of polysomes does not impact the lateral diffusion coefficients of proteins. However, the degradation of mRNA induced by rifampicin treatment leads to an increase in protein mobility within the cytoplasm. Additionally, we establish a significant correlation between cell aging, the asymmetric localization of protein aggregates and reduced diffusion coefficients at the cell poles. Notably, we observe variations in the hindrance of diffusion at the poles and the central nucleoid region for small and large proteins, and we reveal differences between the old and new pole of the cell. Collectively, our research highlights cellular processes and mechanisms responsible for spatially organizing the bacterial cytoplasm into domains with different structural features and apparent viscosity., (© 2024. The Author(s).)

Transmembrane transporter proteins are essential for maintaining cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the wealth of detailed structural and functional data available for these systems, our understanding of their dynamics and, consequently, how they function is generally limited. We introduce an innovative approach that enables us to directly measure the dynamics and stability of interdomain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally relevant motions and interdomain interactions of individual transmembrane transporter proteins in real time in a lipid bilayer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are trapped in microfluidic devices for analysis by optical microscopy.

Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic strength, and the energy status, influences the structure of the cytoplasm and thereby indirectly macromolecular function. Notably, crowding also promotes the formation of biomolecular condensates by phase separation, initially identified in eukaryotic cells but more recently discovered to play key functions in bacteria. Bacterial cells require a variety of mechanisms to maintain physicochemical homeostasis, in particular in environments with fluctuating conditions, and the formation of biomolecular condensates is emerging as one such mechanism. In this work, we connect physicochemical homeostasis and macromolecular crowding with the formation and function of biomolecular condensates in the bacterial cell and compare the supramolecular structures found in bacteria with those of eukaryotic cells. We focus on the effects of crowding and phase separation on the control of bacterial chromosome replication, segregation, and cell division, and we discuss the contribution of biomolecular condensates to bacterial cell fitness and adaptation to environmental stress.

The width of the periplasmic space of Gram-negative bacteria is only about 25-30 nm along the long axis of the cell, which affects free diffusion of (macro)molecules. We have performed single-particle displacement measurements and diffusion simulation studies to determine the impact of confinement on the apparent mobility of proteins in the periplasm of Escherichia coli. The diffusion of a reporter protein and of OsmY, an osmotically regulated periplasmic protein, is characterized by a fast and slow component regardless of the osmotic conditions. The diffusion coefficient of the fast fraction increases upon osmotic upshift, in agreement with a decrease in macromolecular crowding of the periplasm, but the mobility of the slow (immobile) fraction is not affected by the osmotic stress. We observe that the confinement created by the inner and outer membranes results in a lower apparent diffusion coefficient, but this can only partially explain the slow component of diffusion in the particle displacement measurements, suggesting that a fraction of the proteins is hindered in its mobility by large periplasmic structures. Using particle-based simulations, we have determined the confinement effect on the apparent diffusion coefficient of the particles for geometries akin the periplasmic space of Gram-negative bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

We have developed Simulation-based Reconstructed Diffusion (SbRD) to determine diffusion coefficients corrected for confinement effects and for the bias introduced by two-dimensional models describing a three-dimensional motion. We validate the method on simulated diffusion data in three-dimensional cell-shaped compartments. We use SbRD, combined with a new cell detection method, to determine the diffusion coefficients of a set of native proteins in Escherichia coli. We observe slower diffusion at the cell poles than in the nucleoid region of exponentially growing cells, which is independent of the presence of polysomes. Furthermore, we show that the newly formed pole of dividing cells exhibits a faster diffusion than the old one. We hypothesize that the observed slowdown at the cell poles is caused by the accumulation of aggregated or damaged proteins, and that the effect is asymmetric due to cell aging., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Mantovanelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In living cells, the biochemical processes such as energy provision, molecule synthesis, gene expression, and cell division take place in a confined space where the internal chemical and physical conditions are different from those in dilute solutions. The concentrations of specific molecules and the specific reactions and interactions vary for different types of cells, but a number of factors are universal and kept within limits, which we refer to as physicochemical homeostasis. For instance, the internal pH of many cell types is kept within the range of 7.0 to 7.5, the fraction of macromolecules occupies 15%-20% of the cell volume (also known as macromolecular crowding) and the ionic strength is kept within limits to prevent salting-in or salting-out effects. In this article we summarize the generic physicochemical properties of the cytoplasm of bacteria, how they are connected to the energy status of the cell, and how they affect biological processes (Fig. 1). We describe how the internal pH and proton motive force are regulated, how the internal ionic strength is kept within limits, what the impact of macromolecular crowding is on the function of enzymes and the interaction between molecules, how cells regulate their volume (and turgor), and how the cytoplasm is structured. Physicochemical homeostasis is best understood in Escherichia coli, but pioneering studies have also been performed in lactic acid bacteria., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metabolism in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochemical homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein composition of a cell. We compare our bottom-up design with the equivalent essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixture of membrane proteins into lipid bilayers and provide a semiquantitative estimate of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal number of membrane proteins) that are required for the construction of a synthetic cell.

The human pathogen Listeria monocytogenes can cope with severe environmental challenges, for which the high molecular weight stressosome complex acts as the sensing hub in a complicated signal transduction pathway. Here, we show the dynamics and functional roles of the stressosome protein RsbR1 and its paralogue, the blue-light receptor RsbL, using photo-activated localization microscopy combined with single-particle tracking and single-molecule displacement mapping and supported by physiological studies. In live cells, RsbR1 is present in multiple states: in protomers with RsbS, large clusters of stressosome complexes, and in connection with the plasma membrane via Prli42. RsbL diffuses freely in the cytoplasm but forms clusters upon exposure to light. The clustering of RsbL is independent of the presence of Prli42. Our work provides a comprehensive view of the spatial organization and intracellular dynamics of the stressosome proteins in L. monocytogenes, which paves the way towards uncovering the stress-sensing mechanism of this signal transduction pathway., (© 2023. The Author(s).)

Natamycin is a polyene macrolide, widely employed to treat fungal keratitis and other yeast infections as well as to protect food products against fungal molds. In contrast to other polyene macrolides, such as nystatin or amphotericin B, natamycin does not form pores in yeast membranes, and its mode of action is not well understood. Here, we have employed a variety of spectroscopic methods, computational modeling, and membrane reconstitution to study the molecular interactions of natamycin underlying its antifungal activity. We find that natamycin forms aggregates in an aqueous solution with strongly altered optical properties compared to monomeric natamycin. Interaction of natamycin with model membranes results in a concentration-dependent fluorescence increase which is more pronounced for ergosterol- compared to cholesterol-containing membranes up to 20 mol% sterol. Evidence for formation of specific ergosterol-natamycin complexes in the bilayer is provided. Using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy, we find that natamycin sequesters sterols, thereby interfering with their well-known ability to order acyl chains in lipid bilayers. This effect is more pronounced for membranes containing the sterol of fungi, ergosterol, compared to those containing mammalian cholesterol. Natamycin interferes with ergosterol-dependent transport of lysine by the yeast transporter Lyp1, which we propose to be due to the sequestering of ergosterol, a mechanism that also affects other plasma membrane proteins. Our results provide a mechanistic explanation for the selective antifungal activity of natamycin, which can set the stage for rational design of novel polyenes in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The passive permeability of cell membranes is of key importance in biology, biomedical research and biotechnology as it determines the extent to which various molecules such as drugs, products of metabolism, and toxins can enter or leave the cell unaided by dedicated transport proteins. The quantification of passive solute permeation is possible with radio-isotope distribution experiments, spectroscopic measurements and molecular dynamics simulations. This protocol describes stopped-flow fluorimetry measurements performed on lipid vesicles and living yeast cells to estimate the osmotic permeability of water and solutes across (bio)membranes. Encapsulation of the fluorescent dye calcein into lipid vesicles allows monitoring of volume changes upon osmotic shifts of the medium via (de)quenching of the fluorophore, which we interpret using a well-defined physical model that takes the dynamics of the vesicles into account to calculate the permeability coefficients of solutes. We also present analogous procedures to probe weak acid and base permeability in vesicles and cells by using the read-out of encapsulated or expressed pH-sensitive probes. We describe the preparation of synthetic vesicles of varying lipid composition and determination of vesicle size distribution by dynamic light scattering. Data on membrane permeation are obtained using either conventional or stopped-flow kinetic fluorescence measurements on instruments available in most research institutes and are analyzed with a suite of user-friendly MATLAB scripts ( https://doi.org/10.5281/zenodo.6511116 ). Collectively, these procedures provide a comprehensive toolbox for determining membrane permeability coefficients in a variety of experimental systems, and typically take 2-3 d., (© 2022. Springer Nature Limited.)

We analyze the structure of the cytoplasm by performing single-molecule displacement mapping on a diverse set of native cytoplasmic proteins in exponentially growing Escherichia coli . We evaluate the method for application in small compartments and find that confining effects of the cell membrane affect the diffusion maps. Our analysis reveals that protein diffusion at the poles is consistently slower than in the center of the cell, i.e., to an extent greater than the confining effect of the cell membrane. We also show that the diffusion coefficient scales with the mass of the used probes, taking into account the oligomeric state of the proteins, while parameters such as native protein abundance or the number of protein-protein interactions do not correlate with the mobility of the proteins. We argue that our data paint the prokaryotic cytoplasm as a compartment with subdomains in which the diffusion of macromolecules changes with the perceived viscosity.

The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biology. Synthetic cellular systems are envisioned as out-of-equilibrium enzymatic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metabolism. Importantly, gaining tight control over the external medium is essential to avoid thermodynamic equilibrium due to nutrient depletion or waste buildup in a closed compartment ( e.g. , a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, e.g. , of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymatic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the Escherichia coli glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable l-arginine breakdown. In addition, we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium composition and to achieve sustainable glycerol 3-phosphate synthesis.

Cell membranes provide a selective semi-permeable barrier to the passive transport of molecules. This property differs greatly between organisms. While the cytoplasmic membrane of bacterial cells is highly permeable for weak acids and glycerol, yeasts can maintain large concentration gradients. Here we show that such differences can arise from the physical state of the plasma membrane. By combining stopped-flow kinetic measurements with molecular dynamics simulations, we performed a systematic analysis of the permeability of a variety of small molecules through synthetic membranes of different lipid composition to obtain detailed molecular insight into the permeation mechanisms. While membrane thickness is an important parameter for the permeability through fluid membranes, the largest differences occur when the membranes transit from the liquid-disordered to liquid-ordered and/or to gel state, which is in agreement with previous work on passive diffusion of water. By comparing our results with in vivo measurements from yeast, we conclude that the yeast membrane exists in a highly ordered and rigid state, which is comparable to synthetic saturated DPPC-sterol membranes., (© 2022. The Author(s).)

Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzymatic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD + to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equivalents in the lumen. A soluble transhydrogenase subsequently utilizes NADH for reduction of NADP + thereby making NAD + available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diameter (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochemical process of reduction of glutathione disulfide can be driven by the transfer of reducing equivalents from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metabolism., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MBP) from Escherichia coli. We used single-molecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The motion in the cytosol of microorganisms such as bacteria and yeast has been observed to undergo a dramatic slowing down upon cell energy depletion. These observations have been interpreted as the motion being "glassy," but whether this notion is useful also for active, motor-protein-driven transport in eukaryotic cells is less clear. Here, we use fluorescence microscopy of beads in human (HeLa) cells to probe the motion of membrane-surrounded structures that are carried along the cytoskeleton by motor proteins. Evaluating several hallmarks of glassy dynamics, we show that at short length scales, the motion is heterogeneous, is nonergodic, is well described by a model for the displacement distribution in glassy systems, and exhibits a decoupling of the exchange and persistence times. Overall, these results suggest that the short length scale behavior of objects that can be transported actively by motor proteins in human cells shares features with the motion in glassy systems., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions.

Proton coupled transport of α-glucosides via Mal11 into Saccharomyces cerevisiae costs one ATP per imported molecule. Targeted mutation of all three acidic residues in the active site resulted in sugar uniport, but expression of these mutant transporters in yeast did not enable growth on sucrose. We then isolated six unique transporter variants of these mutants by directed evolution of yeast for growth on sucrose. In three variants, new acidic residues emerged near the active site that restored proton-coupled sucrose transport, whereas the other evolved transporters still catalysed sucrose uniport. The localization of mutations and transport properties of the mutants enabled us to propose a mechanistic model of proton-coupled sugar transport by Mal11. Cultivation of yeast strains expressing one of the sucrose uniporters in anaerobic, sucrose-limited chemostat cultures indicated an increase in the efficiency of sucrose dissimilation by 21% when additional changes in strain physiology were taken into account. We thus show that a combination of directed and evolutionary engineering results in more energy efficient sucrose transport, as a starting point to engineer yeast strains with increased yields for industrially relevant products., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE-FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE-FRET to monitor protein-protein interactions and conformational states simultaneously.

Obtaining (dynamic) structure related information on proteins is key for understanding their function. Methods as single-molecule Förster Resonance Energy Transfer (smFRET) and Electron Paramagnetic Resonance (EPR) that measure distances between labeled residues to obtain dynamic information rely on selection of suitable residue pairs for chemical modification. Selection of pairs of amino acids, that show sufficient distance changes upon activity of the protein, can be a tedious process. Here we present an in silico approach that makes use of two or more structures (or structure models) to filter suitable residue pairs for FRET or EPR from all possible pairs within the protein. We apply the method for the study of the conformational dynamics of the substrate-binding domain of the osmoregulatory ATP-Binding Cassette transporter OpuA. This method speeds up the process of designing mutants, and because of its systematic nature, the chances of missing promising candidates are reduced.

Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient ( D L ) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that D L is much less affected by hyperosmotic stress in L. monocytogenes than under similar conditions in Lactococcus lactis and Escherichia coli . We find a temperature optimum for protein diffusion in L. monocytogenes at 30°C, which deviates from predicted trends from the generalized Stokes-Einstein equation under dilute conditions and suggests that the structure of the cytoplasm and macromolecular crowding vary as a function of temperature. The turgor pressure of L. monocytogenes is comparable to other Gram-positive bacteria like Bacillus subtilis and L. lactis but higher in a knockout strain lacking the stress-inducible sigma factor SigB. We discuss these findings in the context of how L. monocytogenes survives during environmental transmission and interaction with the human host., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tran, Prabha, Iyer, O’Byrne, Abee and Poolman.)

In cells, the breakdown of arginine to ornithine and ammonium ion plus carbon dioxide is coupled to the generation of metabolic energy in the form of ATP. The arginine breakdown pathway is minimally composed of arginine deiminase, ornithine transcarbamoylase, carbamate kinase, and an arginine/ornithine antiporter; ammonia and carbon dioxide most likely diffuse passively across the membrane. The genes for the enzymes and transporter have been cloned and expressed, and the proteins have been purified from Lactococcus lactis IL1403 and incorporated into lipid vesicles for sustained production of ATP. Here, we study the kinetic parameters and biochemical properties of the individual enzymes and the antiporter, and we determine how the physicochemical conditions, effector composition, and effector concentration affect the enzymes. We report the K M and V MAX values for catalysis and the native oligomeric state of all proteins, and we measured the effect of pathway intermediates, pH, temperature, freeze-thaw cycles, and salts on the activity of the cytosolic enzymes. We also present data on the protein-to-lipid ratio and lipid composition dependence of the antiporter., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Fluorescence-based sensors play a fundamental role in biological research. These sensors can be based on fluorescent proteins, fluorescent probes or they can be hybrid systems. The availability of a very large dataset of fluorescent molecules, both genetically encoded and synthetically produced, together with the structural insights on many sensing domains, allowed to rationally design a high variety of sensors, capable of monitoring both molecular and global changes in living cells or in in vitro systems. The advancements in the fluorescence-imaging field helped researchers to obtain a deeper understanding of how and where specific changes occur in a cell or in vitro by combining the readout of the fluorescent sensors with the spatial information provided by fluorescent microscopy techniques. In this review we give an overview of the state of the art in the field of fluorescent biosensors and fluorescence imaging techniques, and eventually guide the reader through the choice of the best combination of fluorescent tools and techniques to answer specific biological questions. We particularly focus on sensors for probing the bioenergetics and physicochemical status of the cell., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Amino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced downregulation of amino acid transporters in Saccharomyces cerevisiae is thought to be a mechanism to avoid this toxicity. It has been shown that unregulated uptake by the general amino acid permease Gap1 causes cells to become sensitive to amino acids. Here, we show that overexpression of eight other amino acid transporters (Agp1, Bap2, Can1, Dip5, Gnp1, Lyp1, Put4, or Tat2) also induces a growth defect when specific single amino acids are present at concentrations of 0.5-5 mM. We can now state that all proteinogenic amino acids, as well as the important metabolite ornithine, are growth inhibitory to S. cerevisiae when transported into the cell at high enough levels. Measurements of initial transport rates and cytosolic pH show that toxicity is due to amino acid accumulation and not to the influx of co-transported protons. The amino acid sensitivity phenotype is a useful tool that reports on the in vivo activity of transporters and has allowed us to identify new transporter-specific substrates.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

(Micro)organisms are exposed to fluctuating environmental conditions, and adaptation to stress is essential for survival. Increased osmolality (hypertonicity) causes outflow of water and loss of turgor and is dangerous if the cell is not capable of rapidly restoring its volume. The osmoregulatory adenosine triphosphate-binding cassette transporter OpuA restores the cell volume by accumulating large amounts of compatible solute. OpuA is gated by ionic strength and inhibited by the second messenger cyclic-di-AMP, a molecule recently shown to affect many cellular processes. Despite the master regulatory role of cyclic-di-AMP, structural and functional insights into how the second messenger regulates (transport) proteins on the molecular level are lacking. Here, we present high-resolution cryo-electron microscopy structures of OpuA and in vitro activity assays that show how the osmoregulator OpuA is activated by high ionic strength and how cyclic-di-AMP acts as a backstop to prevent unbridled uptake of compatible solutes., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Yeast amino acid transporters of the APC superfamily are responsible for the proton motive force-driven uptake of amino acids into the cell, which for most secondary transporters is a reversible process. The l-lysine proton symporter Lyp1 of Saccharomyces cerevisiae is special in that the Michaelis constant from out-to-in transport ( K m out → in ) is much lower than K m in → out , which allows accumulation of l-lysine to submolar concentration. It has been proposed that high intracellular lysine is part of the antioxidant mechanism of the cell. The molecular basis for the unique kinetic properties of Lyp1 is unknown. We compared the sequence of Lyp1 with APC para- and orthologues and find structural features that set Lyp1 apart, including differences in extracellular loop regions. We screened the extracellular loops by alanine mutagenesis and determined Lyp1 localization and activity and find positions that affect either the localization or activity of Lyp1. Half of the affected mutants are located in the extension of extracellular loop 3 or in a predicted α-helix in extracellular loop 4. Our data indicate that extracellular loops not only connect the transmembrane helices but also serve functionally important roles., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Prokaryotic ATP-binding cassette (ABC) importers require a substrate-binding protein (SBP) for the capture and delivery of the cognate substrate to the transmembrane domain (TMD) of the transporter. Various biochemical compounds have been identified that bind to the SBP but are not transported. The mechanistic basis for the "non-cognate" substrates not being transported differs. Some non-cognate substrates fail to trigger the appropriate conformational change in the SBP, resulting in loss of affinity for the TMD or the inability to allosterically activate transport. In another mechanism, the SBP cannot release the bound non-cognate substrate. Here, we used rate equations to derive the steady-state transport rate of cognate substrates of an ABC importer and investigated how non-cognate substrates influence this rate. We found that under limiting non-cognate substrate concentrations, the transport rate remains unaltered for each of the mechanisms. In contrast, at saturating substrate and SBP concentrations, the effect of the non-cognate substrate depends heavily on the respective mechanism. For instance, the transport rate becomes zero when the non-cognate substrate cannot be released by the SBP. Yet it remains unaffected when substrate release is possible but the SBP cannot dock onto the TMDs. Our work shows how the different mechanisms of substrate inhibition impact the transport kinetics, which is relevant for understanding and manipulating solute fluxes and hence the propagation of cells in nutritionally complex milieus., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Unprotected 3-keto-saccharides have become readily accessible via site-selective oxidation, but their protection-free functionalization is relatively unexplored. Here we show that protecting groups are obsolete in a variety of stereoselective modifications of our model substrate methyl α-glucopyranoside. This allows the preparation of rare sugars and the installation of click handles and reactive groups. To showcase the applicability of the methodology, maltoheptaose has been converted into a chemical probe, and the rare sugar evalose has been synthesized.

Membrane lipids act as solvents and functional cofactors for integral membrane proteins. The yeast plasma membrane is unusual in that it may have a high lipid order, which coincides with low passive permeability for small molecules and a slow lateral diffusion of proteins. Yet, membrane proteins whose functions require altered conformation must have flexibility within membranes. We have determined the molecular composition of yeast plasma membrane lipids located within a defined diameter of model proteins, including the APC-superfamily lysine transporter Lyp1. We now use the composition of lipids that naturally surround Lyp1 to guide testing of lipids that support the normal functioning of the transporter, when reconstituted in vesicles of defined lipid composition. We find that phosphatidylserine and ergosterol are essential for Lyp1 function, and the transport activity displays a sigmoidal relationship with the concentration of these lipids. Non-bilayer lipids stimulate transport activity, but different types are interchangeable. Remarkably, Lyp1 requires a relatively high fraction of lipids with one or more unsaturated acyl chains. The transport data and predictions of the periprotein lipidome of Lyp1 support a new model in which a narrow band of lipids immediately surrounding the transmembrane stalk of a model protein allows conformational changes in the protein., Competing Interests: Conflict of Interest The authors declare no conflict of interest, (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Attachment of lipophilic groups is an important post-translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids, or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine-based trifunctional linker. The linker allows the facile incorporation of two different functionalities at a cysteine residue in a single step. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α-helical model peptide WALP with or without here and in all cases below; palmitoyl groups in giant unilamellar vesicles that contain a liquid-ordered (L o ) and liquid-disordered (L d ) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the L d phase. These data demonstrate that the L o domain in model membranes is highly unfavorable for a single membrane-spanning peptide., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Yeast tolerates a low pH and high solvent concentrations. The permeability of the plasma membrane (PM) for small molecules is low and lateral diffusion of proteins is slow. These findings suggest a high degree of lipid order, which raises the question of how membrane proteins function in such an environment. The yeast PM is segregated into the Micro-Compartment-of-Can1 (MCC) and Pma1 (MCP), which have different lipid compositions. We extracted proteins from these microdomains via stoichiometric capture of lipids and proteins in styrene-maleic-acid-lipid-particles (SMALPs). We purified SMALP-lipid-protein complexes by chromatography and quantitatively analyzed periprotein lipids located within the diameter defined by one SMALP. Phospholipid and sterol concentrations are similar for MCC and MCP, but sphingolipids are enriched in MCP. Ergosterol is depleted from this periprotein lipidome, whereas phosphatidylserine is enriched relative to the bulk of the plasma membrane. Direct detection of PM lipids in the 'periprotein space' supports the conclusion that proteins function in the presence of a locally disordered lipid state., Competing Interests: Jv, TC, HS, AJ, BM, BP No competing interests declared

The nuclear pore complex (NPC) is embedded in the nuclear envelope and forms the main gateway to the nuclear interior including the inner nuclear membrane (INM). Two INM proteins in yeast are selectively imported. Their sorting signals consist of a nuclear localization signal, separated from the transmembrane domain by a long intrinsically disordered (ID) linker. We used computational models to predict the dynamic conformations of ID linkers and analyzed the INM targeting efficiency of proteins with linker regions with altered Stokes radii and decreased flexibilities. We find that flexibility, Stokes radius, and the frequency at which the linkers are at an extended end-to-end distance larger than 25 nm are good predictors for the targeting of the proteins. The data are consistent with a transport mechanism in which INM targeting of Heh2 is dependent on an ID linker that facilitates the crossing of the approximately 25-nm thick NPC scaffold., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We modeled the relaxation dynamics of (lipid) vesicles upon osmotic upshift, taking into account volume variation, chemical reaction kinetics, and passive transport across the membrane. We focused on the relaxation kinetics upon addition of impermeable osmolytes such as KCl and membrane-permeable solutes such as weak acids. We studied the effect of the most relevant physical parameters on the dynamic behavior of the system, as well as on the relaxation rates. We observe that 1) the dynamic complexity of the relaxation kinetics depends on the number of permeable species; 2) the permeability coefficients (P) and the weak acid strength (pK a -values) determine the dynamic behavior of the system; 3) the vesicle size does not affect the dynamics, but only the relaxation rates of the system; and 4) heterogeneities in the vesicle size provoke stretching of the relaxation curves. The model was successfully benchmarked for determining permeability coefficients by fitting of our experimental relaxation curves and by comparison of the data with literature values (in this issue of Biophysical Journal). To describe the dynamics of yeast cells upon osmotic upshift, we extended the model to account for turgor pressure and nonosmotic volume., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). We observe a relation between the molecule permeability and the saturation of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, we conclude that the yeast plasma membrane is impermeable to lactic acid on timescales up to ∼2.5 h., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The bottom-up construction of synthetic cells from molecular components is arguably one of the most challenging areas of research in the life sciences. We review the impact of confining biological systems in synthetic vesicles. Complex cell-like systems require control of the internal pH, ionic strength, (macro)molecular crowding, redox state and metabolic energy conservation. These physicochemical parameters influence protein activity and need to be maintained within limits to ensure the system remains in steady-state. We present the physicochemical considerations for building synthetic cells with dimensions ranging from the smallest prokaryotes to eukaryotic cells., (© 2019 The Author(s).)

We review the mechanisms responsible for amino acid homeostasis in Saccharomyces cerevisiae and other fungi. Amino acid homeostasis is essential for cell growth and survival. Hence, the de novo synthesis reactions, metabolic conversions, and transport of amino acids are tightly regulated. Regulation varies from nitrogen pool sensing to control by individual amino acids and takes place at the gene (transcription), protein (posttranslational modification and allostery), and vesicle (trafficking and endocytosis) levels. The pools of amino acids are controlled via import, export, and compartmentalization. In yeast, the majority of the amino acid transporters belong to the APC ( a mino acid- p olyamine-organo c ation) superfamily, and the proteins couple the uphill transport of amino acids to the electrochemical proton gradient. Although high-resolution structures of yeast amino acid transporters are not available, homology models have been successfully exploited to determine and engineer the catalytic and regulatory functions of the proteins. This has led to a further understanding of the underlying mechanisms of amino acid sensing and subsequent downregulation of transport. Advances in optical microscopy have revealed a new level of regulation of yeast amino acid transporters, which involves membrane domain partitioning. The significance and the interrelationships of the latest discoveries on amino acid homeostasis are put in context., (Copyright © 2019 American Society for Microbiology.)

We are aiming for a blue print for synthesizing (moderately complex) subcellular systems from molecular components and ultimately for constructing life. However, without comprehensive instructions and design principles, we rely on simple reaction routes to operate the essential functions of life. The first forms of synthetic life will not make every building block for polymers de novo according to complex pathways, rather they will be fed with amino acids, fatty acids and nucleotides. Controlled energy supply is crucial for any synthetic cell, no matter how complex. Herein, we describe the simplest pathways for the efficient generation of ATP and electrochemical ion gradients. We have estimated the demand for ATP by polymer synthesis and maintenance processes in small cell-like systems, and we describe circuits to control the need for ATP. We also present fluorescence-based sensors for pH, ionic strength, excluded volume, ATP/ADP, and viscosity, which allow the major physicochemical conditions inside cells to be monitored and tuned., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One of the grand challenges in chemistry is the construction of functional out-of-equilibrium networks, which are typical of living cells. Building such a system from molecular components requires control over the formation and degradation of the interacting chemicals and homeostasis of the internal physical-chemical conditions. The provision and consumption of ATP lies at the heart of this challenge. Here we report the in vitro construction of a pathway in vesicles for sustained ATP production that is maintained away from equilibrium by control of energy dissipation. We maintain a constant level of ATP with varying load on the system. The pathway enables us to control the transmembrane fluxes of osmolytes and to demonstrate basic physicochemical homeostasis. Our work demonstrates metabolic energy conservation and cell volume regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.