Proceedings of the National Academy of Sciences最新文献

{"title":"The crucial role of genome-wide genetic variation in conservation","authors":"Marty Kardos, E. Armstrong, S. Fitzpatrick, Samantha S Hauser, P. Hedrick, Joshua M. Miller, D. Tallmon, W. Funk","doi":"10.1101/2021.07.05.451163","DOIUrl":"https://doi.org/10.1101/2021.07.05.451163","url":null,"abstract":"The unprecedented rate of extinction calls for efficient use of genetics to help conserve biodiversity. Several recent genomic and simulation-based studies have argued that the field of conservation biology has placed too much focus on conserving genome-wide genetic variation, and that the field should instead focus on managing the subset of functional genetic variation that is thought to affect fitness. Here, we critically evaluate the feasibility and likely benefits of this approach in conservation. We find that population genetics theory and empirical results show that conserving genome-wide genetic variation is generally the best approach to prevent inbreeding depression and loss of adaptive potential from driving populations toward extinction. Focusing conservation efforts on presumably functional genetic variation will only be feasible occasionally, often misleading, and counterproductive when prioritized over genome-wide genetic variation. Given the increasing rate of habitat loss and other environmental changes, failure to recognize the detrimental effects of lost genome-wide genetic variation on long-term population viability will only worsen the biodiversity crisis.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"25 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83286040","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain","authors":"Evan R. Semenza, Maged M. Harraz, Efrat Abramson, Adarsha P. Malla, C. Vasavda, Moataz M. Gadalla, M. Kornberg, S. Snyder, Robin Roychaudhuri","doi":"10.1101/2021.07.05.451211","DOIUrl":"https://doi.org/10.1101/2021.07.05.451211","url":null,"abstract":"Significance d-amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. Cysteine is the amino acid with the fastest in vitro spontaneous racemization rate, but its d-stereoisomer has not been examined. Here, we establish the presence of endogenous d-cysteine in the mammalian brain. Using sensitive and specific assays, we delineate its actions as a negative regulator of growth factor signaling during cortical development and identify a putative binding partner mediating these effects. By describing the newest member of the d-amino acid family, we open an avenue of research into the functions of these multifaceted signaling molecules. d-amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. However, the d-stereoisomer of the amino acid with the fastest spontaneous racemization ratein vitro in vitro, cysteine, has not been examined in mammals. Using chiral high-performance liquid chromatography and a stereospecific luciferase assay, we identify endogenous d-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the N-methyl-d-aspartate (NMDA) glutamate receptor coagonist d-serine, as a candidate biosynthetic enzyme for d-cysteine. d-cysteine is enriched more than 20-fold in the embryonic mouse brain compared with the adult brain. d-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by ∼50%, effects not shared with d-serine or l-cysteine. The antiproliferative effect of d-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of d-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for d-cysteine–binding proteins in NPCs by immunoprecipitation with a d-cysteine–specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative d-cysteine–binding protein. Together, these results establish endogenous mammalian d-cysteine and implicate it as a physiologic regulator of NPC homeostasis in the developing brain.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78079659","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Gene drive that results in addiction to a temperature-sensitive version of an essential gene triggers population collapse in Drosophila","authors":"Georg Oberhofer, Tobin Ivy, B. Hay","doi":"10.1101/2021.07.03.451005","DOIUrl":"https://doi.org/10.1101/2021.07.03.451005","url":null,"abstract":"One strategy for population suppression seeks to use gene drive to spread genes that confer conditional lethality or sterility, providing a way of combining population modification with suppression. Stimuli of potential interest could be introduced by humans, such as an otherwise benign virus or chemical, or occur naturally on a seasonal basis, such as a change in temperature. Cleave and Rescue (ClvR) selfish genetic elements use Cas9 and guide RNAs (gRNAs) to disrupt endogenous versions of an essential gene while also including a Rescue version of the essential gene resistant to disruption. ClvR spreads by creating loss-of-function alleles of the essential gene that select against those lacking it, resulting in populations in which the Rescue provides the only source of essential gene function. As a consequence, if function of the Rescue, a kind of Trojan horse now omnipresent in a population, is condition dependent, so too will be the survival of that population. To test this idea, we created a ClvR in Drosophila in which Rescue activity of an essential gene, dribble, requires splicing of a temperature-sensitive intein (TS-ClvRdbe). This element spreads to transgene fixation at 23 °C, but when populations now dependent on Ts-ClvRdbe are shifted to 29 °C, death and sterility result in a rapid population crash. These results show that conditional population elimination can be achieved. A similar logic, in which Rescue activity is conditional, could also be used in homing-based drive and to bring about suppression and/or killing of specific individuals in response to other stimuli.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80294479","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance","authors":"Agatha Lyczek, B. Berger, Aziz M. Rangwala, YiTing Paung, Jessica Tom, Hannah Philipose, Jiaye Guo, Steven K. Albanese, M. Robers, S. Knapp, J. Chodera, M. Seeliger","doi":"10.1101/2021.06.28.449968","DOIUrl":"https://doi.org/10.1101/2021.06.28.449968","url":null,"abstract":"Significance We performed an in-cell screen of imatinib binding against a library of Abl kinase mutants derived from patients with imatinib-resistant chronic myeloid leukemia. The majority of mutations readily bind imatinib, posing the question of how these mutations cause resistance in patients. We identified several kinetic mutants, one of which binds imatinib with wild-type affinity but dissociates considerably faster from the mutant kinase. Using NMR and molecular dynamics, we found that this mutation increases the conformational dynamics of the mutant protein, linking conformational dynamics of the protein to drug dissociation. The results underline the importance of drug dissociation kinetics for drug efficacy and propose a kinetic resistance mechanism that may be targetable by altering drug treatment schedules. Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type–like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"20 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87621166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Accurate model of liquid–liquid phase behavior of intrinsically disordered proteins from optimization of single-chain properties","authors":"G. Tesei, Thea K. Schulze, R. Crehuet, K. Lindorff-Larsen","doi":"10.1101/2021.06.23.449550","DOIUrl":"https://doi.org/10.1101/2021.06.23.449550","url":null,"abstract":"Significance Cells may compartmentalize proteins via a demixing process known as liquid–liquid phase separation (LLPS), which is often driven by intrinsically disordered proteins (IDPs) and regions. Protein condensates arising from LLPS may develop into insoluble protein aggregates, as in neurodegenerative diseases and cancer. Understanding the process of formation, dissolution, and aging of protein condensates requires models that accurately capture the underpinning interactions at the residue level. In this work, we leverage data from biophysical experiments on IDPs in dilute solution to develop a sequence-dependent model which predicts conformational and phase behavior of diverse and unrelated protein sequences with good accuracy. Using the model, we gain insight into the coupling between chain compaction and LLPS propensity. Many intrinsically disordered proteins (IDPs) may undergo liquid–liquid phase separation (LLPS) and participate in the formation of membraneless organelles in the cell, thereby contributing to the regulation and compartmentalization of intracellular biochemical reactions. The phase behavior of IDPs is sequence dependent, and its investigation through molecular simulations requires protein models that combine computational efficiency with an accurate description of intramolecular and intermolecular interactions. We developed a general coarse-grained model of IDPs, with residue-level detail, based on an extensive set of experimental data on single-chain properties. Ensemble-averaged experimental observables are predicted from molecular simulations, and a data-driven parameter-learning procedure is used to identify the residue-specific model parameters that minimize the discrepancy between predictions and experiments. The model accurately reproduces the experimentally observed conformational propensities of a set of IDPs. Through two-body as well as large-scale molecular simulations, we show that the optimization of the intramolecular interactions results in improved predictions of protein self-association and LLPS.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"31 13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84633324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Wildfire smoke impacts on indoor air quality assessed using crowdsourced data in California","authors":"Yutong Liang, Deep Sengupta, M. Campmier, David M. Lunderberg, J. Apte, A. Goldstein","doi":"10.26434/CHEMRXIV.14739219.V1","DOIUrl":"https://doi.org/10.26434/CHEMRXIV.14739219.V1","url":null,"abstract":"Significance Wildfires are an increasingly large source of particulate matter (PM2.5) in the western United States. Previous characterizations of exposure to wildfire smoke particles were based mainly on outdoor concentrations of PM2.5. Since people mainly shelter indoors during smoke events, the infiltration of wildfire PM2.5 into buildings determines exposure. We present analysis of infiltration of wildfire PM2.5 into more than 1,400 buildings in California using more than 2.4 million sensor hours of data from the PurpleAir sensor network. Our study reveals that infiltration of PM2.5 during wildfire days was substantially reduced compared with non-fire days, due to people’s behavioral changes. These results improve understanding of exposure to wildfire particles and facilitate informing the public about effective ways to reduce their exposure. Wildfires have become an important source of particulate matter (PM2.5 < 2.5-µm diameter), leading to unhealthy air quality index occurrences in the western United States. Since people mainly shelter indoors during wildfire smoke events, the infiltration of wildfire PM2.5 into indoor environments is a key determinant of human exposure and is potentially controllable with appropriate awareness, infrastructure investment, and public education. Using time-resolved observations outside and inside more than 1,400 buildings from the crowdsourced PurpleAir sensor network in California, we found that the geometric mean infiltration ratios (indoor PM2.5 of outdoor origin/outdoor PM2.5) were reduced from 0.4 during non-fire days to 0.2 during wildfire days. Even with reduced infiltration, the mean indoor concentration of PM2.5 nearly tripled during wildfire events, with a lower infiltration in newer buildings and those utilizing air conditioning or filtration.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"330 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80490281","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Amphiphilic proteins coassemble into multiphasic condensates and act as biomolecular surfactants","authors":"F. Kelley, Bruna Favetta, R. M. Regy, J. Mittal, Benjamin S. Schuster","doi":"10.1101/2021.05.28.446223","DOIUrl":"https://doi.org/10.1101/2021.05.28.446223","url":null,"abstract":"Significance Membraneless organelles are assemblies of highly concentrated biomolecules that condense through liquid–liquid phase separation. One major question in the field is how proteins assemble into multilayered condensates. Understanding mechanisms of formation of these systems is important for understanding the function and regulation of multiphasic organelles, such as P granules and nucleoli. A second outstanding question is how the size of biomolecular condensates is controlled. In this work, we generated amphiphilic proteins that localize to the surface of condensates. We observed diverse assemblies, including condensates enveloped by surfactant-like films, as well as complex multiphasic morphologies. In some configurations, these surfactant-like proteins influence condensate size. Our results suggest an important role of protein amphiphiles in establishing membraneless organelle structure and function. Cells contain membraneless compartments that assemble due to liquid–liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bioinspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non–phase-separating domain. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and influence droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations coassemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"142 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73242269","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metapopulations with habitat modification","authors":"Z. Miller, S. Allesina","doi":"10.1101/2021.05.27.446046","DOIUrl":"https://doi.org/10.1101/2021.05.27.446046","url":null,"abstract":"Significance Beavers build dams, which dramatically alter the local landscape and ecological community. Bacteria modify the chemistry of their environment, changing its suitability for other microbes. Viral infections induce adaptive immunity, blunting future infection by similar strains. These apparently dissimilar situations share common features: An organism causes lasting changes to the environment that affect other species—even after the beavers emigrate, the bacterial colony collapses, or the infection is cleared. To understand the dynamics of these systems, we extend a metapopulation model (in which local populations inhabit patches connected by dispersal) to incorporate “patch memory,” modeling environmental modification. This model can produce complex dynamics and illuminates mechanisms that promote diversity in the meta-ecosystem and affect its robustness to changing environmental conditions. Across the tree of life, organisms modify their local environment, rendering it more or less hospitable for other species. Despite the ubiquity of these processes, simple models that can be used to develop intuitions about the consequences of widespread habitat modification are lacking. Here, we extend the classic Levins metapopulation model to a setting where each of n species can colonize patches connected by dispersal, and when patches are vacated via local extinction, they retain a “memory” of the previous occupant—modeling habitat modification. While this model can exhibit a wide range of dynamics, we draw several overarching conclusions about the effects of modification and memory. In particular, we find that any number of species may potentially coexist, provided that each is at a disadvantage when colonizing patches vacated by a conspecific. This notion is made precise through a quantitative stability condition, which provides a way to unify and formalize existing conceptual models. We also show that when patch memory facilitates coexistence, it generically induces a positive relationship between diversity and robustness (tolerance of disturbance). Our simple model provides a portable, tractable framework for studying systems where species modify and react to a shared landscape.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"54 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86650424","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"COVID-19 and mental health of individuals with different personalities","authors":"E. Proto, A. Zhang","doi":"10.1101/2021.05.24.21257581","DOIUrl":"https://doi.org/10.1101/2021.05.24.21257581","url":null,"abstract":"Significance Analyzing how personality affects mental health deterioration during the COVID-19 pandemic is important because it can lead to more personalized psychological or psychiatric treatments. Drawing on a longitudinal dataset representative of the UK population before and during the pandemic, we document that personality can be an important factor. In particular, agreeableness is a negative predictor, while openness and, to a lower extent, extraversion are positive predictors; the effect of neuroticism is surprisingly weak. In female respondents, cognitive skills and openness, and in non-British White respondents, extraversion and openness are particularly strong predictors of mental health deterioration. The fact that neuroticism has an effect that is weaker than expected represents an interesting puzzle. Several studies have been devoted to establishing the effects of the COVID-19 pandemic on mental health across gender, age, and ethnicity. However, much less attention has been paid to the differential effect of COVID-19 according to different personalities. We do this using the UK Household Longitudinal Study (UKHLS), a large-scale panel survey representative of the UK population. The UKHLS allows us to assess the mental health of the same respondent before and during the COVID-19 period based on their “Big Five” personality traits and cognitive skills. We find that during the COVID-19 period, individuals who have more extravert and open personality traits report a higher mental health deterioration, while those scoring higher in agreeableness are less affected. The effect of openness is particularly strong: One more SD predicts up to 0.23 more symptoms of mental health deterioration in the 12-item General Health Questionnaire (GHQ-12) test during the COVID-19 period. In particular, for females, cognitive skills and openness are strong predictors of mental health deterioration, while for non-British White respondents, these predictors are extraversion and openness. Neuroticism strongly predicts worse mental health cross-sectionally, but it does not lead to significantly stronger deterioration during the pandemic. The study’s results are robust to the inclusion of potential confounding variables such as changes in physical health, household income, and job status (like unemployed or furloughed).","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"36 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85538816","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry","authors":"Wen-Yi Low, Shuhua Thong, Shu-Sin Chng","doi":"10.1101/2021.05.25.445566","DOIUrl":"https://doi.org/10.1101/2021.05.25.445566","url":null,"abstract":"Significance Biological membranes define cellular boundaries, allow compartmentalization, and represent a prerequisite for life. In gram-negative bacteria, the outer membrane (OM) prevents entry of toxic substances, conferring intrinsic resistance against many antibiotics. This barrier function requires unequal distribution of lipids across the OM bilayer, yet how such lipid asymmetry is maintained is not well understood. In this study, we established the directionality of lipid transport for a conserved membrane protein complex and uncovered mechanistic insights into how ATP powers such transport from the OM to the inner membrane. Our work provides fundamental understanding of lipid trafficking within the gram-negative double-membrane envelope in the context of OM lipid asymmetry and highlights the importance of targeting lipid transport processes for future antibiotics development. The hallmark of the gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ATP-binding cassette transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point in vitro lipid transfer assay that allows direct tracking of [14C]-labeled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid-binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts toward discovering new antibiotics against gram-negative pathogens.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89004494","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}