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Lipids are distributed in a highly heterogeneous fashion in different cellular membranes. Only a minority of lipids achieve their final intracellular distribution through transport by vesicles. Instead, the bulk of lipid traffic is mediated by a large group of lipid transfer proteins (LTPs), which move small numbers of lipids at a time using hydrophobic cavities that stabilize lipid molecules outside membranes. Although the first LTPs were discovered almost 50 years ago, most progress in understanding these proteins has been made in the past few years, leading to considerable temporal and spatial refinement of our understanding of the function of these lipid transporters. The number of known LTPs has increased, with exciting discoveries of their multimeric assembly. Structural studies of LTPs have progressed from static crystal structures to dynamic structural approaches that show how conformational changes contribute to lipid handling at a sub-millisecond timescale. A major development has been the finding that many intracellular LTPs localize to two organelles at the same time, forming a shuttle, bridge or tube that links donor and acceptor compartments. The understanding of how different lipids achieve their final destination at the molecular level allows a better explanation of the range of defects that occur in diseases associated with lipid transport and distribution, opening up the possibility of developing therapies that specifically target lipid transfer.

The noradrenaline transporter has a pivotal role in regulating neurotransmitter balance and is crucial for normal physiology and neurobiology 1 . Dysfunction of noradrenaline transporter has been implicated in numerous neuropsychiatric diseases, including depression and attention deficit hyperactivity disorder 2 . Here we report cryo-electron microscopy structures of noradrenaline transporter in apo and substrate-bound forms, and as complexes with six antidepressants. The structures reveal a noradrenaline transporter dimer interface that is mediated predominantly by cholesterol and lipid molecules. The substrate noradrenaline binds deep in the central binding pocket, and its amine group interacts with a conserved aspartate residue. Our structures also provide insight into antidepressant recognition and monoamine transporter selectivity. Together, these findings advance our understanding of noradrenaline transporter regulation and inhibition, and provide templates for designing improved antidepressants to treat neuropsychiatric disorders. Cryo-electron microscopy structures of the noradrenaline transporter in the apo state, bound to noradrenaline and bound to various antidepressants shed light on the substrate transport, molecular recognition and dimeric architecture of this protein.

BTR1 (SLC4A11) is a NH 3 stimulated H + (OH - ) transporter belonging to the SLC4 family. Dysfunction of BTR1 leads to diseases such as congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy (FECD). However, the mechanistic basis of BTR1 activation by alkaline pH, transport activity regulation and pathogenic mutations remains elusive. Here, we present cryo-EM structures of human BTR1 in the outward-facing state in complex with its activating ligands PIP 2 and the inward-facing state with the pathogenic R125H mutation. We reveal that PIP 2 binds at the interface between the transmembrane domain and the N-terminal cytosolic domain of BTR1. Disruption of either the PIP 2 binding site or protonation of PIP 2 phosphate groups by acidic pH can transform BTR1 into an inward-facing conformation. Our results provide insights into the mechanisms of how the transport activity and conformation changes of BTR1 are regulated by PIP 2 binding and interaction of TMD and NTD. BTR1 is an electrogenic H+ transporter belonging to the bicarbonate transporter family. Here, the authors present structures of BTR1 in different conformations and uncover PIP2 as the activator of BTR1.

Cation/proton antiporters (CPAs) play a major role in maintaining living cells’ homeostasis. CPAs are commonly divided into two main groups, CPA1 and CPA2, and are further characterized by two main phenotypes: ion selectivity and electrogenicity. However, tracing the evolutionary relationships of these transporters is challenging because of the high diversity within CPAs. Here, we conduct comprehensive evolutionary analysis of 6537 representative CPAs, describing the full complexity of their phylogeny, and revealing a sequence motif that appears to determine central phenotypic characteristics. In contrast to previous suggestions, we show that the CPA1/CPA2 division only partially correlates with electrogenicity. Our analysis further indicates two acidic residues in the binding site that carry the protons in electrogenic CPAs, and a polar residue in the unwound transmembrane helix 4 that determines ion selectivity. A rationally designed triple mutant successfully converted the electrogenic CPA, EcNhaA, to be electroneutral. Cation/proton antiporters (CPAs) play a major role in maintaining living cells’ homeostasis and are divided in two main groups: CPA1 and CPA2. Here authors use a comprehensive evolutionary analysis of 6537 representative CPAs and reveal a sequence motif that determines central phenotypic characteristics.

T-cell-mediated immune responses aim to protect mammals against cancers and infections, and are also involved in the pathogenesis of various inflammatory or autoimmune diseases. Cellular uptake and the utilization of nutrients is closely related to the T-cell fate decision and function. Research in this area has yielded surprising findings in the importance of amino-acid transporters for T-cell development, homeostasis, activation, differentiation and memory. In this review, we present current information on amino-acid transporters, such as LAT1 ( l -leucine transporter), ASCT2 ( l -glutamine transporter) and GAT-1 ( γ -aminobutyric acid transporter-1), which are critically important for mediating peripheral naive T-cell homeostasis, activation and differentiation, especially for Th1 and Th17 cells, and even memory T cells. Mechanically, the influence of amino-acid transporters on T-cell fate decision may largely depend on the mechanistic target of rapamycin complex 1 (mTORC1) signaling. These discoveries remarkably demonstrate the role of amino-acid transporters in T-cell fate determination, and strongly indicate that manipulation of the amino-acid transporter-mTORC1 axis could ameliorate many inflammatory or autoimmune diseases associated with T-cell-based immune responses.

Glucose is the primary source of energy for many organisms and is efficiently taken up by bacteria through a dedicated transport system that exhibits high specificity. In Escherichia coli , the glucose-specific transporter IICB Glc serves as the major glucose transporter and functions as a component of the phosphoenolpyruvate-dependent phosphotransferase system. Here, we report cryo-electron microscopy (cryo-EM) structures of the glucose-bound IICB Glc protein. The dimeric transporter embedded in lipid nanodiscs was captured in the occluded, inward- and occluded, outward-facing conformations. Together with biochemical and biophysical analyses, and molecular dynamics (MD) simulations, we provide insights into the molecular basis and dynamics for substrate recognition and binding, including the gates regulating the binding sites and their accessibility. By combination of these findings, we present a mechanism for glucose transport across the plasma membrane. Overall, this work provides molecular insights into the structure, dynamics, and mechanism of the IICB Glc transporter in a native-like lipid environment. Glucose is a key energy source for many organisms, efficiently transported in bacteria by specific systems. Here, the authors reveal cryo-EM structures of the glucose transporter IICB from E. coli, providing insights into its mechanism and dynamics.

The presynaptic serotonin transporter (SERT) clears extracellular serotonin following vesicular release to ensure temporal and spatial regulation of serotonergic signalling and neurotransmitter homeostasis. Prescription drugs used to treat neurobehavioral disorders, including depression, anxiety, and obsessive-compulsive disorder, trap SERT by blocking the transport cycle. In contrast, illicit drugs of abuse like amphetamines reverse SERT directionality, causing serotonin efflux. Both processes result in increased extracellular serotonin levels. By combining molecular dynamics simulations with biochemical experiments and using a homologous series of serotonin analogues, we uncovered the coupling mechanism between the substrate and the transporter, which triggers the uptake of serotonin. Free energy analysis showed that only scaffold-bound substrates could initiate SERT occlusion through attractive long-range electrostatic interactions acting on the bundle domain. The associated spatial requirements define substrate and inhibitor properties, enabling additional possibilities for rational drug design approaches. The serotonin transporter, targeted by several medications, terminates neurotransmission by clearing serotonin from the synaptic cleft. Combining biochemical results with in silico data, the authors show the key interactions that initiate substrate transport.

Eukaryotic cells possess a universal repair machinery that ensures rapid resealing of plasma membrane disruptions. Before resealing, the torn membrane is submitted to considerable tension, which functions to expand the disruption. Here we show that annexin-A5 (AnxA5), a protein that self-assembles into two-dimensional (2D) arrays on membranes upon Ca 2+ activation, promotes membrane repair. Compared with wild-type mouse perivascular cells, AnxA5-null cells exhibit a severe membrane repair defect. Membrane repair in AnxA5-null cells is rescued by addition of AnxA5, which binds exclusively to disrupted membrane areas. In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair. We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca 2+ , AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing. Eukaryotic cell plasma membranes possess a mechanism to repair tears caused by stimuli such as mechanical stress. The authors demonstrate that annexin-A5, when assembled into two-dimensional arrays in the presence of calcium, is required for membrane repair.

Intraflagellar transport proteins (IFT) are required for hedgehog (Hh) signalling transduction that is essential for bone development, however, how IFT proteins regulate Hh signalling in osteoblasts (OBs) remains unclear. Here we show that deletion of ciliary IFT80 in OB precursor cells (OPC) in mice results in growth retardation and markedly decreased bone mass with impaired OB differentiation. Loss of IFT80 blocks canonical Hh–Gli signalling via disrupting Smo ciliary localization, but elevates non-canonical Hh–Gαi–RhoA–stress fibre signalling by increasing Smo and Gαi binding. Inhibition of RhoA and ROCK activity partially restores osteogenic differentiation of IFT80 -deficient OPCs by inhibiting non-canonical Hh–RhoA–Cofilin/MLC2 signalling. Cytochalasin D, an actin destabilizer, dramatically restores OB differentiation of IFT80 -deficient OPCs by disrupting actin stress fibres and promoting cilia formation and Hh–Gli signalling. These findings reveal that IFT80 is required for OB differentiation by balancing between canonical Hh–Gli and non-canonical Hh–Gαi–RhoA pathways and highlight IFT80 as a therapeutic target for craniofacial and skeletal abnormalities. Primary cilia are highly conserved microtubule-based organelles that play essential roles in several cellular processes including osteogenesis. Here the authors show that intraflagellar protein IFT80 regulates osteoblast differentiation by balancing signalling though the canonical and non-canonical Hedgehog pathways.