Biology subjects

Parker, J. L.

Publications and source records attributed to Parker, J. L..

7 recordsLinked to original sources

bioRxiv2026

Cornichon receptors couple membrane adaptation to cargo selection during ER export.

Selective export of membrane proteins from the endoplasmic reticulum (ER) is fundamental for eukaryotic cell biology, yet how trafficking receptors coordinate cargo recognition with membrane adaptation and COPII recruitment remains unknown. Cornichon homolog (CNIH) proteins comprise a conserved family of trafficking receptors that mediate ER export of ion channels, G protein-coupled receptors (GPCRs), ATP-binding cassette (ABC) and solute carrier (SLC) transporters. Here, we determine cryo-electron microscopy structures of the prototypical cornichon receptor Erv14 bound to an SLC transporter in detergent and lipid nanodiscs. We show that cargo recognition is mediated by a dynamic network of interactions, in which structural lipids stabilize the receptor-cargo interface. Nanodisc structures reveal the assembly of a second Erv14 receptor that remodels the receptor-cargo interface in response to membrane architecture, thereby reducing local membrane thickness and providing direct structural evidence that cornichon receptors buffer hydrophobic mismatch during membrane protein biogenesis. Structural and trafficking analyses further show that the second receptor recruits the COPII adaptor Sec24, coupling membrane remodelling to cargo export. Together, our findings establish that cornichon receptors couple lipid-mediated membrane adaptation with cargo selection through sequential receptor assembly, linking membrane protein folding to selective COPII-mediated ER export. One sentence summaryCornichon receptors integrate membrane adaptation with cargo recognition to coordinate membrane protein quality control and selective ER export.

cell biology↗

bioRxiv2025

Mechanism of phosphoinositide regulation of lysosomal pH via inhibition of CLC-7

AbstractLysosomes process cellular waste and coordinate responses to metabolic challenge. Central to lysosomal homeostasis are phosphoinositide lipids, key signaling molecules which establish organelle identity, regulate membrane dynamics and are tightly linked to the pathophysiology and therapy of lysosomal storage disorders, neurodegeneration, and cancer. Phosphatidylinositol 3,5- bisphosphate (PI(3,5)P2) interacts with multiple lysosomal membrane proteins and plays a critical role in regulating lysosomal pH by directly inhibiting the chloride/proton antiporter ClC-7, though the molecular mechanism of this inhibition remains unclear. Here, using a combination of functional, structural, and computational analysis, we demonstrate that PI(3,5)P2 binding dramatically remodels the structure of ClC-7 by inducing close association between cytosolic and transmembrane domains. Disease-causing mutations show increased transport activity through loss of PI(3,5)P2 binding and subsequent inhibition. Conversely, ClC-7 activation is correlated with dissociation and increased disorder of the cytoplasmic domain along with novel transmembrane domain conformations, revealing a mechanistic link between specific lysosomal lipids, transporter regulation, and the enigmatic basis of the ClC-7 slow gate.

biochemistry↗

bioRxiv2024

Distinct roles for the Staphylococcus aureus POT transporter DtpT in di/tripeptide uptake and glutathione utilisation inside human macrophages

Peptides available in biological niches inhabited by the human pathogen Staphylococcus aureus serve as a rich source of amino acids required for growth and successful host colonisation. Uptake of peptides by S. aureus involves at least two transport systems: the di/tripeptide permease DtpT and the oligopeptide ABC transporter Opp3. Here we study the individual and combined functions of DtpT and Opp3 in enabling utilisation of diverse di-/tripeptides via a high-throughput phenotypic screen. We reveal that DtpT is the primary route of uptake for dipeptides, and although many peptides can be utilised via either of the two transport systems, we demonstrate a clear preference for Asp/Glu-containing peptides among DtpT substrates. To better understand the substrate preferences of DtpT, the protein was purified and reconstituted into proteoliposomes. Active transport of diverse di- and tripeptides was demonstrated, supporting the conclusions of the phenotypic screen. During this in vitro analysis, we discovered that DtpT could transport the biologically prevalent tripeptide glutathione (GSH). Bacterial growth assays demonstrate that dtpT is essential for GSH utilisation in the absence of the known glutathione transporter, Gis, identifying DtpT as the second GSH uptake system of S. aureus. We demonstrate that GSH transport is required by S. aureus for complete fitness during in vitro macrophage infection experiments. Finally, based on analysis of the DtpT structure and identification of key residues needed for GSH binding and transport, we suggest that GSH transport may be conserved in the DtpT orthologue of Listeria monocytogenes. Together, these data reveal important new functions for DtpT in the utilisation of diverse peptides and point toward a novel role for DtpT (and, potentially, other bacterial POT proteins) in glutathione acquisition during intracellular infection. Author summaryThe environments where bacterial pathogens thrive are often rich in proteins and their degradation products, including oligopeptides, which can be taken up by the bacterium and used as nutrients. Understanding how this occurs could help us find ways to tackle the growth of these pathogens during infection. Here we examine how the major human pathogen Staphylococcus aureus takes up oligopeptides. We demonstrate that a membrane transporter protein, DtpT, is the major route of dipeptide uptake and reveal over 100 new di-/tripeptide targets for this protein. Our findings highlight a defined role for DtpT in the accumulation of Aspartate- and Glutamate-containing peptides, which may serve as relevant nitrogen sources during infection. We also provide the first evidence that DtpT transports the prevalent human metabolite reduced glutathione, and demonstrate that DtpT functions alongside the previously identified Gis glutathione transport system to support intracellular survival of this pathogen inside macrophages. Overall, our findings provide a clear example of how substrate selectivity allows DtpT to fulfil specific biological roles in S. aureus, and this functional specialisation may be a common feature of homologous peptide transporters in other bacterial pathogens and across the Tree of Life.

microbiology↗

bioRxiv2024

Identification of SLC45A4 as a pain gene encoding a neuronal polyamine transporter.

Polyamines are regulatory metabolites with key roles in transcription, translation, cell signalling and autophagy1. They are implicated in multiple neurological disorders including stroke, epilepsy and neurodegeneration and can regulate neuronal excitability through interactions with ion channels2. Polyamines have been linked to pain showing altered levels in human persistent pain states and modulation of pain behaviour in animal models3. However, the systems governing polyamine transport within the nervous system remain unclear. In undertaking a Genome Wide Association Study (GWAS) of chronic pain intensity in the UK-Biobank we found significant association with variants mapping to the SLC45A4 gene locus. In the mouse nervous system SLC45A4 expression is enriched in all sensory neuron sub-types within the dorsal root ganglion including nociceptors. Cell-based assays show that SLC45A4 is a selective plasma membrane polyamine transporter, whilst the cryo-EM structure reveals a novel regulatory domain and basis for polyamine recognition. Mice lacking SLC45A4 show normal mechanosensitivity but reduced sensitivity to noxious heat and algogen induced tonic pain that is associated with reduced excitability of peptidergic nociceptors. Our findings thus establish a role for neuronal polyamine transport in pain perception and identify a new target for therapeutic intervention in pain treatment.

neuroscience↗

bioRxiv2024

The mechanism of mammalian proton-coupled peptide transporters

Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1 and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

biophysics↗

bioRxiv2021

Molecular basis for redox control by the human cystine/glutamate antiporter System xc-.

Cysteine plays an essential role in cellular redox homeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc- is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc- is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc- in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells.

biochemistry↗

bioRxiv2021

A baton-relay mechanism for ER retrieval signal capture and proofreading by the KDEL receptor

The KDEL-retrieval pathway captures escaped ER proteins with a KDEL or variant C-terminal signal at acidic pH in the Golgi and releases them at neutral pH in the ER. To address the mechanism of signal binding and the molecular basis for differences in signal affinity, we determined the HDEL and RDEL bound structures of the KDEL-receptor. Affinity differences are explained by interactions between the variable -4 position of the signal and W120, whereas initial capture of retrieval signals by their carboxyl-terminus is mediated by a baton-relay mechanism involving a series of conserved arginine residues in the receptor. This explains how the signal is first captured and then pulled into the binding cavity. During capture, retrieval signals undergo a selective proofreading step involving two gatekeeper residues D50 and E117 in the receptor. These mechanisms operate upstream of the pH-dependent closure of the receptor and explain the selectivity of the KDEL-retrieval pathway.

cell biology↗