Biology subjects

Hesketh, G. G.

Publications and source records attributed to Hesketh, G. G..

9 recordsLinked to original sources

bioRxiv2026

SCM-1/SCAMP Maintains Microdomain Boundaries and Cargo Sorting within the Endosomal System

After endocytosis, transmembrane cargo reaches sorting endosomes where it is partitioned into physically distinct recycling or degradative microdomains. While the J-domain protein RME-8/DNAJC13 is known to maintain these boundaries by actively removing degradative machinery from the recycling microdomain, other factors that contribute to this spatial organization remain poorly defined. Here, we identify the conserved tetraspan protein SCM-1/SCAMP as a key microdomain organizer, discovered through RME-8 proximity-dependent biotinylation screens in C. elegans and human cells. Leveraging the large endosomes of C. elegans coelomocytes, we show that SCM-1 is selectively enriched within the recycling microdomain. In scm-1 mutants, recycling and degradative microdomains still assemble but fail to remain spatially distinct, resulting in inappropriate microdomain overlap. This loss of boundary integrity occurs without increasing the recruitment of sorting machineries, indicating a mechanism distinct from the RME-8-mediated uncoating pathway. scm-1 mutants exhibit significant sorting defects, including misrouting of recycling cargo MIG-14/Wls and v-SNARE SNB-2/VAMP3 to late endosomes and lysosomes. We find that snb-2 mutants themselves missort MIG-14 to late endosomes and lysosomes, suggesting that SNB-2 sorting is key for recycling function. Our data suggest that both microdomains lose efficiency in scm-1 mutants, as cargo missorted into late endosomes and lysosomes is not depleted overall, and degradation of an independent ESCRT-dependent cargo is delayed. We conclude that SCM-1 ensures endosomal sorting fidelity by stabilizing microdomain boundary integrity, a process required for efficient recycling and degradation of transmembrane cargo.

cell biology↗

bioRxiv2024

Cargo-selective regulation of clathrin-mediated endocytosis by AMP-activated protein kinase

The cell surface abundance of many proteins is controlled by clathrin-mediated endocytosis (CME). CME is driven by the assembly of clathrin and other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). Regulation of CCP dynamics allows for control of the function of specific cell surface proteins, impacting a range of cellular outcomes. AMP-activated protein kinase (AMPK) becomes activated upon metabolic insufficiency and facilitates cellular adaptation to nutrient stress. Here, we examined how AMPK regulates CME and the cell surface membrane traffic of {beta}1-integrin. We find that AMPK controls CCP dynamics and regulates the abundance of the endocytic adaptor protein Dab2 within CCPs in a manner that requires the GTPase Arf6, thus selectively promoting the CCP recruitment and internalization of {beta}1-integrin. This reveals a novel signaling pathway for cargo-selective metabolic regulation of CME by AMPK, impacting the function of cell surface proteins such as integrins.

cell biology↗

bioRxiv2023

Repression of mRNA translation initiation by GIGYF1 via blocking the eIF3-eIF4G1 interaction

Viruses commonly interfere with the function of the eukaryotic translation initiation factor 4G1 (eIF4G1), a pivotal factor in the recruitment of the eIF3 complex and ribosome to the mRNA. This results in the inhibition of general host protein synthesis and redirecting ribosomes toward viral mRNAs. Certain viruses also selectively repress the translation of mRNAs involved in the host antiviral response. GIGYF2 and its interacting cap-binding protein 4EHP enable the transcript-specific repression of mRNA translation mediated by microRNAs and RNA-binding proteins (RBPs). RNA viruses, such as SARS-CoV-2, exploit the GIGYF2/4EHP complex to selectively repress the translation of transcripts such as Ifnb1 mRNA, which encodes the antiviral cytokine Interferon {beta} (IFN-{beta}). Herein, we reveal that GIGYF1, a paralogue of GIGYF2, robustly represses cellular mRNA translation through a distinct mechanism independent of 4EHP. Upon recruitment to a target mRNA by RBPs, the C-terminal region of GIGYF1 binds to subunits of eIF3 at the interaction interface of eIF3-eIF4G1. This disrupts binding of eIF3 to eIF4G1, resulting in mRNA-specific translational repression. This mechanism exerts profound influences on the host cells response to viral infection. Depletion of GIGYF1 induces a robust immune response by derepressing Ifnb1 mRNA translation. Overall, our study highlights a unique mechanism of translational regulation by GIGYF1 that involves sequestering eIF3 and abrogating its binding to eIF4G1. This mechanism can be utilized by RBPs that interact with GIGYF1 to specifically repress the translation of their target mRNAs, significantly affecting critical biological processes, including host-pathogen interactions.

cell biology↗

bioRxiv2022

SLC3A2 N-glycosylation and alternate evolutionary trajectories for amino acid metabolism

SLC3A2 (4F2hc, CD98) is an adaptor to the SLC7A exchangers and has undergone extensive repositioning of N-glycosylation sites with vertebrate evolution, presumably in synchrony with the species-specific demands of metabolism. The SLC3A2*SLC7A5 heterodimer imports essential amino acids (AA) and thereby stimulates mTOR signaling, while SLC3A2*SLC7A11 imports cystine required for glutathione synthesis and mitigation of oxidative stress. Analysis of SLC3A2 N-glycans revealed stable site-specific profiles of Golgi remodeling, apart from the conserved N365 site where branching and poly-N-acetylglucosamine content were sensitive to the insertion of lost ancestral sites and to metabolism. N-glycans at N381 and N365 stabilized SLC3A2 in the galectin lattice and opposed endocytosis, while N365 which is nearest the membrane, also promoted down-regulation by galectin-driven clathrin-independent endocytosis (glycolipid-lectin GL-Lect). This is the first report of both positive and negative regulation by galectin binding to N-glycans that are strategically positioned in the same membrane glycoproteins. Proteomics analysis in SLC3A2 mutant HeLa cells with induced re-expression of SLC3A2 as bait revealed the canonical non-N-glycosylated interactors, SLC7A5 and SLC7A11 exchangers, but also AA transporters that were dependent on SLC3A2 N-glycosylation, and are themselves, N-glycosylated AA/Na+ symporters (SLC7A1, SLC38A1, SLC38A2, SLC1A4, SLC1A5). The results suggest that the N-glycans on SLC3A2 regulate clustering of SLC7A exchangers with AA/Na+ symporters, thereby promoting Gln/Glu export-driven import of essential AA and cystine, with the potential to adversely impact redox balance. The evolution of modern birds (Neoaves) led to improved control of bioenergetics with the loss of genes including SLC3A2, SLC7A-5, -7, -8, -10, BCAT2, KEAP1, as well as duplications of SLC7A9, SLC7A11 and the Golgi branching enzymes MGAT4B and MGAT4C known to enhance affinities for galectins. Analyzing the fate of these and other genes in the down-sized genomes of birds, spanning [~]10,000 species and >100 Myr of evolution, may reveal the mystery of their longevity with prolonged vitality. Key PointsO_LIGolgi N-glycan remodeling at each site on SLC3A2 differs with the microenvironment. C_LIO_LIThe galectin lattice and GL-Lect mediated endocytosis act as opposing forces on trafficking, controlled by N-glycans at the distal N381 and membrane proximal N365 sites, respectively. C_LIO_LIMutation at N381 or N365 decreased SLC3A2 association with SLC7A5, SLC7A11 and N-glycosylated AA/Na+ symporters as well as the capacity to mitigate stress. C_LIO_LIClustering of SLC3A2*SLC7A exchangers, with AA/Na+ symporter and ATPase Na+/K+ exchanger promotes growth but continuously consumes ATP in non-proliferating cells. C_LIO_LIBird evolution has improved bioenergetics with the deletion of SLC3A2 and associated transporters; - replaced by transporters of keto acids and a re-enforced galectin lattice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/516651v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1cb7958org.highwire.dtl.DTLVardef@18ba32dorg.highwire.dtl.DTLVardef@861269org.highwire.dtl.DTLVardef@22df0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Structure of mammalian V-ATPase with the TLDc domain protein mEAK7 bound

V-ATPases are rotary proton pumps that serve as signaling hubs with numerous proposed binding partners in cells. We used cryoEM combined with exhaustive focused classification to detect endogenous proteins that associate with V-ATPase from porcine kidney. A super-stoichiometric copy of subunit C was found in ~3% of complexes, while an additional ~1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 shows that mEAK-7s TLDc domain interacts with V-ATPases stator while its C-terminal helix binds V-ATPases rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike inhibition of yeast V-ATPase by the TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that interaction of mEAK-7 with V-ATPase is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. Together, these results show that differences in V-ATPase binding by TLDc domain-containing proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzymes activity.

biochemistry↗

bioRxiv2020

Rag GTPases and Phosphatidylinositol 3-Phosphate Mediate Recruitment of the AP-5/SPG11/SPG15 Complex

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells, and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which on its own localises to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15 while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway, and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation. SummaryThe AP-5/SPG11/SPG15 complex is recruited onto late endosomes/lysosomes, and contributes to lysosomal homeostasis and autophagic lysosome reformation. Hirst et al. show that recruitment is by coincidence detection, requiring both phosphatidylinositol 3-phosphate and Rag GTPases, thus uncovering a link between AP-5/SPG11/SPG15 and the mTORC1 pathway.

cell biology↗

bioRxiv2019

A proximity biotinylation map of a human cell

Compartmentalization is a defining characteristic of eukaryotic cells, partitioning cellular processes into discrete subcellular locations. High throughput microscopy1 and biochemical fractionation coupled with mass spectrometry2-6 have helped to define the proteomes of a variety of organelles and macromolecular structures. However, many other intracellular compartments have remained refractory to such approaches, due for example to difficulty in purifying non-membrane bound structures. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach for defining the composition of cellular compartments in living cells7-10. Here we present a BioID-based map of a human cell based on 192 markers from 32 different subcellular compartments, comprising 35,902 high confidence proximity interactions, and defining the intracellular locations of 4,145 unique proteins in HEK 293 cells. Our localization predictions meet or exceed previous-approaches, with higher specificity, and enabled the discovery of proteins at the mitochondrial outer membrane-endoplasmic reticulum (ER) interface that are critical for mitochondrial homeostasis. Based on this dataset, we have established humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to better understand BioID datasets.

molecular biology↗

bioRxiv2018

The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that plays an important role in regulating plasma cholesterol and cardiovascular disease risk. PCSK9 secretion uniquely depends on the cytoplasmic COPII protein SEC24A, suggesting the presence of a transmembrane ER cargo receptor mediating this interaction. Here, we report a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify proteins that facilitate the efficient secretion of PCSK9 heterologously expressed in HEK293T cells. We first identified 35 candidate proteins that were labeled by BirA* fusions to PCSK9 and either COPII component SAR1A or SAR1B. We then performed genome-scale pooled CRISPR mutagenesis to identify genes whose perturbation resulted in intracellular accumulation of PCSK9-eGFP but not the control A1AT-mCherry. The 4 most enriched sgRNAs in this screen all targeted SURF4, a homologue of the yeast endoplasmic reticulum (ER) cargo receptor Erv29p and the only candidate also identified by proximity-dependent biotinylation. The functional contribution of SURF4 to PCSK9 secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. Compatible with a function of SURF4 as a cargo receptor for PCSK9, fluorescence microscopy localized SURF4 to the early secretory pathway, coimmunoprecipitation revealed a physical interaction between SURF4 and PCSK9, and SURF4 deletion resulted in decreased extracellular secretion of PCSK9 and PCSK9 accumulation in the ER. Taken together, these findings support a model in which SURF4 functions as an ER cargo receptor for the efficient cellular secretion of PCSK9.

cell biology↗

bioRxiv2017

Proteomic and biochemical comparison of the cellular interaction partners of human VPS33A and VPS33B

Multi-subunit tethering complexes control membrane fusion events in eukaryotic cells. CORVET and HOPS are two such multi-subunit tethering complexes, both containing the Sec1/Munc18 protein subunit VPS33A. Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. It has been recently suggested that VPS33B and VIPAR comprise two subunits of a novel multi-subunit tethering complex (named CHEVI), analogous in configuration to CORVET and HOPS. We utilised the BioID proximity biotinylation assay to compare and contrast the interactomes of VPS33A and VPS33B. Overall, few proteins were identified as associating with both VPS33A and VPS33B, suggesting these proteins have distinct sub-cellular localisations. Consistent with previous reports, we observed that VPS33A was co-localised with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although in this assay VPS33A clearly co-localised with several subunits of CORVET and HOPS, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localise with VPS33B. Instead, we identified two novel VPS33B-interacting proteins, VPS53 and CCDC22. CCDC22 co-immunoprecipitated with VPS33B and VIPAR in over-expression conditions and interacts directly with the VPS33B-VIPAR complex in vitro. However, CCDC22 does not appear to co-fractionate with VPS33B and VIPAR in gel filtration of human cell lysates. We also observed that the protein complex in HEK293T cells which contained VPS33B and VIPAR was considerably smaller than CORVET/HOPS, suggesting that, unlike VPS33A, VPS33B does not assemble into a large stable multi-subunit tethering complex.

biochemistry↗