Biology subjects

Freund, C.

Publications and source records attributed to Freund, C..

At least 19 recordsLinked to original sources

bioRxiv2026

A general role for GGA adaptors in the modulation of AP-1-dependent trafficking

The Golgi-localized, {gamma}-ear containing, ADP-ribosylation factor binding proteins (GGAs) are a family of adaptor proteins that regulate transport of specific cargo receptors from the Golgi to endosomes. For many years it was assumed that GGAs transport cargo via interaction with the adaptor complex AP-1. However, recent findings suggest that GGA and AP-1 may have opposing roles, with GGAs facilitating forward transport between Golgi and endosomes, and AP-1 mediating the opposite trafficking step. To shed light on the functional connection of GGAs with AP-1, we combined CRISPR-Cas9 gene editing with live-cell imaging and TurboID-based proximity labelling. We find that GGAs localize not only to the Golgi apparatus but also, to a greater extent, to peripheral ARF1-positive compartments responsible for secretory trafficking and endocytic recycling. At both, the Golgi and peripheral sites, we observe distinct sorting domains containing either AP-1 or GGAs alone, as well as domains in which both adaptors are present. Interestingly, GGAs can recruit clathrin lattices independently of AP-1. Proximome mapping shows that AP-1 specific cargoes only localize to AP-1 domains in the absence of GGAs. These findings point to a regulatory role of GGAs in AP-1 transport. We speculate that GGAs prevent binding of AP-1 to its cargo clients to avoid premature retrieval and to modulate bi-directional trafficking between the Golgi and endosomes.

cell biology↗

bioRxiv2025

Multi-omics reveals global signaling rewiring and identifies Activin A-induced dysregulation of FOS/Activator Protein 1 as a novel target in Fibrodysplasia ossificans progressiva

BackgroundFibrodysplasia ossificans progressiva (FOP) is caused by an activating mutation (p.R206H) in the type I BMP receptor ALK2, leading to heterotopic ossification (HO) in soft connective tissues. While aberrant Activin A-induced SMAD signaling is central in FOP pathogenesis, global signaling alterations remain poorly understood. MethodsWe performed phosphoproteomics, transcriptomics and biochemical analyses in mesenchymal cells (MSCs) overexpressing wild-type ALK2WT or mutant ALK2R206H receptors and in induced-MSCs derived from FOP patient iPSCs. Findings were validated in vivo using FOP-like mouse models and in vitro via pharmacological interventions. ResultsMulti-omics analyses revealed previously unrecognized signaling networks in ALK2R206H cells, including enhanced MAPK, mTOR, RUNX2 and RHO-mediated mechanotransduction pathways. Notably, we identified dysregulated Activator Protein-1 (AP-1) expression and function as a novel contributor to FOP. AP-1 factors were highly enriched in HO lesions in FOP-like animals. Pharmacological inhibition of AP-1 significantly reduced osteochondrogenic differentiation in vitro. ConclusionThis study highlights global signaling dysregulation in FOP and identifies AP-1 as a critical driver and potential therapeutic target for FOP.

molecular biology↗

bioRxiv2024

T cell interaction partners of DHHC20

Reversible palmitoylation of proteins at cysteine residues represents a post-translational modification that can alter the cellular localization of proteins, change their distribution within lipid membranes or modulate their conformation and molecular interaction patterns. DHHC enzymes catalyze protein palmitoylation, while thioesterases or hydrolases can rapidly remove the acyl-chain. In human T cells, DHHC proteins have been shown to modify proteins that are involved in major signaling pathways such as Ca2+-signaling or kinase-dependent activation of transcription factors for cytokines. For DHHC20, a role in the palmitoylation of the Orai1 Ca2+-channel has been demonstrated, but otherwise its T cell interaction partners are largely unknown. Here, we show that recombinantly expressed DHHC20 robustly interacts with 28 proteins from Jurkat T cells, as shown by affinity enrichment combined with mass spectrometric analysis. We find a robust interaction between DHHC20 and the {beta}-subunit of the trimeric G protein Gs{beta}1{gamma}2, while the typically palmitoylated Ga subunit is not identified. Cross-linking mass spectrometry with purified DHHC20 and Gs{beta}1{gamma}2 then confirms a direct interaction between the {beta}1{gamma}2 domains and the enzyme, while rigid docking offers structural poses that are in agreement with the observed intermolecular cross-linking constraints. Thus, we suggest a model where the {beta}1{gamma}2 subunits of a trimeric G protein serve as a stable interaction partner of a DHHC enzyme, presumably acting as a landing platform for the G subunit that is subsequently palmitoylated by the enzyme.

biochemistry↗

bioRxiv2024

Quantitative analysis of MHC class II peptide exchange reveals pivotal role of peptide association rate

MHC-II presents antigenic peptides to T helper cells, thus shaping adaptive immune responses. Peptide loading of MHC-II in endosomes is shaped by the susceptibility of the peptide-MHC-II complex to dissociation by the catalyst HLA-DM. For a given MHC-II allotype, experimental data reveal an enormous range of HLA-DM susceptibilities of different peptides - more than five orders of magnitude. To understand the underlying mechanisms, we develop a coarse-grained kinetic model and confront it with experimental data. The model explains the observed variation of HLA-DM susceptibility with the peptide-MHC-II binding energy by an allosteric competition mechanism. Paradoxically, however, certain peptides are resistant to dissociation by HLA-DM regardless of their binding energy. Our model predicts that this resistance is linked with fast peptide association to MHC-II in the absence of HLA-DM. In sum, our data-based theoretical analysis identifies two distinct molecular mechanisms that shape antigen presentation by MHC-II.

systems biology↗

bioRxiv2024

Effective assessment of CD4+ T cell Immunodominance patterns: impact of antigen processing and HLA restriction

CD4+ T cell responses to viral infections are driven by immunodominant determinants. Accessing the peptides defining these determinants in humans is essential for advancing our understanding and potentially tuning immune responses to pathogens. State of the art methods identifying CD4+ T cell immunodominant epitopes are constrained by limitations in throughput, performance or both. Here, we leverage on the combined use of a reconstituted antigen processing system and of in silico prediction tools to query and study CD4+ T cell immunodominance of two model SARS-CoV-2 antigens. We applied this combined platform over a DRB1* panel with broad population and functional coverage to gain mechanistic insights beyond single allotypes. This approach delineates a minimalistic peptide pool (59 candidates) featuring a high immunogenic profile (similar response to 10-fold larger pools) through a representative human sample. Analysis of antigen-encoded and processing-related features on IEDB curated immunodominant peptides reveal that distinct antigen processing mechanisms apply for the Nucleocapsid and Spike model antigens. Notably, the First Bind and then cut mechanism was favored for Nucleocapsid-derived epitopes (up to 55 %), whereas the First Cut and then bind predominated for the Spike (80 %). Together, our results highlight differential processing pathways underlying CD4 T cell immunodominance for distinct viral antigens providing a mechanistic foundation to improve epitope prediction algorithms. Significance StatementEfficient access to immunodominant CD4+ T cell epitopes will inform improved peptide pool design for studying or tuning immunity towards any pathogen. Despite advances in the field, in silico epitope prediction remains error-prone and experimental efforts are logistically challenging. Epitope prediction tools are mainly focused on the identification of binding motifs while neglecting key processing steps. Considering antigen processing factors and constraints is expected to improve their performance. We describe and apply a platform for the systematic investigation of antigen processing mechanisms and how they impinge on the selection of CD4+ T cell immunodominance. Our results validate distinct immunodominant epitope selection pathways for two model antigens at the human population level.

immunology↗

bioRxiv2023

Population context drives cell-to-cell variability in interferon response in epithelial cells

Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to the neighboring cells) of human intestinal epithelial cells affects single cell response to interferons. We identified that cells at the edge of a cellular colony are significantly more responsive than cells embedded within this colony. We determined that this spatial heterogeneity in IFN response was the result of the polarized basolateral distribution of the IFN receptors making cells located in the center of a cellular colony not responsive to ectopic IFN stimulation. We could demonstrate that this population context driven cell-to-cell variability influences the outcome of viral infection as cells embedded in a cellular colony are not protected by interferons and therefore more susceptible to infection. Our data highlights that the behavior of individual isolated cells does not directly translate to their behavior in a population, placing the population context as a key driver of cell-to-cell heterogeneity in IFN response.

immunology↗

bioRxiv2023

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space

Fibroblast Growth Factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by PI(4,5)P2-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the protein-protein interaction interface through which FGF2 binds to the 1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we provide insights into a FGF2 dimerization interface that brings C95 residues in close proximity, resulting in disulfide bridged FGF2 dimers. We propose a mechanism by which they bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95 bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

biochemistry↗

bioRxiv2022

Efficient and scalable generation of primordial germ cells in 2D culture using basement membrane extract overlay

Current human primordial germ cell like cells (hPGCLCs) differentiation methods from human pluripotent stem cells (hPSCs) are inefficient, and it is challenging to generate sufficient hPGCLCs to optimize in vitro gametogenesis. We present a new differentiation method that uses diluted basement membrane extract (BME) and low BMP4 concentration to efficiently induce hPGCLC differentiation in scalable 2D cell culture. We show that BME overlay potentiated BMP/SMAD signaling, induced lumenogenesis and increased expression of key hPGCLC-progenitor markers such as TFAP2A and EOMES. These findings highlight the importance of (factors in) BME during hPGCLC differentiation, and demonstrate the potential of the BME-overlay method to interrogate the formation of PGCs and amnion in humans as well to investigate the next steps to achieve in vitro gametogenesis. HIGHLIGHTSO_LIhPGCLCs can be generated efficiently in 2D from hPSCs with treatment of BMP4 and BME overlay C_LIO_LIBME overlay method is highly scalable, cost-effective and simple to perform C_LIO_LIhPGCLCs differentiate together with amniotic ectoderm- and mesoderm-like cells from a TFAP2A+/CDX2+/EOMES+/GATA3+ common progenitor population C_LIO_LIBME overlay enables robust hPGCLC formation by potentiating BMP/SMAD signaling in the common progenitor population C_LI

developmental biology↗

bioRxiv2021

ADAPs intrinsically disordered region is an actin sponge regulating T cell motility

Intrinsically disordered proteins (IDPs) play a vital role in biological processes that rely on transient molecular compartmentation1. In T cells, the dynamic switching between migration and adhesion mandates a high degree of plasticity in the interplay of adhesion and signaling molecules with the actin cytoskeleton2,3. Here, we show that the N-terminal intrinsically disordered region (IDR) of adhesion- and degranulation-promoting adapter protein (ADAP) acts as a multipronged scaffold for G- and F-actin, thereby promoting actin polymerization and bundling. Positively charged motifs, along a sequence of at least 200 amino acids, interact with both longitudinal sides of G-actin in a promiscuous manner. These polymorphic interactions with ADAP become constrained to one side once F-actin is formed. Actin polymerization by ADAP acts in synergy with a capping protein but competes with cofilin. In T cells, ablation of ADAP impairs adhesion and migration with a time-dependent reduction of the F-actin content in response to chemokine or T cell receptor (TCR) engagement. Our data suggest that IDR-assisted molecular crowding of actin above the critical concentration defines a new mechanism to regulate cytoskeletal dynamics. The principle of IDRs serving as molecular sponges to facilitate regulated self-assembly of filament-forming proteins might be a general phenomenon.

biochemistry↗

bioRxiv2021

Target recognition in tandem WW domains: complex structures for parallel and antiparallel ligand orientation in h-FBP21 tandem WW

Protein-protein interactions often rely on specialized recognition domains, such as WW domains, which bind to specific proline-rich sequences. The specificity of these protein-protein interactions can be increased by tandem repeats, i.e. two WW domains connected by a linker. With a flexible linker, the WW domains can move freely with respect to each other. Additionally, the tandem WW domains can bind in two different orientations to their target sequences. This makes the elucidation of complex structures of tandem WW domains extremely challenging. Here, we identify and characterize two complex structures of the tandem WW domain of human formin-binding protein 21 and a peptide sequence from its natural binding partner, the core-splicing protein SmB/B. The two structures differ in the ligand orientation, and consequently also in the relative orientation of the two WW domains. We analyze and probe the interactions in the complexes by molecular simulations and NMR experiments. The workflow to identify the complex structures uses molecular simulations, density-based clustering and peptide docking. It is designed to systematically generate possible complex structures for repeats of recognition domains. These structures will help us to understand the synergistic and multivalency effects that generate the astonishing versatility and specificity of protein-protein interactions.

molecular biology↗

bioRxiv2021

When less is more - Endogenous tagging with TurboID increases the sensitivity of proximity labelling-based experiments

In recent years, proximity labelling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. While physiological expression of the labelling enzyme is beneficial for the mapping of interactors, generation of the desired cell lines remains time-consuming and challenging. Using our established pipeline for the rapid generation of C- and N-terminal CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labelling enzymes when endogenously expressed. Endogenous tagging of the subunit of the AP-1 complex with TurboID allowed identification of known interactors and cargo proteins that simple overexpression of a labelling enzyme fusion protein could not reveal. We used the KI-strategy to compare the interactome of the different adaptor protein (AP) complexes and clathrin and were able to assemble lists of potential interactors and cargo proteins that are specific for each sorting pathway. Our approach greatly simplifies the execution of proximity labelling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry analysis and interactome data in just over a month.

cell biology↗

bioRxiv2021

Inability to switch from ARID1A-BAF to ARID1B-BAF impairs exit from pluripotency and commitment towards neural crest differentiation in ARID1B-related neurodevelopmental disorders

The BAF complex modulates chromatin accessibility. Specific BAF configurations have functional consequences, and subunit switches are essential for cell differentiation. ARID1B and its paralog ARID1A encode for mutually exclusive BAF subunits. De novo ARID1B haploinsufficient mutations cause a neurodevelopmental disorder spectrum, including Coffin-Siris syndrome, which is characterized by neurological and craniofacial features. Here, we reprogrammed ARID1B+/- Coffin-Siris patient-derived skin fibroblasts into iPSCs and modeled cranial neural crest cell (CNCC) formation. We discovered that ARID1B is active only during the first stage of this process, coinciding with neuroectoderm specification, where it is part of a lineage-specific BAF configuration (ARID1B-BAF), which includes SMARCA4 and nine additional subunits. ARID1B-BAF acts as a gatekeeper, ensuring exit from pluripotency and lineage commitment, by attenuating NANOG, SOX2 and thousands of enhancers directly regulated by these two pluripotency factors at the iPSC stage. In iPSCs, these enhancers are maintained active by an ARID1A-containing BAF. At the onset of differentiation, cells transition from ARID1A-BAF to ARID1B-BAF, eliciting attenuation of the NANOG/SOX2 networks, and triggering pluripotency exit. Coffin-Siris patient cells fail to perform the ARID1A/ARID1B switch and maintain ARID1A-BAF at pluripotency enhancers throughout all stages of CNCC formation. This leads to a persistent and aberrant SOX2 and NANOG activity, which impairs CNCC formation. In fact, despite showing the typical neural crest signature (TFAP2A+, SOX9+), ARID1B-haploinsufficient CNCCs are also NANOG/OCT4-positive, in stark contrast with the ARID1B-wt CNCCs, which are NANOG/OCT4-negative. These findings suggest a connection between ARID1B mutations, neuroectoderm formation, and a pathogenic mechanism for Coffin-Siris syndrome.

genomics↗

bioRxiv2021

Spatial and temporal plasticity of neoantigen-specific T-cell responses bases on characteristics associated to antigen and TCR

Neoantigens derived from somatic mutations have been demonstrated to correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of personalized medicine approaches although their quality and associated immune responses is not yet well understood. In a case study of metastatic malignant melanoma, we performed an in-depth characterization of neoantigens and respective T-cell responses in the context of immunotherapy with Ipilimumab. Three neoantigens identified either by immunopeptidomics or in silico prediction were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient immune repertoire recognizing these antigens. TCRs were compared in-vitro and in-vivo with multi-parametric analyses. Identified immunogenic peptides showed similar binding affinities to the human leukocyte antigen (HLA) complex and comparable differences to their wildtype counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs differed substantially in functionality and frequency. In fact, TCRs with comparably lower functional avidity and higher potential for cross-reactivity provided at least equal anti-tumor immune responses in vivo. Of note, these TCRs showed a reduced activation pattern upon primary in vitro stimulation. Exploration of the TCR-{beta} repertoire in blood and in different tumor-related tissues over three years, offered insights on the high frequency and particular long-term persistence of low-avidity TCRs. These data indicate that qualitative differences of neoantigen-specific TCRs and their impact on function and longevity need to be considered for neoantigen targeting by adoptive T-cell therapy using TCR-transgenic T cells. Statement of translational relevanceImmunotherapy has demonstrated high efficacy in diverse malignancies. Neoantigens derived from mutations provide promising targets for safe and highly tumor-specific therapeutic approaches. Yet, single determinants of an effective and enduring T-cell mediated tumor rejection are still not well understood. We analyzed in detail seven neoantigen-specific T-cell receptors (TCRs) derived from a melanoma patient targeting three different altered peptide ligands identified by mass spectrometry and prediction analyses. Functional characterization of these TCRs revealed potent anti-tumor reactivity of all TCRs. Of special interest, TCRs with comparably lower affinity demonstrated effective in vivo activity as well as dominant spatial and temporal distribution in blood and tissue. Functional differences of TCR may require further T-cell and/or TCR engineering and should be considered for future clinical trial designs.

immunology↗

bioRxiv2020

Human mobility at Tell Atchana (Alalakh) during the 2nd millennium BC: integration of isotopic and genomic evidence

The Middle and Late Bronze Age Near East, a period roughly spanning the second millennium BC (ca. 2000-1200 BC), is frequently referred to as the first international age, characterized by intense and far-reaching contacts between different entities from the eastern Mediterranean to the Near East and beyond. In a large-scale tandem study of stable isotopes and ancient DNA of individuals excavated at Tell Atchana (Alalakh), situated in the northern Levant, we explore the role of mobility at the capital of a regional kingdom. We generated strontium isotope data for 53 individuals, oxygen isotope data for 77 individuals, and added ancient DNA data from 9 new individuals to a recently published dataset of 28 individuals. A dataset like this, from a single site in the Near East, is thus far unparalleled in terms of both its breadth and depth, providing the opportunity to simultaneously obtain an in-depth view of individual mobility and also broader demographic insights into the resident population. The DNA data reveals a very homogeneous gene pool, with only one outlier. This picture of an overwhelmingly local ancestry is consistent with the evidence of local upbringing in most of the individuals indicated by the isotopic data, where only five were found to be non-local. High levels of contact, trade, and exchange of ideas and goods in the Middle and Late Bronze Ages, therefore, seem not to have translated into high levels of individual mobility detectable at Tell Atchana.

genetics↗

bioRxiv2020

The postsynaptic MAGUK scaffold protein MPP2 organises a distinct interactome that incorporates GABAA receptors at the periphery of excitatory synapses

Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in sub-synaptic nanodomains. The synaptic MAGUK scaffold protein MPP2 is a component of AMPA receptor-associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM1. Using super-resolution imaging, we now show that MPP2 and SynCAM1 are situated at the periphery of the postsynaptic density. In order to explore MPP2-associated protein complexes, we used a quantitative comparative mass spectrometry approach and identified multiple GABAA receptor subunits among the novel synaptic MPP2 interactors. We further show that GABAA receptors are found together with MPP2 in a subset of dendritic spines and thus highlight MPP2 as a scaffold molecule capable of acting as an adaptor molecule that links peripheral synaptic elements critical for inhibitory regulation to central structures at the PSD of glutamatergic synapses.

neuroscience↗

bioRxiv2020

Genomic and dietary transitions during the Mesolithic and Early Neolithic in Sicily

Southern Italy is a key region for understanding the agricultural transition in the Mediterranean due to its central position. We present a genomic transect for 19 prehistoric Sicilians that covers the Early Mesolithic to Early Neolithic period. We find that the Early Mesolithic hunter-gatherers (HGs) are a highly drifted sister lineage to Early Holocene western European HGs, whereas a quarter of the Late Mesolithic HGs ancestry is related to HGs from eastern Europe and the Near East. This indicates substantial gene flow from (south-)eastern Europe between the Early and Late Mesolithic. The Early Neolithic farmers are genetically most similar to those from the Balkan and Greece, and carry only a maximum of [~]7% ancestry from Sicilian Mesolithic HGs. Ancestry changes match changes in dietary profile and material culture, except for two individuals who may provide tentative initial evidence that HGs adopted elements of farming in Sicily. One-sentence summaryGenome-wide and isotopic data from prehistoric Sicilians reveal a pre-farming connection to (south-) eastern Europe, and tentative initial evidence that hunter-gatherers adopted some Neolithic aspects prior to near-total replacement by early farmers.

genetics↗

bioRxiv2019

Dynamic Palmitoylation Events Following T-Cell Receptor Signaling

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi.

biochemistry↗

bioRxiv2019

The α1 subunit of the Na,K-ATPase acts upstream of PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2 from tumor cells

Fibroblast Growth Factor 2 (FGF2) is a tumor cell survival factor that is exported from cells by an unconventional secretory pathway. This process is based on direct translocation of FGF2 across the plasma membrane. FGF2 membrane translocation depends on PI(4,5)P2-induced formation of membrane-inserted FGF2 oligomers followed by extracellular trapping of FGF2 at the outer leaflet mediated by cell surface heparan sulfate proteoglycans. Beyond the well-characterized core mechanism of FGF2 membrane translocation, the Na,K-ATPase has been proposed to play a so far unknown role in unconventional secretion of FGF2. Here, we define a direct physical interaction of FGF2 with a subdomain of the cytoplasmic part of the 1 subunit of the Na,K-ATPase. Employing NMR spectroscopy and molecular dynamics simulations, we identified two lysine residues on the molecular surface of FGF2 that are shown to be essential for its interaction with 1. In intact cells, the corresponding lysine-to-glutamate variants of FGF2 were characterized by inefficient secretion and reduced recruitment to the inner plasma membrane leaflet as shown by single molecule TIRF microscopy. Our findings suggest that 1 acts upstream of PI(4,5)P2 facilitating efficient membrane translocation of FGF2 to the cell surface of tumor cells.

biochemistry↗