Cellular and Molecular Life Sciences

In mammalian cells one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61-channel. While the Sec61-complex facilitates ER-import of most precursor polypeptides, the Sec61-associated Sec62/Sec63-complex supports ER-import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63-complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER-import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified twenty-two novel Sec62/Sec63-substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides with comparatively longer but less hydrophobic H-region and lower C-region polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly-gating signal peptide and a downstream translocation-disruptive positively charged cluster of amino acid residues as decisive for the Sec62-/Sec63-requirement. In the case of ERj3, these features were found to be responsible for an additional BiP-requirement and to correlate with sensitivity towards the Sec61-channel inhibitor CAM741. Thus, the human Sec62/Sec63-complex may support Sec61-channel opening for precursor polypeptides with slowly-gating signal peptides by direct interaction with the cytosolic amino-terminal peptide of Sec61 or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked Polycystic Liver Disease. DatabasesThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data under the DOI:10.17632/6s5hn73jcv.1 (http://dx.doi.or/10.17632/6s5hn73jcv.1).

Growth hormone (GH) receptor (GHR) and (full-length) prolactin (PRL) receptor (PRLR) are transmembrane class I cytokine receptors that co-exist in various normal and cancerous cells. Both receptors respond to their associated ligands predominantly by activating the Janus Kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) signaling pathways, and both are also known to initiate receptor-specific JAK2-independent signaling. Together with their cognate ligands, these receptors have been associated with pro-tumorigenic effects in various cancers, including breast cancer (BC). Human GH is known to bind GHR and PRLR, while PRL can only bind PRLR. A growing body of work suggests that GHR and PRLR can form heteromers in BC cells, modulating GH signal transduction. However, the dynamics of PRLR and GHR on the plasma membrane and how these could affect their respective signaling still need to be understood. To this end, we set out to unravel the spatiotemporal dynamics of GHR and PRLR on the surface of human T47D breast cancer cells and {gamma}2A-JAK2 cells. We applied direct stochastic optical reconstruction microscopy (dSTORM) and quantified the colocalization and availability of both receptors on the plasma membrane at the nanometer scale at different time points following treatment with GH and PRL. In cells co-expressing GHR and PRLR, we surprisingly observed that not only GH but also PRL treatment induces a significant loss of surface GHR. In cells lacking PRLR or expressing a mutant PRLR deficient in JAK2 binding, we observed that GH induces downregulation of cell surface membrane-bound GHR, but PRL no longer induces loss of surface GHR. Colocalizations of GHR and PRLR were confirmed by proximity ligation (PL) assay. Our results suggest that PRLR-GHR interaction, direct or indirect, is indispensable for PRL- but not GH- induced loss of surface GHR and for both GH-induced and PRL-induced increase of surface PRLR, with potential consequences for downstream signaling. Furthermore, our results suggest that JAK2 binding via the receptor intracellular domains Box1 element is crucial for the observed regulation of one class I cytokine receptors cell surface availability via ligand-induced activation of another class I cytokine receptor. Our findings shed new light on the reciprocal and collective role that PRLR and GHR play in regulating cell signaling.

The organization and dynamic regulation of the vimentin intermediate filament network are essential for diverse cellular processes, including cell division, cytoskeletal crosstalk, and organelle positioning. In particular, the vimentin network is required to maintain a compact perinuclear endoplasmic reticulum (ER) that supports the ER quality-control compartment. Here, we show that the vimentin variant c.1160T>C (L387P), identified in a progeroid syndrome, disrupts ER organization and function. In SW13/cl.2 cells, vimentin L387P forms aggregates containing cisternal-like structures of predominant perinuclear localization that associate with pronounced nuclear distortion. These aggregates sequester chaperones, the SEL1L/HRD1 complex, and the 20S proteasomal subunit, while excluding RNF26 and VCP/p97. This extensive redistribution of ER-associated degradation components coincides with impaired proteostasis and reduced free Ca2+ in intra-organelle compartments, indicating compromised ER functionality. Altogether, these findings suggest that the L387P mutation drives extensive ER disorganization and highlight the importance of an intact vimentin network for ER homeostasis.

Kv1.3 is the main voltage-gated potassium channel in T cells. At the immunological synapse (IS), it sustains Ca{superscript 2} signaling and facilitates T cell activation. Aberrant Kv1.3 expression or activity is linked to autoimmune disorders, yet the mechanisms regulating its targeting and organization at the IS remain unclear. We show that Kv1.3 palmitoylation is a dynamic process mediating channel rearrangement at the IS. The ZDHHC21 acyltransferase, which also S-acylates the TCR, palmitoylates Kv1.3, positioning this enzyme as a potential therapeutic target. Palmitoylation promotes channel migration to the synapse center for removal from the surface. A nonpalmitoylated mutant (Cysless Kv1.3) accumulated at the distal IS and was excluded from lipid raft-enriched domains. Mislocalization and reduced current hindered lymphocyte activation. Moreover, Cysless Kv1.3 showed stronger interaction with PSD95 and cortactin, stabilizing the channel at the surface. These findings highlight S-palmitoylation as a crucial regulator of Kv1.3 during immune responses and a promising target in autoimmune disease therapy.

Clathrin-mediated endocytosis (CME) internalizes cell-surface receptors via clathrin-coated invaginations of the plasma membrane. Both clathrin and endocytic cargo are recruited to the these sites by the adaptor protein complex AP2. AP2 cycles between a closed cytoplasmic conformation and an open membrane-bound state, and efficient CME requires both conformations and their dynamic interconversion. The mechanisms regulating these conformational changes, which include post-translational modifications of the AP2, remain incompletely understood. Here, we report that p70S6 kinase phosphorylates the {micro}2 subunit of the AP2 and that the phosphorylation of serine 45 (S45) depends on p70S6K activity. Loss of S45-{micro}2 phosphorylation results in decreased internalization of canonical CME cargo such as transferrin and PDGF receptors. In Caenorhabditis elegans, lack of S45-{micro}2 phosphorylation produces directionally similar but markedly weaker phenotypes than AP2 loss of function. Live imaging and in silico dynamic modelling suggest that S45-2 phosphorylation has impact on the conformational changes of the AP2 complex. These findings identify a p70S6K-dependent mechanism that modulates AP2 function and highlight the importance of post-translational regulation in controlling CME.

The Spike protein from SARS-CoV-2 mediates docking of the virus onto cells and contributes to viral invasion. Several cellular receptors are involved in SARS-CoV-2 Spike docking at the cell surface, including ACE2 and neuropilin. The intermediate filament protein vimentin has been reported to be present at the surface of certain cells and act as a co-receptor for several viruses; furthermore, its potential involvement in interactions with Spike proteins has been proposed. Here we have explored the binding of Spike protein constructs to several cell types using low-temperature immunofluorescence approaches in live cells, to minimize internalization. Incubation of cells with tagged Spike S or Spike S1 subunit led to discrete dotted patterns at the cell surface, which showed scarce colocalization with a lipid raft marker, but consistent coincidence with ACE2. Under our conditions, vimentin immunoreactivity appeared as spots or patches unevenly distributed at the surface of diverse cell types. Remarkably, several observations including potential antibody internalization and adherence to cells of vimentin-positive structures present in the extracellular medium exposed the complexity of vimentin cell surface immunoreactivity, which requires careful assessment. Notably, overall colocalization of Spike and vimentin signals markedly varied with the cell type and the immunodetection sequence. In turn, vimentin-positive spots moderately colocalized with ACE2; however, a particular enrichment was detected at elongated structures positive for acetylated tubulin, consistent with primary cilia, which also showed Spike binding. Thus, these results suggest that vimentin-ACE2 interaction could occur at selective locations near the cell surface, including ciliated structures, which can act as platforms for SARS-CoV-2 docking.

Endoplasmic reticulum (ER) retention of mis-folded glycoproteins is mediated by the ER- localised eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognises a mis-folded glycoprotein and flags it for ER retention by reglucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease even if the mutant glycoprotein retains activity ("responsive mutant"). Here, we investigated the subcellular localisation of the human Trop-2 Q118E variant, which causes gelatinous drop- like corneal dystrophy (GDLD). Compared with the wild type Trop-2, which is correctly localised at the plasma membrane, the Trop-2-Q118E variant is found to be heavily retained in the ER. Using Trop-2-Q118E, we tested UGGT modulation as a rescue-of-secretion therapeutic strategy for congenital rare disease caused by responsive mutations in genes encoding secreted glycoproteins. We investigated secretion of a EYFP-fusion of Trop-2-Q118E by confocal laser scanning microscopy. As a limiting case of UGGT inhibition, mammalian cells harbouring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 gene expressions were used. The membrane localisation of the Trop-2-Q118E-EYFP mutant was successfully rescued in UGGT1-/-and UGGT1/2-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation constitutes a novel therapeutic strategy for the treatment of Trop-2-Q118E associated GDLD, and it encourages the testing of modulators of ER glycoprotein folding Quality Control (ERQC) as broad-spectrum rescue- of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants. SynopsisDeletion of the UGGT1 and UGGT1/2 genes in HEK 293T cells rescues secretion of an EYFP-fusion of the human Trop-2-Q118E glycoprotein mutant. The mutant is retained in the secretory pathway in wild type cells and it localises to the cell membrane in UGGT1-/- single and UGGT1/2-/- double knock-out cells. The Trop-2-Q118E glycoprotein disease mutant is efficiently glucosylated by UGGT1 in human cells demonstrating that it is a bona fide cellular UGGT1 substrate. O_FIG O_LINKSMALLFIG WIDTH=120 HEIGHT=200 SRC="FIGDIR/small/542711v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@ac29f1org.highwire.dtl.DTLVardef@f5bcbforg.highwire.dtl.DTLVardef@13ab5a1org.highwire.dtl.DTLVardef@16a9406_HPS_FORMAT_FIGEXP M_FIG C_FIG

In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred - either in a membrane-bound melanosome or as a melanosome core, i.e. melanocore. Here we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1 and CtBP1/BARS, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Furthermore, we confirmed that melanocore, but not melanosome uptake is dependent on the Protease-activated receptor-2 (PAR-2) and found that PAR-2 can be specifically activated by melanocores. As skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.

Macropinocytosis is an endocytic process that allows cells to respond to changes in their environment by internalizing nutrients and cell surface proteins, as well as modulating cell size. Here, we identify that adenosine triphosphate (ATP) triggers macropinocytosis in murine neuroblastoma cells, thereby internalizing the ATP release channel pannexin 1 (PANX1) while concurrently increasing cross-sectional cellular area. Amiloride, a potent inhibitor of macropinocytosis-associated GTPases, abolished ATP-induced PANX1 internalization and cell area expansion. Transient expression of the GTP-hydrolysis resistant GTPase ARF6 Q67L led to increased PANX1 internalization and increased cell area equivalent to levels seen with ATP stimulation. Mutation of an extracellular tryptophan (W74) in PANX1 abolished ATP-evoked cell area enlargement suggesting that PANX1 regulates this form of macropinocytosis. This novel role of PANX1 in macropinocytosis could be particularly important for disease states implicating PANX1, such as cancer, where ATP can act as a purinergic regulator of cell growth/metastasis and as a supplementary energy source following internalization.

Tetraspanin proteins play an important role in many cellular processes as they are key organizers of different receptors on the plasma membrane. Most tetraspanins are highly glycosylated at their large extracellular loop, but the function of this post-translational modification remains largely unstudied. In this study we investigated the effects of glycosylation of CD37 and CD53, two tetraspanins important for cellular and humoral immunity. Broad and cell-specific repertoires of N-glycosylated CD37 and CD53 were observed in human B cells. We generated different glycosylation mutants of CD37 and CD53 and analyzed their localization, nanoscale organization and partner protein interaction capacity. Abrogation of glycosylation in CD37 revealed the importance of this modification for CD37 surface expression, whereas neither surface expression nor nanoscale organization of CD53 was affected by its glycosylation. CD37 interaction with its known partner proteins, CD20 and IL-6R, was not affected by glycosylation, other than via its changed subcellular localization. Surprisingly, glycosylation was found to inhibit the interaction between CD53 and its partner proteins CD45 and CD20. Together, our data show that tetraspanin glycosylation affects their function in immune cells, which adds another layer of regulation to tetraspanin-mediated membrane organization.

V-ATPase is an important factor in synaptic vesicle acidification and is implicated in synaptic transmission. Rotation of the extra-membranous V1 sector drives proton transfer through the membrane-embedded multi-subunit V0 sector of the V-ATPase. Intra-vesicular protons are then used to drive neurotransmitter uptake by synaptic vesicles. V0a and V0c, two membrane subunits of the V0 sector have been shown to interact with SNARE proteins and their photo-inactivation rapidly impairs synaptic transmission. V0d, a soluble subunit of the V0 sector strongly interacts with its membrane embedded subunits and is crucial for the canonic proton transfer activity of the V-ATPase. Our investigations show that the loop 1.2 of V0c interacts with complexin, a major partner of the SNARE machinery and that V0d1 binding to V0c inhibits this interaction, as well as V0c association with SNARE complex. Injection of recombinant V0d1 in rat superior cervical ganglion neurons rapidly reduced neurotransmission. In chromaffin cells, V0d1 overexpression and V0c silencing modified in a comparable manner several parameters of unitary exocytotic events. Our data suggest that V0c subunit promotes exocytosis via interactions with complexin and SNAREs and that this activity can be antagonized by exogenous V0d.

Derailed signaling originating from the plasma membrane is associated with many types of cancer. Different human cancers and thrombocytopenia are linked to ANKRD26 mutations. We unveil that Ankrd26 is a plasma membrane-localized protein forming nanoclusters and that Ankrd26 is critical for retinoic acid/BDNF-induced neuroblastoma differentiation. An N-terminal amphipathic structure lacking in an AML-associated Ankrd26 mutant is indispensable for membrane binding and bending by partial membrane insertion and renders Ankrd26 inactive in both gain-of-function and loss-of- function/rescue studies addressing cellular differentiation. In a papillary thyroid carcinoma-linked mutant, truncated Ankrd26 is fused with the kinase domain of the protooncogene RET. Our data show that the Ankrd26 part of this fusion mutant mediates anchoring of the RET kinase domain to the plasma membrane and self-association by the coiled coil domain of Ankrd26. Ankrd26-RET fusion led to massively increased ERK1/2 activity and RET autophosphorylation at both Y905 and Y1015, i.e. caused aberrant RET signaling. Our results highlight the importance and molecular details of Ankrd26-mediated organizational platforms for cellular differentiation and signaling pathways from the plasma membrane, which, if derailed, lead to cancer-associated pathomechanisms involving the unveiled Ankrd26 properties.

The pathogenesis of influenza A viruses (IAVs) is influenced by several factors, including IAV strain origin and reassortment, tissue tropism and host type. While such factors were mostly investigated in the context of virus entry, fusion and replication, little is known about the viral-induced changes to the host lipid membranes which might be relevant in the context of virion assembly. In this work, we applied several biophysical fluorescence microscope techniques (i.e., Forster energy resonance transfer, generalized polarization imaging and scanning fluorescence correlation spectroscopy) to quantify the effect of infection by two IAV strains of different origin on the plasma membrane (PM) of avian and human cell lines. We found that IAV infection affects the membrane charge of the inner leaflet of the PM. Moreover, we showed that IAV infection impacts lipid-lipid interactions by decreasing membrane fluidity and increasing lipid packing. Because of such alterations, diffusive dynamics of membrane-associated proteins are hindered. Taken to-gether, our results indicate that the infection of avian and human cell lines with IAV strains of different origins had similar effects on the biophysical properties of the PM.

CD4+ T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented on the surface of antigen presenting cells (APCs) by the MHC-II molecules. The pathways leading to endogenous MHC-II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy-dependent and -independent endogenous presentation of HIV- and HCMV-derived peptides. By studying the immunopeptidome of MHC-II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC-II-loading compartments and a rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC-II molecules. We determined the interactome of T6BP, identified calnexin as a T6BP partner and show that CANX cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4+ T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC-II-restricted endogenous presentation pathway and we propose one potential mechanism of action.

Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2{+/-}0.9 proteins/m2. Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

Transient receptor potential melastatin 8 (TRPM8), the cold and menthol receptor is essential to thermosensation, although its roles in organs within the body are still unclear. Besides TRPM8, we previously cloned several isoforms, like 4TM-TRPM8, which can be expressed with or without TRPM8. In this study, we characterize the human TRPM8(85) in ER membranes in the vicinity of Golgi apparatus (GA) and mitochondria in prostate epithelial cells. Silencing of TRPM8(85) induces lipid droplet accumulation, GA expansion and fragmentation associated with a drop in the vesicular trafficking to plasmalemma. Furthermore, lipidomic analysis reveals a strong shift in unsaturated fatty acids (UFAs), induced by TRPM8(85) silencing and to a lesser extent silencing of TRPM8. UFAs increase is caused by the induction of {Delta}9 stearoyl desaturase (Scd1) gene. Silencing SCD1 or palmitate incubation prevent GA expansion in TRPM8(85)-silenced cells. Finally, we demonstrated that TRPM8 regulates SCD1 via the androgen receptor.

Ceramides are essential but potentially toxic intermediates of sphingolipid metabolism that can act directly on mitochondria to trigger apoptotic cell death, but the underlying mechanism is unclear. While one model postulates that ceramides form stable channels in the outer mitochondrial membrane that induce cell death through direct release of cytochrome c, an alternative view is that ceramides self-assemble into microdomains that facilitate membrane insertion and oligomerization of the pro-apoptotic Bcl-2 protein Bax into cytochrome c-conducting pores. To challenge these models, we here analyzed the influence of ceramides in combination with recombinant Bax on the leakiness of model membranes. We show that ceramides on their own are unable to support membrane passage of even the smallest fluorescence markers. Moreover, we find that ceramides cannot substitute for cardiolipin in facilitating membrane recruitment of Bax and its subsequent assembly into functional pores. Our data argue against a direct role of ceramides in apoptotic pore formation and indicate that the mechanism by which ceramides initiate permeabilization of the outer mitochondrial membrane is independent of ceramide channels or ceramide acting autonomously as translocation platform for Bax.

Upregulated global protein synthesis is associated with the development and progression of several diseases and disorders. Strategies like calorie restriction and pharmacological inhibition of protein synthesis, have exhibited health-promoting effects. However, the complex molecular events that regulate global protein synthesis are not completely understood. Here, we report that SIRT2, a histone deacylase, negatively regulates global protein synthesis by inhibiting the mTORC1 pathway via deacetylating Rheb and promoting Rheb degradation. Our in vitro results suggest that SIRT2 deficiency increases protein synthesis, whereas SIRT2 overexpression suppresses protein synthesis. SIRT2-deficient mice exhibit age-associated and neurohormone-induced cardiac hypertrophy. Here, we report increased global protein synthesis in the hearts of young SIRT2-deficient mice, which may contribute to the development of cardiac hypertrophy. Conversely, cardiac-specific overexpression of SIRT2 reduces global protein synthesis in mice hearts. Mechanistically, SIRT2 binds to and deacetylates Rheb at K151 residue to enhance ubiquitin-proteosome-mediated degradation of Rheb. Depletion of Rheb rescues the increased protein synthesis in SIRT2-inhibited conditions. Our findings suggest that SIRT2 activation can be a potential therapeutic for treating diseases associated with increased protein synthesis.

Cellular stress activates mechanisms such as autophagy and vesicular trafficking to maintain homeostasis in pathological conditions like acute pancreatitis. Vacuole Membrane Protein 1 (VMP1), an autophagy-related protein implicated in pancreatitis, diabetes, and pancreatic cancer, triggers autophagy through ubiquitination and interaction with BECN1. Here, we show that VMP1 is secreted into the extracellular medium and incorporated into extracellular vesicle (EV) membranes. Using cells expressing VMP1-tagged plasmids, we isolated VMP1- containing EVs (VMP1-EVs) by ultracentrifugation and immunoisolation. VMP1-EV secretion decreased with mTOR inhibition and in Atg5-deficient cells. In pancreatic acinar cells, endogenous VMP1 secretion increased under stress, including blocked autophagic flux and experimental pancreatitis. In a rat pancreatitis model, VMP1 secretion in pancreatic juice was also elevated. TEM and DLS analyses revealed VMP1-EVs of [~]150 nm. LC3-II was detected in VMP1-EVs, and its release increased under lysosomal blockade. VMP1 downregulation reduced LC3 and p62 secretion, demonstrating that VMP1 drives a secretory autophagy pathway relevant to pancreatic pathophysiology.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in numerous cell types in vitro and in models of neurodegeneration and diabetes in vivo. So far, the exact mode of its action has remained elusive and plasma membrane or ER-located receptors of MANF have not been identified. We have found that MANF can directly interact with transmembrane unfolded protein response (UPR) receptor IRE1 and compete with the major ER chaperone BiP (GRP78) for the interaction with IRE1. With lower affinities MANF can also interact with other UPR receptors, PERK and ATF6. Using molecular modeling and mutagenesis analysis, we have identified the exact structural MANF regions involved in its binding to the luminal domain of IRE1. MANF attenuates UPR signaling by decreasing IRE1 oligomerization and IRE1 phosphorylation. MANF mutant deficient in IRE1 binding cannot regulate IRE1 oligomerization and fails to protect neurons from ER stress induced death. Importantly, we found that MANF-IRE1 interaction is also crucial for the survival promoting action of MANF for dopamine neurons in an animal model of Parkinsons disease. Our data reveal a novel mechanism of IRE1 regulation during ER stress and demonstrate the intracellular mode of action of MANF as a modulator of UPR and neuronal cell survival through the direct interaction with IRE1 and regulation of its activity. Furthermore, our data explain why MANF in contrast to other growth factors has no effects on naive cells and rescues only ER stressed or injured cells.