Biology subjects

Molinari, M.

Publications and source records attributed to Molinari, M..

18 recordsLinked to original sources

bioRxiv2026

A reversible temperature block captures pre-lysosomal intermediates of starvation-induced ERphagy

The selective lysosomal turnover of endoplasmic reticulum (ER) portions, termed ERphagy, removes aged, excess or damaged ER domains. The intermediates forming between receptor activation and lysosomal delivery are short-lived and remain poorly resolved. Here we identify a reversible temperature block at 25{degree sign}C that arrests ERphagy before lysosomal delivery. At this temperature, nutrient restriction still drives focal accumulation of the endogenously Halo-tagged ERphagy receptor TEX264 (puncta formation), segregation of ER cargo, remodelling of the ER membrane, whereas delivery of this material to degradative endolysosomes is strongly reduced. Rewarming above 27{degrees} restores lysosomal delivery, showing that the accumulated eTEX264-Halo puncta correspond to pre-lysosomal intermediates of the ERphagy program. Their formation requires ULK1/2 activity, the ATG7-dependent conjugation machinery, ATG9A, ATG13 and FIP200, coupling receptor clustering to autophagosome biogenesis. Consistently, expansion microscopy and electron tomography reveal curved, cradle-like TEX264-positive ER domains containing ER cargo, co-localizing with autophagy markers and lying adjacent to p62-cargo condensates.

cell biology↗

bioRxiv2026

Epstein Barr virus exploits ER stress and TMX4 dependent nuclear envelope remodeling to enable capsid egress

Herpesvirus capsids assemble within the nucleoplasm of infected cells in highly ordered icosahedral structures with a diameter of about 100nm. Despite the small distance between outer and inner nuclear membrane, which is fixed between the 25 and the 50nm by disulfide bonded LINC complexes, the capsid particles cross the barrier and are delivered into the cytoplasm, where viral particle assembly continues. How Epstein-Barr virus (EBV) overcomes the spatial constraints imposed by the narrow perinuclear space for nuclear egress of the viral capsids remains unclear. Here, we show that EBV exploits an ER-stress-responsive nuclear envelope (NE) remodeling pathway to promote capsid egress. Induction of EBV lytic replication activates the IRE1 branch of the unfolded protein response and triggers TMX4-dependent remodeling of the NE. Inhibition of IRE1 signaling or depletion of TMX4 prevents efficient redistribution of viral capsid proteins from the nucleus to the cytoplasm, causes accumulation of unused viral glycoprotein GP350 in Golgi-derived membranes, and markedly reduces production of infectious viral particles. Thus, EBV hijacks a host NE adaptation pathway to overcome a fundamental physical barrier during viral maturation.

cell biology↗

bioRxiv2026

The role of N-glycans and their processing in ER-to-lysosome-associated degradation of disease-causing mutant Neuroserpin

Most proteins synthesized in the endoplasmic reticulum (ER) are covalently modified upon addition of pre-assembled oligosaccharides to side chains of asparagine (N) residues. Processing of N-linked oligosaccharides by ER-resident glucosidases, mannosidases and glucosyltransferases determines the fate of the associated polypeptides. Terminally glucose residues are removed from N-glycans to hamper engagement of ER-resident glucose-binding chaperones and promote secretion of native polypeptides. Mannose residues are removed to target terminally misfolded proteins for dislocation across the ER membrane and clearance by the cytoplasmic ubiquitin proteasome system (ER-associated degradation, ERAD). Recent evidence highlights the role of persistent N-glycan glucosylation as a signal that promotes segregation of misfolded proteins in ER subdomains that are eventually delivered to endolysosomal compartments for ER-to-Lysosome-Associated Degradation (ERLAD). Here we show that the polymerization-prone Portland variant of Neuroserpin (NS_PL) associated with familial encephalopathy with NS inclusion bodies (FENIB) is a client of the ERLAD machinery. Its lysosomal clearance relies on the LC3-dependent delivery branch of ERLAD involving the lectin chaperone Calnexin (CNX), the ERphagy receptor FAM134B and the SNARE protein Syntaxin17 (STX17), which is engaged upon persistent glucosylation of the NS_PL oligosaccharide linked at the asparagine residue at position 321.

cell biology↗

bioRxiv2026

Serum modulates the aggregation - toxicity landscape of the staphylococcal toxin PSMα3

The pathogenicity of Staphylococcus aureus relies on the secretion of various toxins, including phenol-soluble modulins 3 (PSM3), which play key roles in invasion and infection through its cytolytic activities. Recent controversies have focused on whether PSM3 propensity to self-assemble into unique amyloid-like cross- structures would underlie its high cytotoxicity. Here, by integrating Thioflavin T fluorescence, electron microscopy, and cell-based assays, we demonstrate that early soluble entities formed upon fibrillation - rather than mature fibrils - drive PSM3 cytotoxicity under near-physiological conditions. This mirrors the behavior of neurodegenerative disease related peptides, thus pointing towards a general mechanism of toxicity by amyloid forming peptides. Importantly, we elucidate the critical role of serum in this process: lipoproteins inhibit the formation of oligomeric and fibrillar entities, both in a time- and concentration-dependent manner, thereby drastically reducing cytotoxicity. We further reveal the uptake of such soluble entities in cells, where membrane interactions likely initiate and trigger cell death. These findings reconcile conflicting interpretations of PSM3 toxicity, highlighting the importance of the cellular environment in shaping peptide aggregation and biological function. By targeting such key virulence determinants, e.g. though the rational design of aggregation inhibitors, this study could open novel avenues in the quest for novel drugs against S. aureus infections.

biophysics↗

bioRxiv2025

ER-phagy and mitophagy programs in human CD4+ memory T cells before and after activation

T lymphocytes can remain in a resting state for very long times. Encountering their cognate antigen triggers their activation, leading to an increase in cell size, proliferation, enhanced protein production, and secretion of signaling molecules. This activation and clonal expansion phase is followed by a contraction phase, with a reduction in cell size, eventually leading to cell death or progression to resting (memory) cells. Here, we show that human resting primary CD4+ memory T cells degrade endoplasmic reticulum (ER) and mitochondrial portions via ER-phagy and mitophagy, respectively. The temporary interruption of ER and mitochondria turnover coupled with a transient induction of the unfolded protein response results in the enlargement of the ER and cellular volume. Cell growth is stabilized and then reversed during the clonal expansion and contraction phases by the resumption of a catabolic phase characterized by the reactivation of ER-phagy and outer mitochondrial membrane (OMM)-phagy. Collectively, our data show that the maintenance and the activity of human CD4+ memory T cells rely on finely tuned execution of anabolic and catabolic programs that regulate mass and activity of the ER and mitochondria.

cell biology↗

bioRxiv2025

The involvement of the ER-phagy receptor FAM134B in membrane contact sites between ER and endolysosomes promotes ERLAD

Membrane contact sites (MCS) between organelles maintain the proximity required for controlled exchange of small molecules and ions yet preventing fusion events that would compromise organelles identity and integrity. Here, by investigating the intracellular fate of the disease-causing Z-variant of alpha1 antitrypsin (ATZ), we report on a novel function of MCS between the endoplasmic reticulum (ER) and RAB7/LAMP1-positive endolysosomes in ER-to-lysosome-associated degradation (ERLAD). For this function, the VAPA:ORP1L:RAB7 multi-protein complex forming MCS between the ER and endolysosomes engages, in an ERLAD client-driven manner, the misfolded protein segregation complex formed by the lectin chaperone Calnexin (CNX), the ER-phagy receptor FAM134B and the ubiquitin-like protein LC3. Generation of this supramolecular complex facilitates the membrane fusion events regulated by the SNARE proteins STX17 and VAMP8 that ensure efficient delivery of ATZ polymers from their site of generation, the ER, to the site of their intracellular clearance, the degradative RAB7/LAMP1-positive endolysosomes.

cell biology↗

bioRxiv2024

A novel class of allosteric glucosylceramidase beta 1 correctors that reduce cellular stress and enhance lysosomal function

Mutations in glucosylceramidase beta 1 (GCase) disrupt the proteins conformational maturation in the endoplasmic reticulum (ER) and hinder its transport to the lysosome. The intralysosomal accumulation of glucocerebrosides, which are substrates of the GCase enzyme, impairs lysosomal function and is linked to Gaucher disease (GD). GCase mutations also increase the risk of Parkinsons disease (PD) and Dementia with Lewy Bodies. We used Site-directed Enzyme Enhancement Therapy (SEE-Tx(R)) technology to design two structurally targeted allosteric regulators (STARs) of GCase. Administration of GT-02287 and GT-02329 to cultured GD patient-derived primary human fibroblasts enhances folding and protects the two most common disease-causing GCase variants, GCaseAsn370Ser and GCaseLeu444Pro, from proteasomal degradation. Mechanistically, these treatments facilitate the lysosomal delivery of enzymatically active forms of mutant GCase, leading to improved lysosomal function and reduced cellular stress in GD patient-derived fibroblasts. The findings suggest that the allosteric pharmacologic regulators GT-02287 and GT-02329 hold promise for further development as potential therapeutic agents for GCase-related disorders, including GD, PD and Dementia with Lewy Bodies.

cell biology↗

bioRxiv2024

Pharmacological GCase Activity Enhancement Inhibits Tau Accumulation

A slow decline in the autophagy-lysosomal pathway is a hallmark of the normal aging brain. Yet, an acceleration of this cellular function may propel neurodegenerative events. In fact, mutations in genes associated with the autophagy-lysosomal pathway can lead to Parkinsons disease. Also, amyloidogenic protein deposition is observed in lysosomal storage disorders, which are caused by genetic mutations representing risk factors for Parkinsons disease. For example, Gauchers disease GBA1 mutations leading to defects in lysosomal sphingolipid metabolism cause -synuclein accumulation. We observed that increased lysosomal Tau accumulation is found in human dermal fibroblasts engineered for inducible Tau expression. Inhibition of the GBA1 product GCase augmented Tau-dependent lysosomal stress and Tau accumulation. Here, we show increased Tau seed-induced Tau accumulation in Gauchers fibroblasts carrying GBA1 mutations when compared to normal fibroblasts. Pharmacological enhancement of GCase reversed this effect, notably, also in normal fibroblasts. This suggests that boosting GCase activity may represent a therapeutic strategy to slow down aging-dependent lysosomal deficits and brain protein deposition.

cell biology↗

bioRxiv2024

ER-phagy Receptors Intrinsically Disordered Modules Drive ER Fragmentation and ER-phagy

Membrane remodeling leading to fragmentation is crucial for autophagy programs that control capture by phagophores or endolysosomes of portions of organelles to be removed from cells. It is driven by membrane-bound autophagy receptors that display cytoplasmic intrinsically disordered modules (IDRs) engaging Atg8/LC3/GABARAP (LC3). Studies on endoplasmic reticulum (ER)-phagy receptors of the FAM134 family revealed the importance of sequential FAM134 proteins phosphorylation, ubiquitylation and clustering for execution of the ER-phagy programs. In this model, ER fragmentation is promoted/facilitated by the membrane-remodeling function of FAM134 reticulon homology domains (RHDs). However, RHDs are not conserved in ER-phagy receptors. The question that we tackle in this work is if activation of ER-phagy receptors anchored at the ER membrane with conventional membrane spanning domains, i.e., most of the ER-phagy receptors known to date, eventually trigger ER remodeling and fragmentation, and how. Here, we show that the membrane-tethering modules of ER-phagy receptors (RHDs for FAM134B, single/multi spanning transmembrane domains for TEX264 and SEC62) determine the sub-compartmental distribution of the receptors but are dispensable for ER fragmentation, regardless of their propensity to remodel the ER membrane. Rather, ER fragmentation is promoted by the ER-phagy receptors intrinsically disordered region (IDR) modules that are a conserved feature of all ER-phagy receptors exposed at the cytoplasmic face of the ER membrane. Since cytoplasmic IDRs with net negative charge are conserved in autophagy receptors at the limiting membrane of other organelles, we anticipate that conserved mechanisms of organelle fragmentaVon driven by cytoplasmic exposed IDRs could operate in eukaryoVc cells.

cell biology↗

bioRxiv2024

TMX5/TXNDC15, a natural trapping mutant of the PDI family is a client of the proteostatic factor ERp44

The endoplasmic reticulum (ER) is the organelle of nucleated cells that produces lipids, sugars and proteins. More than 20 ER-resident members of the Protein Disulfide Isomerase (PDI) family regulate formation, isomerization and disassembly of covalent bonds in newly synthesized polypeptides. The PDI family includes few membrane-bound members. Among these, TMX1, TMX2, TMX3, TMX4 and TMX5 belong to the thioredoxin-related transmembrane (TMX) protein family. TMX5 is the least known member of the family. Here, we establish that TMX5 covalently engages via its active site cysteine residue at position 220 a subset of secretory proteins, mainly single- and multi-pass Golgi-resident polypeptides. TMX5 also interacts non-covalently, and covalently, via non-catalytic cysteine residues, with the PDI family members PDI, ERp57 and ERp44. The association of TMX5 and ERp44 requires formation of a mixed disulfide between the catalytic cysteine residue 29 of ERp44 and the non-catalytic cysteine residues 114 and/or 124 of TMX5 and controls the ER retention of TMX5. Thus, TMX5 belongs to the family of proteins including Ero1, Ero1{beta}, Prx4, ERAP1, SUMF1 that do not display ER retention sequences and rely on ERp44 engagement for proper inter-compartmental distribution.

cell biology↗

bioRxiv2024

5-HT1B receptors mediate dopaminergic inhibition of vesicular fusion and GABA release from striatonigral synapses.

The substantia nigra pars reticulata (SNr), a crucial basal ganglia output nucleus, contains a dense expression of dopamine D1 receptors (D1Rs), along with dendrites belonging to dopaminergic neurons of substantia nigra pars compacta. These D1Rs are primarily located on the terminals of striatonigral medium spiny neurons, suggesting their involvement in the regulation of neurotransmitter release from the direct pathway in response to somatodendritic dopamine release. To explore the hypothesis that D1Rs modulate GABA release from striatonigral synapses, we conducted optical recordings of striatonigral activity and postsynaptic patch-clamp recordings from SNr neurons in the presence of dopamine and D1R agonists. We found that dopamine inhibits optogenetically triggered striatonigral GABA release by modulating vesicle fusion and Ca2+ influx in striatonigral boutons. Notably, the effect of DA was independent of D1R activity but required activation of 5-HT1B receptors. Our results suggest a serotonergic mechanism involved in the therapeutic actions of dopaminergic medications for Parkinsons disease and psychostimulant-related disorders.

neuroscience↗

bioRxiv2024

N-formylation modifies membrane damage associated to PSMα3 interfacial fibrillation

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins 3 (PSM3) peptides, able to self-assemble into amyloid-like cross- fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSM3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining Atomic Force Microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSM3 alters its interactions with model membranes of controlled lipid composition, without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSM3 - membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSM3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains, only in the fluid-phase of membranes, and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSM3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation, and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSM3 with specific membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.

biophysics↗

bioRxiv2023

FAM134B regulates ER-to-lysosome-associated degradation of misfolded proteins upon pharmacologic or genetic inactivation of ER-associated degradation

About 40% of the eukaryotic cells proteome is synthesized and assembled in the endoplasmic reticulum (ER). Native proteins are transported to their intra- or extra-cellular site of activity. Folding-defective polypeptides are dislocated across the ER membrane into the cytoplasm, poly-ubiquitylated and degraded by 26S proteasomes (ER-associated degradation, ERAD). Large misfolded proteins like mutant forms of collagen or aggregation-prone mutant forms of alpha1 antitrypsin cannot be dislocated across the ER membrane for ERAD. Rather, they are segregated in ER subdomains that vesiculate and deliver their cargo to endolysosomal compartments for clearance by ER-to-lysosome-associated degradation (ERLAD). Here, we show the lysosomal delivery of a canonical ERAD substrate upon pharmacologic and genetic inhibition of the ERAD pathways. This highlights the surrogate intervention of ERLAD to remove defective gene products upon dysfunctional ERAD.

cell biology↗

bioRxiv2023

Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells

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

cell biology↗

bioRxiv2022

SEC62 and TMX4 control asymmetric autophagy of the nuclear envelope upon LINC complex disassembly

The endoplasmic reticulum (ER) is a dynamic organelle of nucleated cells that produces proteins, lipids and oligosaccharides. The volume and the activities of the ER are adapted to cellular needs. They are increased upon induction of unfolded protein responses (UPR 1) and are reduced upon activation of ER-phagy programs 2. A specialized domain of the ER, the nuclear envelope (NE), protects the cell genome with two juxtaposed lipid bilayers, the inner and outer nuclear membranes (INM and ONM). SUN proteins in the INM form disulfide-bonded Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes with NESPRIN proteins in the ONM. These complexes set and maintain the width of the periplasmic space (PS), a continuum of the ER lumen, below the 50 nm 3-5 and in yeast prevent transmission of ER volume variations to the PS 6,7. Here we report that expansion of the mammalian ER upon homeostatic perturbations is transmitted to the NE, where the ONM forms large bulges. The process is reverted on recovery of ER homeostasis, by asymmetric vesiculation and autophagic clearance of ONM portions. Remodeling of the mammalian NE requires TMX4-driven reduction of the intermolecular disulfide bond stabilizing LINC complexes, the LC3 lipidation machinery, and the autophagy receptor SEC62, identified here as the first mammalian nucleo-phagy receptor.

cell biology↗

bioRxiv2022

Deficiency of Heterogeneous Nuclear Ribonucleoprotein U leads to delayed neurogenesis

Genetic variants affecting Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU) have been identified in several neurodevelopmental disorders (NDDs). HNRNPU is widely expressed in the human brain and shows the highest postnatal expression in the cerebellum. Recent studies have investigated the role of HNRNPU in cerebral cortical development, but the effects of HNRNPU deficiency on cerebellar development remain unknown. Here, we describe the molecular and cellular outcomes of HNRNPU locus deficiency during in vitro neural differentiation of patient-derived and isogenic neuroepithelial stem cells with a hindbrain profile. We demonstrate that HNRNPU deficiency leads to chromatin remodeling of A/B compartments, and transcriptional rewiring, partly by impacting exon inclusion during mRNA processing. Genomic regions affected by the chromatin restructuring and host genes of exon usage differences show a strong enrichment for genes implicated in epilepsies, intellectual disability, and autism. Lastly, we show that at the cellular level. HNRNPU downregulation leads to altered neurogenesis and an increased fraction of neural progenitors in the maturing neuronal population. We conclude that, HNRNPU locus is involved in delayed commitment of neural progenitors to neuronal maturation in cell types with hindbrain profile.

neuroscience↗

bioRxiv2020

Extracellular Vesicles Hijack the Autophagic Pathway to Induce Tau Accumulation in Endolysosomes

Clinical progression of tauopathies is reflected by the transcellular propagation of pathogenic Tau seeds with the possible involvement of extracellular vesicles as transport vectors. However, the mechanism regulating extracellular vesicle cargo delivery to recipient cells is poorly understood. We established a cell model for investigating extracellular vesicle-delivery of membranes and proteins. In this model, extracellular vesicles are readily internalized and accumulate in endolysosomes. For the first time, we show that in this acidic compartment of recipient cells, extracellular vesicle-delivered Tau seeds cause the accumulation and abnormal folding of normal Tau by a process that requires the participation of autophagy. Endolysomes represent thus a cross-road where Tau seeds released from extracellular vesicles propagate on cellular Tau on its route for autophagy-mediated degradation, ultimately driving its accumulation, endolysosomal stress and cytotoxicity. Whilst, autophagy stimulation is considered as a viable solution to protect neurons from harmful cytosolic protein inclusions, our data suggest that this approach may favour the aberrant accumulation of neurodegeneration-associated proteins induced by exogenous pathogenic protein forms, with possible implications in the spreading of the disease.

cell biology↗

bioRxiv2019

Proteomics identifies signal peptide features determining the substrate specificity in human Sec62/Sec63-dependent ER protein import

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).

biochemistry↗