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Summary To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44’s active cysteine, simultaneously unmask the substrate binding site and −RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles., Graphical Abstract, Highlights • ERp44 governs a pH-regulated assembly control cycle in the early secretory pathway • Accessibility of ERp44’s active site and –RDEL ER retrieval motif is pH dependent • Unmasking of ERp44’s active site likely involves protonation of cysteine 29 • ERp44 captures client proteins at cisGolgi-equivalent pH for retrieval to the ER

Professional secretory cells produce and release abundant proteins. Particularly in case of mutations and/or insufficient chaperoning, these can aggregate and become toxic within or amongst cells. Immunoglobulins (Ig) are no exception. In the extracellular space, certain Ig-L chains form fibrils causing systemic amyloidosis. On the other hand, Ig variants lacking the first constant domain condense in dilated cisternae of the early secretory compartment, called Russell Bodies (RB), frequently observed in plasma cell dyscrasias, autoimmune diseases and chronic infections. RB biogenesis can be recapitulated in lymphoid and non-lymphoid cells by expressing mutant Ig-μ, providing powerful models to investigate the pathophysiology of endoplasmic reticulum storage disorders. Here we analyze the aggregation propensity and the biochemical features of the intra- and extra-cellular Ig deposits in human cells, revealing β-aggregated features for RB.

Background Therapies available for late stage prostate cancer (PCa) patients are limited and mostly palliative. The necessary development of unexplored therapeutic options relies on a deeper knowledge of molecular mechanisms leading to cancer progression. Redox signals are known to modulate the intensity and duration of oncogenic circuits; cues originating from the endoplasmic reticulum (ER) and downstream exocytic organelles are relevant in secretory tumors, including PCa. Ero 1α is a master regulator of redox homeostasis and oxidative folding. Methods We assessed Ero 1α mRNA expression by bioinformatic analysis of three public datasets and protein expression levels in PCa cell lines representing different degrees of tumor progression and different human prostate specimens. Transient Ero 1α knockdown was achieved by RNA interference (siRNA). Consequences of Ero 1α downregulation were monitored by PCa proliferation, migration and invasion properties. Results Ero 1α mRNA and protein levels are upregulated in PCa cell lines compared to non-tumorigenic cells (P=0.0273). Ero 1α expression increases with the grade of malignancy, reaching the highest level in the androgen resistant PC3. In patients' samples from 3 datasets, Ero 1α mRNA expression correlates with pathological Gleason scores. Ero 1α knockdown inhibits proliferation (P=0.0081), migration (P=0.0085) and invasion (P=0.0007) of PC3 cells and alters the levels of integrin β1 (P=0.0024). Conclusions Results indicate that Ero 1α levels correlate with PCa aggressiveness; Ero 1α silencing inhibits key steps over the PCa metastatic process. Therefore, Ero 1α has the potential to be exploited as a novel biomarker and a therapeutic target in PCa.

Antibodies of the immunoglobulin M (IgM) class represent the frontline of humoral immune responses. They are secreted as planar polymers in which flanking µ(2)L(2) “monomeric” subunits are linked by two disulfide bonds, one formed by the penultimate cysteine (C575) in the tailpiece of secretory µ chains (µ(s)tp) and the second by C414 in the Cµ3. The latter bond is not present in membrane IgM. Here, we show that C575 forms a non‐native, intra‐subunit disulfide bond as a key step in the biogenesis of secretory IgM. The abundance of this unexpected intermediate correlates with the onset and extent of polymerization. The rearrangement of the C‐terminal tails into a native quaternary structure is guaranteed by the engagement of protein disulfide isomerase ERp44, which attacks the non‐native C575 bonds. The resulting conformational changes promote polymerization and formation of C414 disulfide linkages. This unusual assembly pathway allows secretory polymers to form without the risk of disturbing the role of membrane IgM as part of the B cell antigen receptor.

Summary The composition of the secretome depends on the combined action of cargo receptors that facilitate protein transport and sequential checkpoints that restrict it to native conformers. Acting after endoplasmic reticulum (ER)-resident chaperones, ERp44 retrieves its clients from downstream compartments. To guarantee efficient quality control, ERp44 should exit the ER as rapidly as its clients, or more. Here, we show that appending ERp44 to different cargo proteins increases their secretion rates. ERp44 binds the cargo receptor ER-Golgi intermediate compartment (ERGIC)-53 in the ER to negotiate preferential loading into COPII vesicles. Silencing ERGIC-53, or competing for its COPII binding with 4-phenylbutyrate, causes secretion of Prdx4, an enzyme that relies on ERp44 for intracellular localization. In more acidic, zinc-rich downstream compartments, ERGIC-53 releases its clients and ERp44, which can bind and retrieve non-native conformers via KDEL receptors. By coupling the transport of cargoes and inspector proteins, cells ensure efficiency and fidelity of secretion., Graphical abstract, Highlights • ERGIC-53 binds ERp44 and accelerates its exit from the ER • Genetic or pharmacological inhibition of ERGIC-53 impairs ERp44 retrieval function • ERp44 and ERGIC-53 form a functional couple needed for post-ER QC, Biological Sciences; Biochemistry; Molecular Biology; Cell Biology

Interactions between protein ligands and receptors are the main language of intercellular communication; hence, how cells select proteins to be secreted or presented on the plasma membrane is a central concern in cell biology. A series of checkpoints are located along the secretory pathway, which ensure the fidelity of such protein signals (quality control). Proteins that pass the checkpoints operated in the endoplasmic reticulum (ER) by the binding immunoglobulin protein (BiP; also known as HSPA5 and GRP78) and the calnexin–calreticulin systems, must still overcome additional scrutiny in the ER-Golgi intermediate compartment (ERGIC) and the Golgi. One of the main players of this process in all metazoans is the ER-resident protein 44 (ERp44); by cycling between the ER and the Golgi, ERp44 controls the localization of key enzymes designed to act in the ER but that are devoid of suitable localization motifs. ERp44 also patrols the secretion of correctly assembled disulfide-linked oligomeric proteins. Here, we discuss the mechanisms driving ERp44 substrate recognition, with important consequences on the definition of ‘thiol-mediated quality control’. We also describe how pH and zinc gradients regulate the functional cycle of ERp44, coupling quality control and membrane trafficking along the early secretory compartment.

Efficiency and fidelity of protein secretion are achieved thanks to the presence of different steps, located sequentially in time and space along the secretory compartment, controlling protein folding and maturation. After entering into the endoplasmic reticulum (ER), secretory proteins attain their native structure thanks to specific chaperones and enzymes. Only correctly folded molecules are allowed by quality control (QC) mechanisms to leave the ER and proceed to downstream compartments. Proteins that cannot fold properly are instead retained in the ER to be finally destined to proteasomal degradation. Exiting from the ER requires, in most cases, the use of coated vesicles, departing at the ER exit sites, which will fuse with the Golgi compartment, thus releasing their cargoes. Protein accumulation in the ER can be caused by a too stringent QC or by ineffective transport: these situations could be deleterious for the organism, due to the loss of the secreted protein, and to the cell itself, because of abnormal increase of protein concentration in the ER. In both cases, diseases can arise. In this review, we will describe the pathophysiology of protein folding and transport between the ER and the Golgi compartment.

Professional secretory cells produce and release abundant proteins. Particularly in case of mutations and/or insufficient chaperoning, these can aggregate and become toxic within or amongst cells. Immunoglobulins (Ig) are no exception. In the extracellular space, certain Ig-L chains form fibrils causing systemic amyloidosis. On the other hand, Ig variants lacking the first constant domain condense in dilated cisternae of the early secretory compartment, called Russell Bodies (RB), frequently observed in plasma cell dyscrasias, autoimmune diseases and chronic infections. RB biogenesis can be recapitulated in lymphoid and non-lymphoid cells by expressing mutant Ig-μ, providing powerful models to investigate the pathophysiology of endoplasmic reticulum storage disorders. Here we analyze the aggregation propensity and the biochemical features of the intra- and extra-cellular Ig deposits in human cells, revealing β-aggregated features for RB.

ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval through interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here, we show that conserved histidine residues in the C-terminal tail also regulate ERp44 in vivo. Mutants lacking these histidine residues retain substrates more efficiently. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon overexpression of different partners. The ensuing gradients might help to optimize folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes.

Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.

In vertebrates, eye development comprises inductive and morphogenetic events that are finely regulated by the coordinated action of many intrinsic and extrinsic factors. Recent evidence suggested that neurotransmitters could be enumerated by the extracellular signals contributing to the retinal and eye development. We showed that, among these neuromodulators, serotonin acting via the 5-HT2B receptor, is involved in the control of retinoblasts proliferation and survival in Xenopus embryogenesis. To further clarify the role of 5-HT2B receptor in ocular development, we performed a gene gain of function analysis in vitro and in vivo in Xenopus embryos. We confirmed that 5-HT2B overexpression is per se sufficient to promote cell proliferation in a neuroblastoma cell line. The in vivo experiments revealed that an over serotonin signaling, via 5-HT2B receptors, resulted in the formation of eyes with an irregular form, position and orientation. Interestingly, we showed 5-HT2B gene expression in periocular mesenchyme that represents a key signaling center required for a correct eye morphogenesis. Moreover, the 5-HT2B receptor overexpressing retina, displays a disorganization of the typical laminar structure with the presence of retinal cells scattered in ectopic positions or forming rosette like structures. On the whole our data support the idea that serotonin signalling has to be finely regulated during eye development to allow a correct retinogenesis and may participate in the correct orchestration and synergism of all the factors and events that regulate eye morphogenesis in ocular and periocular tissues.

Owing to the impossibility of reaching the Golgi for secretion or the cytosol for degradation, mutant Ig-mu chains that lack the first constant domain (muDeltaCH1) accumulate as detergent-insoluble aggregates in dilated endoplasmic reticulum cisternae, called Russell bodies. The presence of similar structures hallmarks many ER storage diseases, but their pathogenic role(s) remain obscure. Exploiting inducible cellular systems, we show here that Russell bodies form when the synthesis of muDeltaCH1 exceeds the degradation capacity. Condensation occurs in different sub-cellular locations, depending on the interacting molecules present in the host cell: if Ig light chains are co-expressed, detergent-insoluble muDeltaCH1-light chain oligomers accumulate in large ribosome-coated structures (rough Russell bodies). In absence of light chains, instead, aggregation occurs in smooth tubular vesicles and is controlled by N-glycan-dependent interactions with ER-Golgi intermediate compartment 53 (ERGIC-53). In cells containing smooth Russell bodies, ERGIC-53 co-localizes with muDeltaCH1 aggregates in a Ca2+ -dependent fashion. Our findings identify a novel ERGIC-53 substrate, and indicate that interactions with light chains or ERGIC-53 seed muDeltaCH1 condensation in different stations of the early secretory pathway.