Early Secretory Pathway Research Articles

TFG binds LC3C to regulate ULK1 localization and autophagosome formation

The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle‐related protein TFG (Trk‐fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C‐ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.

Unscrambling exit site patterns on the endoplasmic reticulum as a quenched demixing process

The endoplasmic reticulum (ER) is a vital organelle in mammalian cells with a complex morphology. Consisting of sheet-like cisternae in the cell center, the peripheral ER forms a vast tubular network on which a dispersed pattern of a few hundred specialized domains (ER exit sites (ERESs)) is maintained. Molecular details of cargo sorting and vesicle formation at individual ERESs, fueling the early secretory pathway, have been studied in some detail. The emergence of spatially extended ERES patterns, however, has remained poorly understood. Here, we show that these patterns are determined by the underlying ER morphology, suggesting ERESs to emerge from a demixing process that is quenched by the ER network topology. In particular, we observed fewer but larger ERESs when transforming the ER network to more sheet-like morphologies. In contrast, little to no changes with respect to native ERES patterns were observed when fragmenting the ER, indicating that hampering the diffusion-mediated coarse graining of domains is key for native ERES patterns. Model simulations support the notion of effective diffusion barriers impeding the coarse graining and maturation of ERES patterns. We speculate that tuning a simple demixing mechanism by the ER topology allows for a robust but flexible adaption of ERES patterns, ensuring a properly working early secretory pathway in a variety of conditions.

Defects in early secretory pathway transport machinery components and neurodevelopmental disorders.

The early secretory pathway, provisionally comprising of vesicular traffic between the endoplasmic reticulum (ER) and the Golgi apparatus, occurs constitutively in mammalian cells. Critical for a constant supply of secretory and plasma membrane (PM) materials, the pathway is presumably essential for general cellular function and survival. Neurons exhibit a high intensity in membrane dynamics and protein/lipid trafficking, with differential and polarized trafficking towards the somatodendritic and axonal PM domains. Mutations in genes encoding early secretory pathway membrane trafficking machinery components are known to result in neurodevelopmental or neurological disorders with disease manifestation in early life. Here, such rare disorders associated with autosomal recessive mutations in coat proteins, membrane tethering complexes and membrane fusion machineries responsible for trafficking in the early secretory pathway are summarily discussed. These mutations affected genes encoding subunits of coat protein complex I and II, subunits of transport protein particle (TRAPP) complexes, members of the YIP1 domain family (YIPF) and a SNAP receptor (SNARE) family member. Why the ubiquitously present and constitutively acting early secretory pathway machinery components could specifically affect neurodevelopment is addressed, with the plausible underlying disease etiologies and neuropathological mechanisms resulting from these mutations explored.

Interleukin-11 (IL-11) receptor cleavage by the rhomboid protease RHBDL2 induces IL-11 trans-signaling.

Interleukin-11 (IL-11) is a pleiotropic cytokine with both pro- and anti-inflammatory properties. It activates its target cells via binding to the membrane-bound IL-11 receptor (IL-11R), which then recruits a homodimer of the ubiquitously expressed, signal-transducing receptor gp130. Besides this classic signaling pathway, IL-11 can also bind to soluble forms of the IL-11R (sIL-11R), and IL-11/sIL-11R complexes activate cells via the induction of gp130 homodimerization (trans-signaling). We have previously reported that the metalloprotease ADAM10 cleaves the membrane-bound IL-11R and thereby generates sIL-11R. In this study, we identify the rhomboid intramembrane protease RHBDL2 as a so far unrecognized alternative sheddase that can efficiently trigger IL-11R secretion. We determine the cleavage site used by RHBDL2, which is located in the extracellular part of the receptor in close proximity to the plasma membrane, between Ala-370 and Ser-371. Furthermore, we identify critical amino acid residues within the transmembrane helix that are required for IL-11R proteolysis. We also show that ectopically expressed RHBDL2 is able to cleave the IL-11R within the early secretory pathway and not only at the plasma membrane, indicating that its subcellular localization plays a central role in controlling its activity. Moreover, RHBDL2-derived sIL-11R is biologically active and able to perform IL-11 trans-signaling. Finally, we show that the human mutation IL-11R-A370V does not impede IL-11 classic signaling, but prevents RHBDL2-mediated IL-11R cleavage.

Tunnels for Protein Export from the Endoplasmic Reticulum.

The functions of coat protein complex II (COPII) coats in cargo packaging and the creation of vesicles at the endoplasmic reticulum are conserved in eukaryotic protein secretion. Standard COPII vesicles, however, cannot handle the secretion of metazoan-specific cargoes such as procollagens, apolipoproteins, and mucins. Metazoans have thus evolved modules centered on proteins like TANGO1 (transport and Golgi organization 1) to engage COPII coats and early secretory pathway membranes to engineer a novel mode of cargo export at the endoplasmic reticulum.

Translational landscape and protein biogenesis demands of the early secretory pathway in Komagataella phaffii

BackgroundEukaryotes use distinct networks of biogenesis factors to synthesize, fold, monitor, traffic, and secrete proteins. During heterologous expression, saturation of any of these networks may bottleneck titer and yield. To understand the flux through various routes into the early secretory pathway, we quantified the global and membrane-associated translatomes of Komagataella phaffii.ResultsBy coupling Ribo-seq with long-read mRNA sequencing, we generated a new annotation of protein-encoding genes. By using Ribo-seq with subcellular fractionation, we quantified demands on co- and posttranslational translocation pathways. During exponential growth in rich media, protein components of the cell-wall represent the greatest number of nascent chains entering the ER. Transcripts encoding the transmembrane protein PMA1 sequester more ribosomes at the ER membrane than any others. Comparison to Saccharomyces cerevisiae reveals conservation in the resources allocated by gene ontology, but variation in the diversity of gene products entering the secretory pathway.ConclusionA subset of host proteins, particularly cell-wall components, impose the greatest biosynthetic demands in the early secretory pathway. These proteins are potential targets in strain engineering aimed at alleviating bottlenecks during heterologous protein production.

Extracellular WNTs: Trafficking, Exosomes, and Ligand-Receptor Interaction.

WNT signaling is a key developmental pathway in tissue organization. A recent focus of research is the secretion of WNT proteins from source cells. Research over the past decade on how WNTs are produced and released into the extracellular space has unravelled very specific control mechanisms in the early secretory pathway, specialized trafficking routes, and redundant forms of packaging for delivery to target cells. In this review I discuss the findings that WNT proteins have been found on extracellular vesicles (EVs) such as exosomes and possible functional implications. There is an ongoing debate in the WNT signaling field whether EV are relevant in vivo and can fulfill specific functions, also fueled by the general preconception of EV secretion as cellular garbage disposal. As part of the EV research community, I want to give an overview of what we know and don't know about WNT secretion on EVs and offer a more unifying model that can explain current discrepancies in observations regarding WNT secretion.

The Functional Classification of ORF8 in SARS-CoV-2 Replication, Immune Evasion, and Viral Pathogenesis Inferred through Phylogenetic Profiling.

ORF8 is a highly variable genomic region of SARS-CoV-2. Although non-essential and the precise functions are unknown, it has been suggested that this protein assists in SARS-CoV-2 replication in the early secretory pathway and in immune evasion. We utilized the binding partners of SARS-CoV-2 proteins in human HEK293T cells and performed genome-wide phylogenetic profiling and clustering analyses in 446 eukaryotic species to predict and discover ORF8 binding partners that share associated functional mechanisms based on co-evolution. Results classified 47 ORF8 binding partner proteins into 3 clusters (groups 1-3), which were conserved in vertebrates (group 1), metazoan (group 2), and eukaryotes (group 3). Gene ontology analysis indicated that group 1 had no significant associated biological processes, while groups 2 and 3 were associated with glycoprotein biosynthesis process and ubiquitin-dependent endoplasmic reticulum-associated degradation pathways, respectively. Collectively, our results classified potential genes that might be associated with SARS-CoV-2 viral pathogenesis, specifically related to acute respiratory distress syndrome, and the secretory pathway. Here, we discuss the possible role of ORF8 in viral pathogenesis and in assisting viral replication and immune evasion via secretory pathway, as well as the possible factors associated with the rapid evolution of ORF8.

Quantitative glycoproteomics reveals cellular substrate selectivity of the ER protein quality control sensors UGGT1 and UGGT2.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) 1 and 2 are central hubs in the chaperone network of the endoplasmic reticulum (ER), acting as gatekeepers to the early secretory pathway, yet little is known about their cellular clients. These two quality control sensors control lectin chaperone binding and glycoprotein egress from the ER. A quantitative glycoproteomics strategy was deployed to identify cellular substrates of the UGGTs at endogenous levels in CRISPR-edited HEK293 cells. The 71 UGGT substrates identified were mainly large multidomain and heavily glycosylated proteins when compared to the general N-glycoproteome. UGGT1 was the dominant glucosyltransferase with a preference toward large plasma membrane proteins whereas UGGT2 favored the modification of smaller, soluble lysosomal proteins. This study sheds light on differential specificities and roles of UGGT1 and UGGT2 and provides insight into the cellular reliance on the carbohydrate-dependent chaperone system to facilitate proper folding and maturation of the cellular N-glycoproteome.

Molecular basis for KDEL-mediated retrieval of escaped ER-resident proteins - SWEET talking the COPs.

ABSTRACTProtein localisation in the cell is controlled through the function of trafficking receptors, which recognise specific signal sequences and direct cargo proteins to different locations. The KDEL receptor (KDELR) was one of the first intracellular trafficking receptors identified and plays an essential role in maintaining the integrity of the early secretory pathway. The receptor recognises variants of a canonical C-terminal Lys-Asp-Glu-Leu (KDEL) signal sequence on ER-resident proteins when these escape to the Golgi, and targets these proteins to COPI- coated vesicles for retrograde transport back to the ER. The empty receptor is then recycled from the ER back to the Golgi by COPII-coated vesicles. Crystal structures of the KDELR show that it is structurally related to the PQ-loop family of transporters that are found in both pro- and eukaryotes, and shuttle sugars, amino acids and vitamins across cellular membranes. Furthermore, analogous to PQ-loop transporters, the KDELR undergoes a pH-dependent and ligand-regulated conformational cycle. Here, we propose that the striking structural similarity between the KDELR and PQ-loop transporters reveals a connection between transport and trafficking in the cell, with important implications for understanding trafficking receptor evolution and function.

Legionella Manipulates Non-canonical SNARE Pairing Using a Bacterial Deubiquitinase

The intracellular bacterial pathogen Legionella pneumophila uses many effector proteins delivered by the bacterial type IV secretion system (T4SS) to hijack the early secretory pathway to establish its replicative niche, known as the Legionella-containing vacuole (LCV). On LCV biogenesis, the endoplasmic reticulum (ER) vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNARE) Sec22b is recruited to the bacterial phagosome and forms non-canonical pairings with target membrane SNAREs (t-SNAREs) from the plasma membrane. Here, we identify a Legionella deubiquitinase (DUB), LotB, that can modulate the early secretory pathway by interacting with coatomer protein complex I (COPI) vesicles when ectopically expressed. We show that Sec22b is ubiquitinated upon L.pneumophila infection in a T4SS-dependent manner and that, subsequently, LotB deconjugates K63-linked ubiquitins from Sec22b. The DUB activity of LotB stimulates dissociation of the t-SNARE syntaxin 3 (Stx3) from Sec22b, which resides on the LCV. Our study highlights a bacterial strategy manipulating the dynamics of infection-induced SNARE pairing using a bacterial DUB.

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway

Sphingolipids play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted human cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as glycosylphosphatidylinositol (GPI) anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway.

Correction: Role of the early secretory pathway in SARS-CoV-2 infection.

Correction: Role of the early secretory pathway in SARS-CoV-2 infection.

Role of the early secretory pathway in SARS-CoV-2 infection.

Similar to other RNA viruses, SARS-CoV-2 must (1) enter a target/host cell, (2) reprogram it to ensure its replication, (3) exit the host cell, and (4) repeat this cycle for exponential growth. During the exit step, the virus hijacks the sophisticated machineries that host cells employ to correctly fold, assemble, and transport proteins along the exocytic pathway. Therefore, secretory pathway-mediated assemblage and excretion of infective particles represent appealing targets to reduce the efficacy of virus biogenesis, if not to block it completely. Here, we analyze and discuss the contribution of the molecular machines operating in the early secretory pathway in the biogenesis of SARS-CoV-2 and their relevance for potential antiviral targeting. The fact that these molecular machines are conserved throughout evolution, together with the redundancy and tissue specificity of their components, provides opportunities in the search for unique proteins essential for SARS-CoV-2 biology that could also be targeted with therapeutic objectives. Finally, we provide an overview of recent evidence implicating proteins of the early secretory pathway as potential antiviral targets with effective therapeutic applications.

Dynamic Capture and Release of Endoplasmic Reticulum Exit Sites by Golgi Stacks in Arabidopsis.

SummaryProtein transport from the endoplasmic reticulum (ER) to Golgi stacks is mediated by the coat protein complex COPII, which is assembled at an ER subdomain called ER exit site (ERES). However, the dynamic relationship between ERESs and Golgi stacks is unknown. Here, we propose a dynamic capture-and-release model of ERESs by Golgi stacks in Arabidopsis thaliana. Using variable-angle epifluorescence microscopy with high-temporal-resolution imaging, COPII-component-bound ERESs were detected as punctate structures with sizes of 300–500 nm. Some punctate ERESs are distributed on ER tubules and sheet rims, whereas others gather around a Golgi stack in an ER-network cavity to form a beaded-ring structure. Free ERESs that wander into an ER cavity are captured by a Golgi stack in a cytoskeleton-independent manner. Then, they are released by the Golgi stack for recycling. The dynamic ERES cycling might contribute to efficient transfer of de novo synthesized cargo proteins from the ER to Golgi stacks.

Loss of Arabidopsis β-COP Function Affects Golgi Structure, Plant Growth and Tolerance to Salt Stress.

The early secretory pathway involves bidirectional transport between the endoplasmic reticulum (ER) and the Golgi apparatus and is mediated by coat protein complex I (COPI)-coated and coat protein complex II (COPII)-coated vesicles. COPII vesicles are involved in ER to Golgi transport meanwhile COPI vesicles mediate intra-Golgi transport and retrograde transport from the Golgi apparatus to the ER. The key component of COPI vesicles is the coatomer complex, that is composed of seven subunits (α/β/β’/γ/δ/ε/ζ). In Arabidopsis two genes coding for the β-COP subunit have been identified, which are the result of recent tandem duplication. Here we have used a loss-of-function approach to study the function of β-COP. The results we have obtained suggest that β-COP is required for plant growth and salt tolerance. In addition, β-COP function seems to be required for maintaining the structure of the Golgi apparatus.

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5-ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5-ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5-ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

Improved secretion of glycoproteins using an N-glycan-restricted passport sequence tag recognized by cargo receptor

MCFD2 and ERGIC-53, which are the products of causative genes of combined factor V and factor VIII deficiency, form a cargo receptor complex responsible for intracellular transport of these coagulation factors in the early secretory pathway. In this study, using an NMR technique, we successfully identified an MCFD2-binding segment from factor VIII composed of a 10 amino acid sequence that enhances its secretion. This prompted us to examine possible effects of attaching this sequence to recombinant glycoproteins on their secretion. We found that the secretion level of recombinant erythropoietin was significantly increased simply by tagging it with the passport sequence. Our findings not only provide molecular basis for the intracellular trafficking of coagulation factors and their genetic deficiency but also offer a potentially useful tool for increasing the production yields of recombinant glycoproteins of biopharmaceutical interest.

Recombinant Expression and Purification of Animal Intracellular L-Type Lectins.

Animal leguminous-type (L-type) lectins, including ERGIC-53 and VIP36 are responsible for intracellular transport and quality control of N-linked glycoproteins in the early secretory pathway. These lectins possess the carbohydrate recognition domain (CRD), which recognizes high-mannose-type glycans in a Ca2+-dependent manner. Here we describe the procedures involved in bacterial overproduction and purification of the CRDs of the animal L-type lectins.

The Early Secretory Pathway in Immunity

The Early Secretory Pathway in Immunity