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Membrane and secretory proteins are essential for almost every aspect of cellular function. These proteins are incorporated into ER-derived carriers and transported to the Golgi before being sorted for delivery to their final destination. Although ER-to-Golgi trafficking is highly conserved among eukaryotes, several layers of complexity have been added to meet the increased demands of complex cell types in metazoans. The specialized morphology of neurons and the necessity for precise spatiotemporal control over membrane and secretory protein localization and function make them particularly vulnerable to defects in trafficking. This review summarizes the general mechanisms involved in ER-to-Golgi trafficking and highlights mutations in genes affecting this process, which are associated with neurological diseases in humans.

Intracellular transport undergoes remodeling upon cell differentiation, which involves cell type-specific regulators. Bone morphogenetic protein 2-inducible kinase (BMP2K) has been potentially implicated in endocytosis and cell differentiation but its molecular functions remained unknown. We discovered that its longer (L) and shorter (S) splicing variants regulate erythroid differentiation in a manner unexplainable by their involvement in AP-2 adaptor phosphorylation and endocytosis. However, both variants interact with SEC16A and could localize to the juxtanuclear secretory compartment. Variant-specific depletion approach showed that BMP2K isoforms constitute a BMP2K-L/S regulatory system that controls the distribution of SEC16A and SEC24B as well as SEC31A abundance at COPII assemblies. Finally, we found L to promote and S to restrict autophagic degradation and erythroid differentiation. Hence, we propose that BMP2K-L and BMP2K-S differentially regulate abundance and distribution of COPII assemblies as well as autophagy, possibly thereby fine-tuning erythroid differentiation.

Despite advances in understanding the STING signaling pathway, mechanisms governing cyclic GMP‐AMP (cGAMP)‐induced STING trafficking out of the endoplasmic reticulum (ER) remain unclear. This study reveals that STING localization is regulated by the balance between coat protein II (COPII)‐ and coat protein I (COPI)‐mediated trafficking, maintaining ER residency in the inactive state or promoting transport to the cis‐Golgi via enhanced COPII‐mediated export upon activation. Two novel TANK‐binding kinase 1 (TBK1)‐regulated phosphorylated COPII sorting signals on STING—a conserved pSGME motif and a primate‐specific pFS motif—are biochemically and structurally identified. These cGAMP‐induced signals drive activated STING toward the ER‐Golgi intermediate compartment (ERGIC) and the cis‐Golgi complex. Using a cell‐free COPII vesicle reconstitution system, TBK1 activation is shown to occur on COPII vesicles, while IRF3 phosphorylation is confined to the ERGIC or the cis‐Golgi complex post‐uncoating, due to the competitive binding of COPII Sec24 and IRF3 to phosphorylated STING. A class of compounds is also identified that attenuates IRF3 phosphorylation by inhibiting phosphorylated STING packaging into COPII vesicles. These findings elucidate STING trafficking mechanisms and offer therapeutic potential for diseases linked to dysregulated STING activation. This schematic outlines STING trafficking and signaling via coat protein II (COPII)‐mediated endoplasmic reticulum (ER) export. Following cyclic GMP‐AMP (cGAMP) stimulation, TANK‐binding kinase 1 (TBK1) activates on COPII vesicles, partially dissociating into the cytosol to phosphorylate ER‐localized STING, creating pSGME/pFS COPII‐sorting motifs. Competition at shared phosphorylation sites limitsinterferon regulatory factor 3 (IRF3) activation to post‐uncoating ER ‐ Golgi intermediate compartment (ERGIC) or cis‐Golgi compartments, emphasizing spatially compartmentalized signaling.

The cytoplasmic coat protein complex-II (COPII) is evolutionarily conserved machinery that is essential for efficient trafficking of protein and lipid cargos. How the COPII machinery is regulated to meet the metabolic demand in response to alterations of the nutritional state remains largely unexplored, however. Here, we show that dynamic changes of COPII vesicle trafficking parallel the activation of transcription factor X-box binding protein 1 (XBP1s), a critical transcription factor in handling cellular endoplasmic reticulum (ER) stress in both live cells and mouse livers upon physiological fluctuations of nutrient availability. Using live-cell imaging approaches, we demonstrate that XBP1s is sufficient to promote COPII-dependent trafficking, mediating the nutrient stimulatory effects. Chromatin immunoprecipitation (ChIP) coupled with high-throughput DNA sequencing (ChIP-seq) and RNA-sequencing analyses reveal that nutritional signals induce dynamic XBP1s occupancy of promoters of COPII traffic-related genes, thereby driving the COPII-mediated trafficking process. Liver-specific disruption of the inositol-requiring enzyme 1α (IRE1α)–XBP1s signaling branch results in diminished COPII vesicle trafficking. Reactivation of XBP1s in mice lacking hepatic IRE1α restores COPII-mediated lipoprotein secretion and reverses the fatty liver and hypolipidemia phenotypes. Thus, our results demonstrate a previously unappreciated mechanism in the metabolic control of liver protein and lipid trafficking: The IRE1α-XBP1s axis functions as a nutrient-sensing regulatory nexus that integrates nutritional states and the COPII vesicle trafficking.

Mutations in the apical Na-K-2Cl co-transporter, NKCC2, cause type I Bartter syndrome (BS1), a life-threatening kidney disease. We have previously demonstrated that the BS1 variant Y998X, which deprives NKCC2 from its highly conserved dileucine-like motifs, compromises co-transporter surface delivery through ER retention mechanisms. However, whether these hydrophobic motifs are sufficient for anterograde trafficking of NKCC2 remains to be determined. Interestingly, sequence analysis of NKCC2 C-terminus revealed the presence of consensus di-acidic (D/E-X-D/E) motifs, 949EEE951 and 1019DAELE1023, located upstream and downstream of BS1 mutation Y998X, respectively. Di-acidic codes are involved in ER export of proteins through interaction with COPII budding machinery. Importantly, whereas mutating 949EEE951 motif to 949AEA951 had no effect on NKCC2 processing, mutating 1019DAE1021 to 1019AAA1021 heavily impaired complex-glycosylation and cell surface expression of the cotransporter in HEK293 and OKP cells. Most importantly, triple mutation of D, E and E residues of 1019DAELE1023 to 1019AAALA1023 almost completely abolished NKCC2 complex-glycosylation, suggesting that this mutant failed to exit the ER. Cycloheximide chase analysis demonstrated that the absence of the terminally glycosylated form of 1019AAALA1023 was caused by defects in NKCC2 maturation. Accordingly, co-immunolocalization experiments revealed that 1019AAALA1023 was trapped in the ER. Finally, overexpression of a dominant negative mutant of Sar1-GTPase abolished NKCC2 maturation and cell surface expression, clearly indicating that NKCC2 export from the ER is COPII-dependent. Hence, our data indicate that in addition to the di-leucine like motifs, NKCC2 uses di-acidic exit codes for export from the ER through the COPII-dependent pathway. We propose that any naturally occurring mutation of NKCC2 interfering with this pathway could form the molecular basis of BS1.

Abstract Curvature-generating proteins that direct membrane trafficking assemble on the surface of lipid bilayers to bud transport intermediates, which move protein and lipid cargoes from one cellular compartment to another. However, it remains unclear what controls the overall shape of the membrane bud once curvature induction has begun. In vitro experiments showed that excessive concentrations of the COPII protein Sar1 promoted the formation of membrane tubules from synthetic vesicles, while COPII-coated transport intermediates in cells are generally more spherical or lobed in shape. To understand the origin of these morphological differences, we employ atomistic, coarse-grained (CG), and continuum mesoscopic simulations of membranes in the presence of multiple curvature-generating proteins. We first characterize the membrane-bending ability of amphipathic peptides derived from the amino terminus of Sar1, as a function of interpeptide angle and concentration using an atomistic bicelle simulation protocol. Then, we employ CG simulations to reveal that Sec23 and Sec24 control the relative spacing between Sar1 protomers and form the inner-coat unit through an attachment with Sar1. Finally, using dynamical triangulated surface simulations based on the Helfrich Hamiltonian, we demonstrate that the uniform distribution of spacer molecules among curvature-generating proteins is crucial to the spherical budding of the membrane. Overall, our analyses suggest a new role for Sec23, Sec24, and cargo proteins in COPII-mediated membrane budding process in which they act as spacers to preserve a dispersed arrangement of Sar1 protomers and help determine the overall shape of the membrane bud.

Normal neural development is essential for the formation of neuronal networks and brain function. Cutaneous T cell lymphoma-associated antigen 5 (cTAGE5)/meningioma expressed antigen 6 (MEA6) plays a critical role in the secretion of proteins. However, its roles in the transport of nonsecretory cellular components and in brain development remain unknown. Here, we show that cTAGE5/MEA6 is important for brain development and function. Conditional knockout of cTAGE5/MEA6 in the brain leads to severe defects in neural development, including deficits in dendrite outgrowth and branching, spine formation and maintenance, astrocyte activation, and abnormal behaviors. We reveal that loss of cTAGE5/MEA6 affects the interaction between the coat protein complex II (COPII) components, SAR1 and SEC23, leading to persistent activation of SAR1 and defects in COPII vesicle formation and transport from the endoplasmic reticulum to the Golgi, as well as disturbed trafficking of membrane components in neurons. These defects affect not only the transport of materials required for the development of dendrites and spines but also the signaling pathways required for neuronal development. Because mutations in cTAGE5/MEA6 have been found in patients with Fahr’s disease, our study potentially also provides insight into the pathogenesis of this disorder.

Membrane proteins are sorted to the plasma membrane via Golgi-dependent trafficking. However, our recent studies challenged the essentiality of Golgi in the biogenesis of specific transporters. Here, we investigate the trafficking mechanisms of membrane proteins by following the localization of the polarized R-SNARE SynA versus the non-polarized transporter UapA, synchronously co-expressed in wild-type or isogenic genetic backgrounds repressible for conventional cargo secretion. In wild-type, the two cargoes dynamically label distinct secretory compartments, highlighted by the finding that, unlike SynA, UapA does not colocalize with the late-Golgi. In line with early partitioning into distinct secretory carriers, the two cargoes collapse in distinct ER-Exit Sites (ERES) in a sec31 ts background. Trafficking via distinct cargo-specific carriers is further supported by showing that repression of proteins essential for conventional cargo secretion does not affect UapA trafficking, while blocking SynA secretion. Overall, this work establishes the existence of distinct, cargo-dependent, trafficking mechanisms, initiating at ERES and being differentially dependent on Golgi and SNARE interactions.

Survival of the apicomplexan parasite Toxoplasma gondii depends on the proper functioning of many glycosylated proteins. Glycosylation is performed in the major membranous organelles ER and Golgi apparatus that constitute a significant portion of the intracellular secretory system. The secretory pathway is bidirectional: cargo is delivered to target organelles in the anterograde direction, while retrograde flow maintains the membrane balance and proper localization of glycosylation machinery. Despite the vital role of the Golgi in parasite infectivity, little is known about its biogenesis in apicomplexan parasites. In this study, we examined the T. gondii conserved oligomeric Golgi (COG) complex and determined that contrary to predictions, T. gondii expresses the entire eight-subunit complex and that each complex subunit is essential for tachyzoite growth. Deprivation of the COG complex induces a pronounced effect on Golgi and ER membranes, which suggests that the T. gondii COG complex has a wider role in intracellular membrane trafficking. We demonstrated that besides its conservative role in retrograde intra-Golgi trafficking, the COG complex also interacted with anterograde and novel transport machinery. Furthermore, we identified coccidian-specific components of the Golgi transport system: TgUlp1 and TgGlp1. Protein structure and phylogenetic analyses revealed that TgUlp1 is an adaptation of the conservative Golgi tethering factor Uso1/p115. TgUlp1 and together with Golgi-localized TgGlp1 showed dominant interactions with the trafficking machinery that was predicted to operate endosome-to-Golgi recycling. Together, our study showed that T. gondii has expanded the function of the conservative Golgi tethering COG complex and evolved additional regulators of transport that are likely to serve parasite-specific secretory organelles. The Golgi is an essential eukaryotic organelle and a major place for protein sorting and glycosylation. Among apicomplexan parasites, Toxoplasma gondii retains the most developed Golgi structure and produces many glycosylated factors necessary for parasite survival. Despite its importance, Golgi function received little attention in the past. In the current study, we identified and characterized the conserved oligomeric Golgi complex and its novel partners critical for protein transport in T. gondii tachyzoites. Our results suggest that T. gondii broadened the role of the conserved elements and reinvented the missing components of the trafficking machinery to accommodate the specific needs of the opportunistic parasite T. gondii .

Dyslipidemia is a well-known risk factor in the development and progression of atherosclerosis. VLDL plays a crucial role in maintaining lipid homeostasis; however, even minor fluctuations in its production, intracellular trafficking, and secretion can contribute to the progression of atherosclerosis. Fenofibrate is an FDA-approved drug that effectively lowers plasma triglycerides and VLDL-associated cholesterol while simultaneously increasing HDL levels. Although fenofibrate is a known PPARα agonist with several proposed mechanisms for its lipid-altering effects, its impact on the intracellular trafficking of VLDL has not yet been investigated. We observed that treatment of HepG2 cells with 50 µM of fenofibrate resulted in a significant reduction in VLDL secretion, as evidenced by a significant decrease in the secretion of 3H-labeled TAG, fluorescent TAG, and ApoB100 protein into the media. Using confocal microscopy to monitor VLDL intracellular trafficking, we observed a distinct change in VLDL triglyceride localization, suggesting delayed transport through the endoplasmic reticulum and Golgi. An immunoblot analysis revealed a decrease in Sar1B protein expression, a key regulator of COPII protein recruitment, which is essential for VTV formation and intracellular VLDL trafficking, the rate-limiting step in VLDL secretion. Our data reveal a novel mechanism by which fenofibrate alters the lipid profile by interfering with intracellular VLDL trafficking in hepatocytes.