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The trans -Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)−1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or “zones”, responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone. The trans -Golgi network (TGN) serves as a platform to sort and transport proteins to their final destinations. Here the authors show that the TGN of Arabidopsis consists of spatially and temporally distinct subregions and propose that these zones may sort cargo to different destinations.

Protein synthesis and autophagic degradation are regulated in an opposite manner by mammalian target of rapamycin (mTOR), whereas under certain conditions it would be beneficial if they occurred in unison to handle rapid protein turnover. We observed a distinct cellular compartment at the trans side of the Golgi apparatus, the TOR-autophagy spatial coupling compartment (TASCC), where (auto)lysosomes and mTOR accumulated during Ras-induced senescence. mTOR recruitment to the TASCC was amino acid— and Rag guanosine triphosphatase—dependent, and disruption of mTOR localization to the TASCC suppressed interleukin-6/8 synthesis. TASCC formation was observed during macrophage differentiation and in glomerular podocytes; both displayed increased protein secretion. The spatial coupling of cells' catabolic and anabolic machinery could augment their respective functions and facilitate the mass synthesis of secretory proteins.

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including ‘Dynamic Organellar Maps’, to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the trans -Golgi network to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the “ATG9A reservoir” required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy. Adaptor protein complex 4 (AP-4) deficiency causes a severe neurological disorder via an unknown mechanism. Here, the authors reveal cargo and machinery of the AP-4 transport pathway, and propose that AP-4 mediates spatial regulation of autophagy through peripheral delivery of ATG9A.

The NLRP3 inflammasome, which has been linked to human inflammatory diseases, is activated by diverse stimuli. How these stimuli activate NLRP3 is unknown. Here we show that different NLRP3 stimuli lead to disassembly of the trans -Golgi network (TGN). NLRP3 is recruited to the dispersed TGN (dTGN) through ionic bonding between its conserved polybasic region and negatively charged phosphatidylinositol-4-phosphate (PtdIns4P) on the dTGN. The dTGN then serves as a scaffold for NLRP3 aggregation into multiple puncta, leading to polymerization of the adaptor protein ASC, thereby activating the downstream signalling cascade. Disruption of the interaction between NLRP3 and PtdIns4P on the dTGN blocked NLRP3 aggregation and downstream signalling. These results indicate that recruitment of NLRP3 to dTGN is an early and common cellular event that leads to NLRP3 aggregation and activation in response to diverse stimuli. Recruitment of NLRP3 to the dispersed trans -Golgi network via binding to PtdIns4P is required for activation of the NLRP3 inflammasome by diverse stimuli.

The plant trans-Golgi network (TGN) is a central trafficking hub where secretory, vacuolar, recycling, and endocytic pathways merge. Among currently known molecular players involved in TGN transport, three different adaptor protein (AP) complexes promote vesicle generation at the TGN with different cargo specificity and destination. Yet, it remains unresolved how sorting into diverging vesicular routes is spatially organized. Here, we study the family of Arabidopsis thaliana Epsin-like proteins, which are accessory proteins to APs facilitating vesicle biogenesis. By comprehensive molecular, cellular, and genetic analysis of the EPSIN gene family, we identify EPSIN1 and MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) as its only TGN-associated members. Despite their large phylogenetic distance, they perform overlapping functions in vacuolar and secretory transport. By probing their relationship with AP complexes, we find that they define two molecularly independent pathways:While EPSIN1 associates with AP-1, MTV1 interacts with AP-4, whose function is required for MTV1 recruitment. Although both EPSIN1/AP-1 and MTV1/AP-4 pairs reside at the TGN, high-resolution microscopy reveals them as spatially separate entities. Our results strongly support the hypothesis of molecularly, functionally, and spatially distinct subdomains of the plant TGN and suggest that functional redundancy can be achieved through parallelization of molecularly distinct but functionally overlapping pathways.

Mitochondrial plasticity is a key regulator of cell fate decisions. Mitochondrial division involves Dynamin-related protein-1 (Drp1) oligomerization, which constricts membranes at endoplasmic reticulum (ER) contact sites. The mechanisms driving the final steps of mitochondrial division are still unclear. Here, we found that microdomains of phosphatidylinositol 4-phosphate [PI(4)P] on trans-Golgi network (TGN) vesicles were recruited to mitochondria–ER contact sites and could drive mitochondrial division downstream of Drp1. The loss of the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) or its effector, phosphatidylinositol 4-kinase IIIβ [PI(4)KIIIβ], in different mammalian cell lines prevented PI(4)P generation and led to a hyperfused and branched mitochondrial network marked with extended mitochondrial constriction sites. Thus, recruitment of TGN-PI(4)P–containing vesicles at mitochondria–ER contact sites may trigger final events leading to mitochondrial scission.

Cell polarity is a fundamental feature of all multicellular organisms. PIN auxin transporters are important cell polarity markers that play crucial roles in a plethora of developmental processes in plants. Here, to identify components involved in cell polarity establishment and maintenance in plants, we performed a forward genetic screening of PIN2:PIN1-HA;pin2 Arabidopsis (Arabidopsis thaliana) plants, which ectopically express predominantly basally localized PIN1 in root epidermal cells, leading to agravitropic root growth. We identified the regulator of PIN polarity 12 (repp12) mutation, which restored gravitropic root growth and caused a switch in PIN1-HA polarity from the basal to apical side of root epidermal cells. Next Generation Sequencing and complementation experiments established the causative mutation of repp12 as a single amino acid exchange in Aminophospholipid ATPase3 (ALA3), a phospholipid flippase predicted to function in vesicle formation. repp12 and ala3 T-DNA mutants show defects in many auxin-regulated processes, asymmetric auxin distribution, and PIN trafficking. Analysis of quintuple and sextuple mutants confirmed the crucial roles of ALA proteins in regulating plant development as well as PIN trafficking and polarity. Genetic and physical interaction studies revealed that ALA3 functions together with the ADP ribosylation factor GTPase exchange factors GNOM and BIG3 in regulating PIN polarity, trafficking, and auxin-mediated development.

Dengue virus has two membrane proteins, E and M, which undergo dramatic structural changes during the life cycle of the virus. The 3.5-Å cryo-EM structure of the mature prefusion Dengue virion reveals the detailed interactions between E and M, providing insight into how conformational changes are triggered. Regulated by pH, membrane-anchored proteins E and M function during dengue virus maturation and membrane fusion. Our atomic model of the whole virion from cryo–electron microscopy at 3.5-Å resolution reveals that in the mature virus at neutral extracellular pH, the N-terminal 20-amino-acid segment of M (involving three pH-sensing histidines) latches and thereby prevents spring-loaded E fusion protein from prematurely exposing its fusion peptide. This M latch is fastened at an earlier stage, during maturation at acidic pH in the trans -Golgi network. At a later stage, to initiate infection in response to acidic pH in the late endosome, M releases the latch and exposes the fusion peptide. Thus, M serves as a multistep chaperone of E to control the conformational changes accompanying maturation and infection. These pH-sensitive interactions could serve as targets for drug discovery.

The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting and transport of cargo. Vacuolar transport from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) is assumed to occur via clathrin-coated vesicles, although direct proof for their participation is missing. Here, we present evidence that post-TGN transport toward lytic vacuoles occurs independently of clathrin and that MVBs/LEs are derived from the TGN/EE through maturation. We show that the V-ATPase inhibitor concanamycin A significantly reduces the number of MVBs and causes TGN and MVB markers to colocalize in Arabidopsis thaliana roots. Ultrastructural analysis reveals the formation of MVBs from the TGN/EE and their fusion with the vacuole.The localization of the ESCRT components VPS28, VPS22, and VPS2 at the TGN/EE and MVBs/LEs indicates that the formation of intraluminal vesicles starts already at the TGN/EE. Accordingly, a dominant-negative mutant of VPS2 causes TGN and MVB markers to colocalize and blocks vacuolar transport. RNA interference-mediated knockdown of the annexin ANNAT3 also yields the same phenotype. Together, these data indicate that MVBs originate from the TGN/EE in a process that requires the action of ESCRT for the formation of intraluminal vesicles and annexins for the final step of releasing MVBs as a transport carrier to the vacuole.

SARS-CoV-2 hijacks multiple organelles for virion assembly, of which the mechanisms have not been fully understood. Here, we identified a SARS-CoV-2-driven membrane structure named the 3a dense body (3DB). 3DBs are unusual electron-dense and dynamic structures driven by the accessory protein ORF3a via remodeling a specific subset of the trans -Golgi network (TGN) and early endosomal membrane. 3DB formation is conserved in related bat and pangolin coronaviruses but was lost during the evolution to SARS-CoV. During SARS-CoV-2 infection, 3DB recruits the viral structural proteins spike (S) and membrane (M) and undergoes dynamic fusion/fission to maintain the optimal unprocessed-to-processed ratio of S on assembled virions. Disruption of 3DB formation resulted in virions assembled with an abnormal S processing rate, leading to a dramatic reduction in viral entry efficiency. Our study uncovers the crucial role of 3DB in maintaining maximal SARS-CoV-2 infectivity and highlights its potential as a target for COVID-19 prophylactics and therapeutics. Hartmann et al. discovered that SARS-CoV-2 constructs a group of dynamic membrane structures named the 3DB. 3DB regulates the processing of the viral spike protein to assemble a highly infectious virus, highlighting the potential of 3DB as a novel COVID-19 therapeutic target.