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In plants, robust defense depends on the efficient and resilient trafficking supply chains to the site of pathogen attack. Though the importance of intracellular trafficking in plant immunity has been well established, a lack of clarity remains regarding the contribution of the various trafficking pathways in transporting immune‐related proteins. We have recently identified a trans‐Golgi network protein, TGN‐ASSOCIATED PROTEIN 1 (TGNap1), which functionally links post‐Golgi vesicles with the cytoskeleton to transport immunity‐related proteins in the model plant species Arabidopsis thaliana. We propose new hypotheses on the various functional implications of TGNap1 and then elaborate on the surprising heterogeneity of TGN vesicles during immunity revealed by the discovery of TGNap1 and other TGN‐associated proteins in recent years. [ABSTRACT FROM AUTHOR]

Zinc (Zn) is an essential element for plants. Numerous proteins in different cellular compartments require Zn for their structure and function. Zn can be toxic when it accumulates in high levels in the cytoplasm. Therefore, Zn homeostasis at tissue, cell, and organelle levels is vital for plant growth. A part of the metal tolerance protein (MTP) / Cation Diffusion Facilitator (CDF) transporters functions as Zn transporters, exporting Zn from the cytosol to various membrane compartments. In Arabidopsis thaliana, MTP1, MTP2, MTP3, MTP4, MTP5, and MTP12 are classified as Zn transporters (Zn-CDF). In this study, we systematically analyzed the localization of GFP-fused Zn-CDFs in the leaf epidermal cells of Nicotiana benthamiana. As previously reported, MTP1 and MTP3 were localized to tonoplast, MTP2 to endoplasmic reticulum, and MTP5 to Golgi. In addition, we identified the localization of MTP4 to trans-Golgi Network (TGN). Since MTP4 is specifically expressed in pollen, we analyzed the localization of MTP4-GFP in the Arabidopsis pollen tubes and confirmed that it is in the TGN. We also showed the Zn transport capability of MTP4 in yeast cells. We then analyzed the phenotype of an mtp4 T-DNA insertion mutant under both limited and excess Zn conditions. We found that their growth and fertility were not largely different from the wild-type. Our study has paved the way for investigating the possible roles of MTP4 in metallating proteins in the secretory pathway or in exporting excess Zn through exocytosis. In addition, our system of GFP-fused MTPs will help study the mechanisms for targeting transporters to specific membrane compartments. [ABSTRACT FROM AUTHOR]

Clathrin-mediated vesicle trafficking plays central roles in post-Golgi transport. In yeast (Saccharomyces cerevisiae), the AP-1 complex and GGA adaptors are predicted to generate distinct transport vesicles at the trans-Golgi network (TGN), and the epsin-related proteins Ent3p and Ent5p (collectively Ent3p/5p) act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN is crucial for yeast Rab5 (Vps21p)-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGA adaptors are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Vps21p activity remains ambiguous. Here, we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Vps21p activation. We show that AP-1 disruption in the ent3Δ5Δ mutant impaired transport of the Vps21p guanine nucleotide exchange factor Vps9p transport to the Vps21p compartment and severely reduced Vps21p activity. Additionally, GGA adaptors, the phosphatidylinositol-4-kinase Pik1p and Rab11 GTPases Ypt31p and Ypt32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway.

Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells, the trans-Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment and function independently of the Sec7p-residing region within the TGN.

Exomer is a protein complex that facilitates trafficking between the Golgi and the plasma membrane (PM). Schizosaccharomyces pombe exomer is composed of Cfr1 and Bch1, and we have found that full activation of the cell integrity pathway (CIP) in response to osmotic stress requires exomer. In the wild-type, the CIP activators Rgf1 (Rho1 GEF) and Pck2 (PKC homologue) and the MEK kinase Mkh1 localize in the PM, internalize after osmotic shock and re-localize after adaptation. This re-localization is inefficient in exomer mutants. Overexpression of the PM-associated 1-phosphatidylinositol 4-kinase stt4+, and deletion of the nem1+ phosphatase suppress the defects in Pck2 dynamics in exomer mutants, but not their defect in CIP activation, demonstrating that exomer regulates CIP in additional ways. Exomer mutants accumulate PI4P in the TGN, and increasing the expression of the Golgi-associated 1-phosphatidylinositol 4-kinase pik1+ suppresses their defect in Pck2 dynamics. These findings suggest that efficient PI4P transport from the Golgi to the PM requires exomer. Mutants lacking clathrin adaptors are defective in CIP activation, but not in Pck2 dynamics or in PI4P accumulation in the Golgi. Hence, traffic from the Golgi regulates CIP activation, and exomer participates in this regulation through an exclusive mechanism.

ABSTRACT Rab GTPases control intracellular vesicular transport, including retrograde trafficking of human papillomavirus (HPV) during cell entry, guiding the virus from the endosome to the trans-Golgi network (TGN), the Golgi apparatus, and eventually the nucleus. Rab proteins have been identified that act prior to the arrival of HPV at the TGN, but Rab proteins operating in later stages of entry remain elusive. Here, we report that knockdown of Rab6a impairs HPV entry by preventing HPV exit from the TGN and impeding intra-Golgi transport of the incoming virus. Rab6a supports HPV trafficking by facilitating the association of HPV with dynein, a motor protein complex, and BICD2, a dynein adaptor, in the TGN. L2 can bind directly to GTP-Rab6a in vitro, and excess of either GTP-Rab6a or GDP-Rab6 inhibits HPV entry, suggesting that cycling between GDP-Rab6 and GTP-Rab6 is critical. Notably, Rab6a is crucial for HPV-BICD2 and HPV-dynein association in the TGN of infected cells but not in the endosome. Our findings reveal important features of the molecular basis of HPV infection, including the discovery that HPV uses different mechanisms to engage dynein at different times during entry, and identify potential targets for therapeutic approaches to inhibit HPV infection.IMPORTANCEHuman papillomaviruses (HPVs) are small, non-enveloped DNA viruses that cause approximately 5% of human cancer. Like most other DNA viruses, HPV traffics to the nucleus during virus entry to successfully infect cells. We show here that HPV utilizes a cellular enzyme, Rab6a, during virus entry to engage the dynein molecular motor for transport along microtubules. Rab6a is required for complex formation between the HPV L2 capsid protein, dynein, and the dynein adaptor BICD2 in the trans-Golgi network (TGN). This complex is required for transport of the incoming virus out of the TGN as it journeys to the nucleus. Our findings identify potential targets for therapeutic approaches.

Membrane trafficking is essential for sculpting neuronal morphology. The GARP and EARP complexes are conserved tethers that regulate vesicle trafficking in the secretory and endolysosomal pathways, respectively. Both complexes contain the Vps51, Vps52, and Vps53 proteins, and a complex-specific protein: Vps54 in GARP and Vps50 in EARP. In Drosophila, we find that both complexes are required for dendrite morphogenesis during developmental remodeling of multidendritic class IV da (c4da) neurons. Having found that sterol accumulates at the trans-Golgi network (TGN) in Vps54KO/KO neurons, we investigated genes that regulate sterols and related lipids at the TGN. Overexpression of oxysterol binding protein (Osbp) or knockdown of the PI4K four wheel drive (fwd) exacerbates the Vps54KO/KO phenotype, whereas eliminating one allele of Osbp rescues it, suggesting that excess sterol accumulation at the TGN is, in part, responsible for inhibiting dendrite regrowth. These findings distinguish the GARP and EARP complexes in neurodevelopment and implicate vesicle trafficking and lipid transfer pathways in dendrite morphogenesis.

Clathrin assembles into honeycomb-like lattices at the plasma membrane but also on internal membranes, such as at the Golgi and tubular endosomes. Clathrin assemblies primarily regulate the intracellular trafficking of different cargoes, but clathrin also has nonendocytic functions in cell adhesion through interactions with specific integrins, contributes to intraluminal vesicle formation by forming flat bilayered coats on endosomes and even assembles on kinetochore k-fibers during mitosis. In this Cell Science at a Glance article and the accompanying poster, we review our current knowledge on the different types of canonical and non-canonical membrane-associated clathrin assemblies in mammalian cells, as observed by thin-section or platinum replica electron microscopy in various cell types, and discuss how the structural plasticity of clathrin contributes to its functional diversity. [ABSTRACT FROM AUTHOR]

In hepatocytes, the Wilson disease protein ATP7B resides on the trans‐Golgi network (TGN) and traffics to peripheral lysosomes to export excess intracellular copper through lysosomal exocytosis. We found that in basal copper or even upon copper chelation, a significant amount of ATP7B persists in the endolysosomal compartment of hepatocytes but not in non‐hepatic cells. These ATP7B‐harbouring lysosomes lie in close proximity of ~10 nm to the TGN. ATP7B constitutively distributes itself between the sub‐domain of the TGN with a lower pH and the TGN‐proximal lysosomal compartments. The presence of ATP7B on TGN‐lysosome colocalising sites upon Golgi disruption suggested a possible exchange of ATP7B directly between the TGN and its proximal lysosomes. Manipulating lysosomal positioning significantly alters the localisation of ATP7B in the cell. Contrary to previous understanding, we found that upon copper chelation in a copper‐replete hepatocyte, ATP7B is not retrieved back to TGN from peripheral lysosomes; rather, ATP7B recycles to these TGN‐proximal lysosomes to initiate the next cycle of copper transport. We report a hitherto unknown copper‐independent lysosomal localisation of ATP7B and the importance of TGN‐proximal lysosomes but not TGN as the terminal acceptor organelle of ATP7B in its retrograde pathway. [ABSTRACT FROM AUTHOR]

Clathrin-mediated vesicle trafficking plays central roles in post-Golgi transport. In yeast (Saccharomyces cerevisiae), the AP-1 complex and GGA adaptors are predicted to generate distinct transport vesicles at the trans-Golgi network (TGN), and the epsin-related proteins Ent3p and Ent5p (collectively Ent3p/5p) act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN is crucial for yeast Rab5 (Vps21p)-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGA adaptors are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Vps21p activity remains ambiguous. Here, we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Vps21p activation. We show that AP-1 disruption in the ent3Δ5Δ mutant impaired transport of the Vps21p guanine nucleotide exchange factor Vps9p transport to the Vps21p compartment and severely reduced Vps21p activity. Additionally, GGA adaptors, the phosphatidylinositol-4-kinase Pik1p and Rab11 GTPases Ypt31p and Ypt32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway. [ABSTRACT FROM AUTHOR]

Cellulose is synthesized at the plasma membrane by cellulose synthase (CESA) complexes (CSCs), which are assembled in the Golgi and secreted to the plasma membrane through the trans-Golgi network (TGN) compartment. However, the molecular mechanisms that guide CSCs through the secretory system and deliver them to the plasma membrane are poorly understood. Here, we identified an uncharacterized gene, TRANVIA (TVA), that is transcriptionally coregulated with the CESA genes required for primary cell wall synthesis. The tva mutant exhibits enhanced sensitivity to cellulose synthesis inhibitors; reduced cellulose content; and defective dynamics, density, and secretion of CSCs to the plasma membrane as compared to wild type. TVA is a plant-specific protein of unknown function that is detected in at least two different intracellular compartments: organelles labeled by markers for the TGN and smaller compartments that deliver CSCs to the plasma membrane. Together, our data suggest that TVA promotes trafficking of CSCs to the plasma membrane by facilitating exit from the TGN and/or interaction of CSC secretory vesicles with the plasma membrane.

A receptor protein called TGN46 has an important role in sorting secretory proteins into vesicles going to different destinations inside cells.

The Golgi is surrounded by a ribosome-excluding matrix. Recently, we reported that the cis-Golgi-localized golgin GM130 can phase-separate to form dynamic, liquid-like condensates in vitro and in vivo. Here, we show that the overexpression of each of the remaining cis (golgin160, GMAP210)- and trans (golgin97, golgin245, GCC88, GCC185)-golgins results in novel protein condensates. Focused ion beam scanning electron microscopy (FIB-SEM) images of GM130 condensates reveal a complex internal organization with branching aqueous channels. Pairs of golgins overexpressed in the same cell form distinct juxtaposed condensates. These findings support the hypothesis that, in addition to their established roles as vesicle tethers, phase separation may be a common feature of the golgin family that contributes to Golgi organization.

The plant trans-Golgi Network/Early Endosome (TGN/EE), as an organizer of vesicle trafficking, fulfills a crucial role for plant development and adaptation. Because it coordinates the transport of cell material along different routes, it is expected that a number of TGN/EE associated factors function in the rapid organization of post-Golgi trafficking to ensure that proteins reach their destination. The roles of Transport Protein Particle (TRAPP) complexes in the regulation of plant post-Golgi trafficking start to emerge. We previously demonstrated that the plant TRAPPIII complex is involved in maintenance of TGN organization and function and has a role in endocytic trafficking mediated by the SYP61 TGN/EE compartment. Here we show that attrappc11 mutants display accumulation of the plasma membrane resident proteins CESA6, BRI1 and PIP1;4 in aberrant intracellular compartments. This adds further insights into the functions of TRAPPIII as a regulators of post-Golgi/endosomal traffic.

Early endosomes, also called sorting endosomes, are known to mature into late endosomes via the Rab5-mediated endolysosomal trafficking pathway. Thus, early endosome existence is thought to be maintained by the continual fusion of transport vesicles from the plasma membrane and the trans-Golgi network (TGN). Here we show instead that endocytosis is dispensable and post-Golgi vesicle transport is crucial for the formation of endosomes and the subsequent endolysosomal traffic regulated by yeast Rab5 Vps21p. Fittingly, all three proteins required for endosomal nucleotide exchange on Vps21p are first recruited to the TGN before transport to the endosome, namely the GEF Vps9p and the epsin-related adaptors Ent3/5p. The TGN recruitment of these components is distinctly controlled, with Vps9p appearing to require the Arf1p GTPase, and the Rab11s, Ypt31p/32p. These results provide a different view of endosome formation and identify the TGN as a critical location for regulating progress through the endolysosomal trafficking pathway.

Clathrin coats drive transport vesicle formation from the plasma membrane and in pathways between the trans-Golgi network (TGN) and endosomes. Clathrin adaptors play central roles orchestrating assembly of clathrin coats. The yeast clathrin adaptor-interacting protein Irc6 is an orthologue of human p34, which is mutated in the inherited skin disorder punctate palmoplantar keratoderma type I. Irc6 and p34 bind to clathrin adaptor complexes AP-1 and AP-2 and are members of a conserved family characterized by a two-domain architecture. Irc6 is required for AP-1-dependent transport between the TGN and endosomes in yeast. Here we present evidence that the C-terminal two amino acids of Irc6 are required for AP-1 binding and transport function. Additionally, like the C-terminal domain, the N-terminal domain when overexpressed partially restores AP-1-mediated transport in cells lacking full-length Irc6. These findings support a functional role for Irc6 binding to AP-1. Negative genetic interactions with irc6∆ are enriched for genes related to membrane traffic and nuclear processes, consistent with diverse cellular roles for Irc6.

Protein kinase D (PKD) is a serine/threonine kinase family that controls important cellular functions, most notably playing a key role in the secretory pathway at the trans-Golgi network. Aberrant expression of PKD isoforms has been found mainly in breast cancer, where it promotes various cellular processes such as growth, invasion, survival and stem cell maintenance. In this review, we discuss the isoform-specific functions of PKD in breast cancer progression, with a particular focus on how the PKD controlled cellular processes might be linked to deregulated membrane trafficking and secretion. We further highlight the challenges of a therapeutic approach targeting PKD to prevent breast cancer progression.

Retrograde transport of WLS (Wntless) from endosomes to trans -Golgi network (TGN) is required for efficient Wnt secretion during development. However, the molecular players connecting endosomes to TGN during WLS trafficking are limited. Here, we identified a role for Eyes Absent (EYA) proteins during retrograde trafficking of WLS to TGN in human cell lines. By using worm, fly, and zebrafish models, we found that the EYA-secretory carrier-associated membrane protein 3 (SCAMP3) axis is evolved in vertebrates. EYAs form a complex and interact with retromer on early endosomes. Retromer-bound EYA complex recruits SCAMP3 to endosomes, which is necessary for the fusion of WLS-containing endosomes to TGN. Loss of EYA complex or SCAMP3 leads to defective transport of WLS to TGN and failed Wnt secretion. EYA mutations found in patients with hearing loss form a dysfunctional EYA-retromer complex that fails to activate Wnt signaling. These findings identify the EYA complex as a component of retrograde trafficking of WLS from the endosome to TGN. [Display omitted] • EYA phosphatase complex associates with the retromer complex • EYA complex recruits SCAMP3 to sorting endosomes and connects it to retromer • Retromer-EYA-SCAMP3 is essential for Wntless trafficking from endosome to TGN • EYA-SCAMP3 axis is an evolved mechanism in vertebrates Reshi et al. demonstrate that the EYA protein complex bridges retromer on endosomes with SCAMP3 vesicles and is necessary for retrograde transport of Wntless from endosomes to the Golgi network. This study helps gain insight into the role of EYA proteins in Wnt signaling and organ development in vertebrates. [ABSTRACT FROM AUTHOR]

In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical-basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 (Fzd6), are located asymmetrically on opposite boundaries of the cell. Examining sorting of these two proteins at the trans-Golgi network (TGN), we demonstrated previously that the GTP-binding protein ADP-ribosylation factor-related protein 1 (Arfrp1) and the clathrin-associated adaptor protein complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled6 does not depend on Arfrp1 or AP-1. Here, to further investigate the TGN sorting process in mammalian cells, we reconstituted release of Vangl2 and Frizzled6 from the TGN into vesicles in vitro Immunoblotting of released vesicles indicated that Vangl2 and Frizzled6 exit the TGN in separate compartments. Knockdown analysis revealed that a clathrin adaptor, epsinR, regulates TGN export of Frizzled6 but not of Vangl2. Protein interaction analysis suggested that epsinR forms a stable complex with clathrin and that this complex interacts with a conserved polybasic motif in the Frizzled6 cytosolic domain to package Frizzled6 into transport vesicles. Moreover, we found that Frizzled6-epsinR binding dissociates epsinR from AP-1, which may separate these two cargo adaptors from each other to perform distinct cargo-sorting functions. Our results suggest that Vangl2 and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations.

The cell wall, a complex macromolecular composite structure surrounding and protecting plant cells, is essential for development, signal transduction, and disease resistance. This structure is also integral to cell expansion, as its tensile resistance is the primary balancing mechanism against internal turgor pressure. Throughout these processes, the biosynthesis, transport, deposition, and assembly of cell wall polymers are tightly regulated. The plant endomembrane system facilitates transport of polysaccharides, polysaccharide biosynthetic and modifying enzymes and glycoproteins through vesicle trafficking pathways. Although a number of enzymes involved in cell wall biosynthesis have been identified, comparatively little is known about the transport of cell wall polysaccharides and glycoproteins by the endomembrane system. This review summarizes our current understanding of trafficking of cell wall components during cell growth and cell division. Emerging technologies, such as vesicle glycomics, are also discussed as promising avenues to gain insights into the trafficking of structural polysaccharides to the apoplast.

The small G protein Arl5b is localised on the trans‐Golgi network (TGN) and regulates endosomes‐to‐TGN transport. Here, we combined in vivo and in vitro techniques to map the interactive partners and near neighbours of Arl5b at the TGN, using constitutively active, membrane‐bound Arl5b(Q70L)‐GFP in stably expressing HeLa cells, and the proximity labelling techniques BioID and APEX2 in parallel with GFP‐Trap pull down. From MS analysis, 22 Golgi proteins were identified; 50% were TGN‐localised Rabs, Arfs and Arls. The scaffold/tethering factors ACBD3 (GCP60) and PIST (GOPC) were also identified, and we show that Arl5b is required for TGN recruitment of ACBD3. Overall, the combination of in vivo labelling and direct pull downs indicates a highly organised complex of small G proteins on TGN membranes. [ABSTRACT FROM AUTHOR]

Cytokinesis in plants is fundamentally different from that in animals and fungi. In plant cells, a cell plate forms through the fusion of cytokinetic vesicles and then develops into the new cell wall, partitioning the cytoplasm of the dividing cell. The formation of the cell plate entails multiple stages that involve highly orchestrated vesicle accumulation, fusion and membrane maturation, which occur concurrently with the timely deposition of polysaccharides such as callose, cellulose and cross‐linking glycans. This review summarizes the major stages in cytokinesis, endomembrane components involved in cell plate assembly and its transition to a new cell wall. An animation that can be widely used for educational purposes further summarizes the process. [ABSTRACT FROM AUTHOR]

Endocytosis, secretion, and endosomal trafficking are key cellular processes that control the composition of the plasma membrane. Through the coordination of these trafficking pathways, cells can adjust the composition, localization, and turnover of proteins and lipids in response to developmental or environmental cues. Upon being incorporated into vesicles and internalized through endocytosis, plant plasma membrane proteins are delivered to the trans‐Golgi network (TGN). At the TGN, plasma membrane proteins are recycled back to the plasma membrane or transferred to multivesicular endosomes (MVEs), where they are further sorted into intralumenal vesicles for degradation in the vacuole. Both types of plant endosomes, TGN and MVEs, act as sorting organelles for multiple endocytic, recycling, and secretory pathways. Molecular assemblies such as retromer, ESCRT (endosomal sorting complex required for transport) machinery, small GTPases, adaptor proteins, and SNAREs associate with specific domains of endosomal membranes to mediate different sorting and membrane‐budding events. In this review, we discuss the mechanisms underlying the recognition and sorting of proteins at endosomes, membrane remodeling and budding, and their implications for cellular trafficking and physiological responses in plants. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) is caused by the infection of a coronavirus, named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses can be replicated in the infected host cells. Coronavirus replication involves various steps, including membrane fusion, peri-nuclear particle formation, and matrix vesicle transport to the cell membrane via the endoplasmic reticulum-Golgi-lysosome route. Recent studies have suggested that protein kinase D (PKD) plays a crucial role in regulation of vesicle formation and trafficking in the trans-Golgi network (TGN). Thus, we hypothesize that inhibiting PKD and its associated pathway could be an effective strategy to limit viral replication. Here, we report that molecular and pharmacological inhibition of PKD and its substrate phosphatidylinositol-4 kinase III beta (PI4KIIIβ) significantly diminishes the replication of common human coronaviruses. Specifically, we found that the PKD-silencing siRNA and the PKD inhibitor CRT0066101 have broad-spectrum antiviral activity against HCoV-OC43, HCoV-NL63, and HCoV-229E in cultured cells. Mechanistic studies revealed that the deactivation of PKD reduced the activation of PI4KIIIβ, thereby blocking the transport of viral particles in the host cells. Furthermore, the PI4KIIIβ inhibitor, BQR695, also exhibited antiviral activity against those coronaviruses. In conclusion, PKD and its substrate, PI4KIIIβ, may serve as novel antiviral targets for human coronaviruses and warrant further investigation., Importance: Human coronaviruses can lead to a range of clinical symptoms, from asymptomatic infection to severe illness and death, with a limited array of antiviral drugs available. Protein kinase D (PKD) is involved in various cellular processes, such as cell proliferation, apoptosis, and membrane fission of the Golgi apparatus. However, the specific role of PKD in the human coronavirus life cycle remains unclear. In this study, we found that PKD inhibitors effectively attenuated human coronavirus replication at the trans-Golgi network (TGN) stage in the viral life cycle. Furthermore, inhibiting PKD reduced PI4KIIIβ activation, thereby blocking viral replication in the host cells. Importantly, PI4KIIIβ inhibitors also blocked human coronavirus replication. Thus, PKD may represent a promising therapeutic target against both current circulating and future emerging coronaviruses.

Rab GTPases control intracellular vesicular transport, including retrograde trafficking of human papillomavirus (HPV) during cell entry, guiding the virus from the endosome to the trans- Golgi network (TGN), the Golgi apparatus, and eventually the nucleus. Rab proteins have been identified that act prior to the arrival of HPV at the TGN, but Rab proteins operating in later stages of entry remain elusive. Here, we report that knockdown of Rab6a impairs HPV entry by preventing HPV exit from the TGN and impeding intra-Golgi transport of the incoming virus. Rab6a supports HPV trafficking by facilitating the association of HPV with dynein, a motor protein complex, and BICD2, a dynein adaptor, in the TGN. L2 can bind directly to GTP-Rab6a in vitro , and excess of either GTP-Rab6a or GDP-Rab6 inhibits HPV entry, suggesting that cycling between GDP-Rab6 and GTP-Rab6 is critical. Notably, Rab6a is crucial for HPV-BICD2 and HPV-dynein association in the TGN of infected cells but not in the endosome. Our findings reveal important features of the molecular basis of HPV infection, including the discovery that HPV uses different mechanisms to engage dynein at different times during entry, and identify potential targets for therapeutic approaches to inhibit HPV infection., Importance: Human papillomaviruses (HPVs) are small, non-enveloped DNA viruses that cause approximately 5% of human cancer. Like most other DNA viruses, HPV traffics to the nucleus during virus entry to successfully infect cells. We show here that HPV utilizes a cellular enzyme, Rab6a, during virus entry to engage the dynein molecular motor for transport along microtubules. Rab6a is required for complex formation between the HPV L2 capsid protein, dynein, and the dynein adaptor BICD2 in the trans -Golgi network (TGN). This complex is required for transport of the incoming virus out of the TGN as it journeys to the nucleus. Our findings identify potential targets for therapeutic approaches., Competing Interests: The authors declare no conflict of interest.

Exomer is a protein complex that facilitates trafficking between the Golgi and the plasma membrane (PM). Schizosaccharomyces pombe exomer is composed of Cfr1 and Bch1, and we have found that full activation of the cell integrity pathway (CIP) in response to osmotic stress requires exomer. In the wild-type, the CIP activators Rgf1 (Rho1 GEF) and Pck2 (PKC homologue) and the MEK kinase Mkh1 localize in the PM, internalize after osmotic shock and re-localize after adaptation. This re-localization is inefficient in exomer mutants. Overexpression of the PM-associated 1-phosphatidylinositol 4-kinase stt4+ , and deletion of the nem1+ phosphatase suppress the defects in Pck2 dynamics in exomer mutants, but not their defect in CIP activation, demonstrating that exomer regulates CIP in additional ways. Exomer mutants accumulate PI4P in the TGN, and increasing the expression of the Golgi-associated 1-phosphatidylinositol 4-kinase pik1+ suppresses their defect in Pck2 dynamics. These findings suggest that efficient PI4P transport from the Golgi to the PM requires exomer. Mutants lacking clathrin adaptors are defective in CIP activation, but not in Pck2 dynamics or in PI4P accumulation in the Golgi. Hence, traffic from the Golgi regulates CIP activation, and exomer participates in this regulation through an exclusive mechanism.

Membrane trafficking contributes to distinct protein compositions of organelles and is essential for proper organellar maintenance and functions. The trans-Golgi network (TGN) acts as a sorting station where various cargo proteins are sorted and directed to post-Golgi compartments, such as the multivesicular body or pre-vacuolar compartment, vacuoles, and plasma membrane. The spatial and temporal segregation of cargo proteins within the TGN, which is mediated with different sets of regulators including small GTPases and cargo adaptors, is a fundamental process in the sorting machinery. Recent studies with powerful imaging technologies have suggested that the TGN possesses spatially distinct subdomains or zones for different trafficking pathways. In this review, we will summarize the spatially and dynamically characteristic features of the plant TGN and their relation to cargo protein trafficking. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) exhibits a complex host-pathogen interaction with peripheral blood monocytes. We have identified a unique, cell-type specific retrograde-like intracellular trafficking pattern that HCMV utilizes to gain access to the monocyte nucleus and for productive infection. We show that infection of primary human monocytes, epithelial cells, and fibroblasts leads to an increase in the amount of the trafficking protein Syntaxin 6 (Stx6). However, only knockdown (KD) of Stx6 in monocytes inhibited viral trafficking to the trans-Golgi network (TGN), a requisite step for nuclear translocation in monocytes. Conversely, KD of Stx6 in epithelial cells and fibroblasts did not change the kinetics of nuclear translocation and productive infection. Stx6 predominantly functions at the level of the TGN where it facilitates retrograde transport, a trafficking pathway used by only a few cellular proteins and seldom by pathogens. We also newly identify that in monocytes, Stx6 exhibits an irregular vesicular localization rather than being concentrated at the TGN as seen in other cell-types. Lastly, we implicate that viral particles that associate with both Stx6 and EEA1 early in infection are the viral population that successfully traffics to the TGN at later time points and undergo nuclear translocation. Additionally, we show for the first time that HCMV enters the TGN, and that lack of Stx6 prevents viral trafficking to this organelle. We argue that we have identified an essential cell-type specific regulator that controls early steps in efficient productive infection of a cell-type required for viral persistence and disease. IMPORTANCE Human cytomegalovirus (HCMV) infection causes severe and often fatal disease in the immunocompromised. It is one of the leading infectious causes of birth defects and causes severe complications in transplant recipients. By uncovering the unique pathways used by the virus to infect key cells, such as monocytes, responsible for dissemination and persistence, we provide new potential targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Main conclusion: AtFTCD-L protein is localized on the TGN vesicles in Arabidopsis root cap cells. AtFTCD-L mutation resulted in slow root growth of Arabidopsis in high-concentration agar culture medium. Arabidopsis formiminotransferase cyclodeaminase-like protein (AtFTCD-L) in Arabidopsis is homologous to the formiminotransferase cyclodeaminase (FTCD) protein in animal cells. However, the localization and function of AtFTCD-L remain unknown in Arabidopsis. In this study, we generated and analyzed a deletion mutant of AtFTCD-L with a T-DNA insertion. We found that the growth of Arabidopsis roots with the T-DNA insertion mutation in AtFTCD-L was slower than that of wild-type roots when grown in high-concentration 1/2 MS agar culture medium. AtFTCD-L-GFP could restore the ftcd-l mutant phenotype. In addition, the AtFTCD-L protein was localized on the trans-Golgi network (TGN) vesicles in Arabidopsis root cap cells. Fluorescence recovery after photobleaching (FRAP) experiment using Arabidopsis pollen-specific receptor-like kinase-GFP (AtPRK1-GFP) stably transformed plants showed that the deficiency of AtFTCD-L protein in Arabidopsis led to slower secretion in the root cap peripheral cells. The AtFTCD-L protein deficiency also resulted in a significantly reduced monosaccharides content in the culture medium. Based on the above results, we speculate that the AtFTCD-L protein may be involved in sorting and/or transportation of TGN vesicles in root cap peripheral cells, thereby regulating the extracellular secretion of mucilage components in the root cap. [ABSTRACT FROM AUTHOR]

Transition metals are essential for a wealth of metabolic reactions, but their concentrations need to be tightly controlled across cells and cell compartments, as metal excess or imbalance has deleterious effects. Metal homeostasis is achieved by a combination of metal transport across membranes and metal binding to a variety of molecules. Gene duplication is a key process in evolution, as the emergence of advantageous mutations on one of the copies can confer a new function. Here, we report that the poplar genome contains two paralogues encoding NRAMP3 metal transporters localized in tandem. All Populus species analyzed had two copies of NRAMP3 , whereas only one could be identified in Salix species indicating that duplication occurred when the two genera separated. Both copies are under purifying selection and encode functional transporters, as shown by expression in the yeast heterologous expression system. However, genetic complementation revealed that only one of the paralogues has retained the original function in the release of metals stored in the vacuole previously characterized in Arabidopsis thaliana. Confocal imaging showed that the other copy has acquired a distinct localization to the Trans-Golgi Network (TGN). Expression in poplar suggested that the copy of NRAMP3 localized on the TGN has a novel function in the control of cell-to-cell transport of manganese. This work provides a clear case of neofunctionalization through a change in the subcellular localization of a metal transporter as well as evidence for the involvement of the secretory pathway in the cell-to-cell transport of manganese. [ABSTRACT FROM AUTHOR]

Yeast Vps13 is a member of a conserved protein family that includes human homologues associated with neurodegenerative and developmental disorders. In this issue, De et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201606078) establish direct roles for Vps13 and its surprising binding partner, the calcium-binding centrin Cdc31, in trans-Golgi network (TGN) to endosome traffic and TGN homotypic fusion.

The recently uncovered role of Fukutin‐related protein (FKRP) in fibronectin glycosylation has challenged our understanding of the basis of disease pathogenesis in the muscular dystrophies. FKRP is a Golgi‐resident glycosyltransferase implicated in a broad spectrum of muscular dystrophy (MD) pathologies that are not fully attributable to the well‐described α‐Dystroglycan hypoglycosylation. By revealing a new role for FKRP in the glycosylation of fibronectin, a modification critical for the development of the muscle basement membrane (MBM) and its associated muscle linkages, new possibilities for understanding clinical phenotype arise. This modification involves an interaction between FKRP and myosin‐10, a protein involved in the Golgi organization and function. These observations suggest a FKRP nexus exists that controls two critical aspects to muscle fibre integrity, both fibre stability at the MBM and its elastic properties. This review explores the new potential disease axis in the context of our current knowledge of muscular dystrophies. [ABSTRACT FROM AUTHOR]

Membrane traffic is a fundamental cellular system to exchange proteins and membrane lipids among single membrane-bound organelles or between an organelle and the plasma membrane in order to keep integrity of the endomembrane system. RAB GTPases and SNARE proteins, the key regulators of membrane traffic, are conserved broadly among eukaryotic species. However, genome-wide analyses showed that organization of RABs and SNAREs that regulate the post-Golgi transport pathways is greatly diversified in plants compared to other model eukaryotes. Furthermore, some organelles acquired unique properties in plant lineages. Like in other eukaryotic systems, the trans-Golgi network of plants coordinates secretion and vacuolar transport; however, uniquely in plants, it also acts as a platform for endocytic transport and recycling. In this review, we focus on RAB GTPases and SNAREs that function at the TGN, and summarize how these regulators perform to control different transport pathways at the plant TGN. We also highlight the current knowledge of RABs and SNAREs' role in regulation of plant development and plant responses to environmental stimuli. [ABSTRACT FROM AUTHOR]

The Golgi apparatus represents a central compartment of membrane traffic. Its apparent architecture, however, differs considerably among species, from unstacked and scattered cisternae in the budding yeast Saccharomyces cerevisiae to beautiful ministacks in plants and further to gigantic ribbon structures typically seen in mammals. Considering the well-conserved functions of the Golgi, its fundamental structure must have been optimized despite seemingly different architectures. In addition to the core layers of cisternae, the Golgi is usually accompanied by next-door compartments on its cis and trans sides. The trans-Golgi network (TGN) can be now considered as a compartment independent from the Golgi stack. On the cis side, the intermediate compartment between the ER and the Golgi (ERGIC) has been known in mammalian cells, and its functional equivalent is now suggested for yeast and plant cells. High-resolution live imaging is extremely powerful for elucidating the dynamics of these compartments and has revealed amazing similarities in their behaviors, indicating common mechanisms conserved along the long course of evolution. From these new findings, I would like to propose reconsideration of compartments and suggest a new concept to describe their roles comprehensively around the Golgi and in the post-Golgi trafficking.

Neurotensin (NT) triggers signaling in human colonic epithelial cells by activating the G protein-coupled receptor, the neurotensin receptor 1 (NTR1). Activated NTR1 traffics from the plasma membrane to early endosomes, and then recycles. Although sustained NT/NTR1 signaling requires efficient NTR1 recycling, little is known about the regulation of NTR1 recycling. We recently showed that NT/NTR1 signaling increases expression of miR-133α. Herein, we studied the mechanism of NT-regulated miR-133α expression and examined the role of miR-133α in intracellular NTR1 trafficking in human NCM460 colonocytes. We found that NT-induced miR-133α upregulation involves the negative transcription regulator, zinc finger E-box binding homeobox 1. Silencing of miR-133α or overexpression of aftiphilin (AFTPH), a binding target of miR-133α, attenuated NTR1 trafficking to plasma membrane in human colonocytes, without affecting NTR1 internalization. We localized AFTPH to early endosomes and the trans-Golgi network (TGN) in unstimulated human colonic epithelial cells. AFTPH overexpression reduced NTR1 localization in early endosomes and increased expression of proteins related to endosomes and the TGN trafficking pathway. AFTPH overexpression and de-acidification of intracellular vesicles increased NTR1 expression. Our results suggest a novel mechanism of GPCR trafficking in human colonic epithelial cells by which a microRNA, miR-133α regulates NTR1 trafficking through its downstream target AFTPH.

Key message: Arabidopsis PLDζ1 and PLDζ2 localize to the trans-Golgi network and to compartments including the trans-Golgi network, multi-vesicular bodies, and the tonoplast, respectively, depending on their N-terminal regions containing PX-PH domains. Phospholipase D (PLD) is involved in dynamic cellular processes, including membrane trafficking, cytoskeletal reorganization, and signal transduction for gene expression, through the production of phosphatidic acid in membrane compartments specific to each process. Although PLD plays crucial roles in various plant phenomena, the underlying processes involving PLD for each phenomenon remain largely elusive, partly because the subcellular localization of PLD remains obscure. In this study, we performed comparative subcellular localization analyses of the Arabidopsis thaliana PX-PH-PLDs PLDζ1 and PLDζ2. In mature lateral root cap cells, own promoter-driven fluorescence protein fusions of PLDζ1 localized to the entire trans-Golgi network (TGN) while that of PLDζ2 localized to punctate structures including part of the TGN and multi-vesicular bodies as well as the tonoplast. These localization patterns were reproduced using N-terminal partial proteins, which contain PX-PH domains. An inducibly overexpressed fluorescence protein fusion of the PLDζ2 partial protein first localized to punctate structures, and then accumulated predominantly on the tonoplast. Further domain dissection analysis revealed that the N-terminal moiety preceding the PX-PH domain of PLDζ2 was required for the tonoplast-predominant accumulation. These findings suggest that PLDζ1 and PLDζ2 play partially overlapping but nonetheless distinctive roles in post-Golgi compartments along the membrane trafficking pathway from the TGN to the tonoplast. [ABSTRACT FROM AUTHOR]

The trans-Golgi network (TGN) and recycling endosome (RE) have been recognized as sorting centers, the former for newly synthesized and the latter for endocytosed proteins. However, recent findings have revealed that TGN also receives endocytosed materials and RE accepts newly synthesized proteins destined to the plasma membrane. Recently, we reported that in both Drosophila and microtubule-disrupted HeLa cells, REs are associated with the trans-side of Golgi stacks. REs are highly dynamic: their separation from and association with Golgi stacks are often observed. Importantly, a newly synthesized cargo, glycosylphosphatidylinositol-anchored-GFP was found to be concentrated in Golgi-associated REs (GA-REs), while another cargo VSVG-GFP was excluded from GA-REs before post-Golgi trafficking to the plasma membrane. This suggested that the sorting of cargos takes place at the interface of Golgi stacks and GA-REs. In this study, we demonstrated that REs could associate with Golgi stacks in sea urchin embryos, further indicating that the association of REs with Golgi stacks is a well-conserved phenomenon in the animal kingdom.

Plants maintain nutrient homeostasis by controlling the activities and abundance of nutrient transporters. In Arabidopsis thaliana, the borate (B) transporter BOR1 plays a role in the efficient translocation of B under low-B conditions. BOR1 undergoes polyubiquitination in the presence of sufficient B and is then transported to the vacuole via multivesicular bodies (MVBs) to prevent B accumulation in tissues at a toxic level. A previous study indicated that BOR1 physically interacts with µ subunits of adaptor protein complexes AP-3 and AP-4, both involved in vacuolar sorting pathways. In this study, we investigated the roles of AP-3 and AP-4 subunits in BOR1 trafficking in Arabidopsis. The lack of AP-3 subunits did not affect either vacuolar sorting or polar localization of BOR1-GFP, whereas the absence of AP-4 subunits resulted in a delay in high-B-induced vacuolar sorting without affecting polar localization. Super-resolution microscopy revealed a rapid sorting of BOR1-GFP into AP-4-positive spots in the trans-Golgi network (TGN) upon high-B supply. These results indicate that AP-4 is involved in sequestration of ubiquitinated BOR1 into a TGN-specific subdomain "vacuolar-trafficking zone," and is required for efficient sorting to MVB and vacuole. Our findings elucidate the rapid vacuolar sorting process facilitated by AP-4 in plant nutrient transporters., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

The plant cell wall provides a strong yet flexible barrier to protect cells from the external environment. Modifications of the cell wall, either during development or under stress conditions, can induce cell wall integrity responses and ultimately lead to alterations in gene expression, hormone production, and cell wall composition. These changes in cell wall composition presumably require remodelling of the secretory pathway to facilitate synthesis and secretion of cell wall components and cell wall synthesis/remodelling enzymes from the Golgi apparatus. Here, we used a combination of live-cell confocal imaging and transmission electron microscopy to examine the short-term and constitutive impact of isoxaben, which reduces cellulose biosynthesis, and Driselase, a cocktail of cell-wall-degrading fungal enzymes, on cellular processes during cell wall integrity responses in Arabidopsis. We show that both treatments altered organelle morphology and triggered rebalancing of the secretory pathway to promote secretion while reducing endocytic trafficking. The actin cytoskeleton was less dynamic following cell wall modification, and organelle movement was reduced. These results demonstrate active remodelling of the endomembrane system and actin cytoskeleton following changes to the cell wall., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

In neuronal cells, many membrane receptors interact via their intracellular, C-terminal tails with PSD-95/discs large/ZO-1 (PDZ) domain proteins. Some PDZ proteins act as scaffold proteins. In addition, there are a few PDZ proteins such as Gopc which bind to receptors during intracellular transport. Gopc is localized at the trans-Golgi network (TGN) and binds to a variety of receptors, many of which are eventually targeted to postsynaptic sites. We have analyzed the role of Gopc by knockdown in primary cultured neurons and by generating a conditional Gopc knockout (KO) mouse line. In neurons, targeting of neuroligin 1 (Nlgn1) and metabotropic glutamate receptor 5 (mGlu5) to the plasma membrane was impaired upon depletion of Gopc, whereas NMDA receptors were not affected. In the hippocampus and cortex of Gopc KO animals, expression levels of Gopc-associated receptors were not altered, while their subcellular localization was disturbed. The targeting of mGlu5 to the postsynaptic density was reduced, coinciding with alterations in mGluR-dependent synaptic plasticity and deficiencies in a contextual fear conditioning paradigm. Our data imply Gopc in the correct subcellular sorting of its associated mGlu5 receptor in vivo. [ABSTRACT FROM AUTHOR]

SUMMARY: The plant hormone auxin controls many aspects of plant development. Membrane trafficking processes, such as secretion, endocytosis and recycling, regulate the polar localization of auxin transporters in order to establish an auxin concentration gradient. Here, we investigate the function of the Arabidopsis thaliana R‐SNAREs VESICLE‐ASSOCIATED MEMBRANE PROTEIN 721 (VAMP721) and VAMP722 in the post‐Golgi trafficking required for proper auxin distribution and seedling growth. We show that multiple growth phenotypes, such as cotyledon development, vein patterning and lateral root growth, were defective in the double homozygous vamp721 vamp722 mutant. Abnormal auxin distribution and root patterning were also observed in the mutant seedlings. Fluorescence imaging revealed that three auxin transporters, PIN‐FORMED 1 (PIN1), PIN2 and AUXIN RESISTANT 1 (AUX1), aberrantly accumulate within the cytoplasm of the double mutant, impairing the polar localization at the plasma membrane (PM). Analysis of intracellular trafficking demonstrated the involvement of VAMP721 and VAMP722 in the endocytosis of FM4‐64 and the secretion and recycling of the PIN2 transporter protein to the PM, but not its trafficking to the vacuole. Furthermore, vamp721 vamp722 mutant roots display enlarged trans‐Golgi network (TGN) structures, as indicated by the subcellular localization of a variety of marker proteins and the ultrastructure observed using transmission electron microscopy. Thus, our results suggest that the R‐SNAREs VAMP721 and VAMP722 mediate the post‐Golgi trafficking of auxin transporters to the PM from the TGN subdomains, substantially contributing to plant growth. [ABSTRACT FROM AUTHOR]

The rapid formation and activation of the NLRP3 inflammasome is induced by co‐stimulation with LPS and nigericin. It requires the LPS‐stimulated activation of IKKβ, which exerts its effects independently of de novo gene transcription, protein translation and other protein kinases activated by IKKβ. IKKβ is not required for the nigericin‐induced dispersion of the trans‐Golgi network (TGN), but to bring NLRP3 in proximity with TGN38. The nigericin‐induced dispersion of the Golgi is enhanced by co‐stimulation with LPS, and this enhancement is IKKβ‐dependent. Prolonged stimulation with LPS to increase the expression of NLRP3, followed by stimulation with nigericin, produced larger TGN38‐positive puncta, and the ensuing activation of the NLRP3 inflammasome was also suppressed by IKKβ inhibitors added prior to stimulation with nigericin. IKKβ therefore has a key role in recruiting NLRP3 to the dispersed TGN, leading to the formation and activation of the NLRP3 inflammasome. Synopsis: Formation of the NLRP3 inflammasome requires LPS to activate IKKβ and nigericin to disperse the trans‐Golgi network (TGN). IKKβ functions to recruit NLRP3 to the dispersed TGN promoting inflammasome formation and activation. The rapid formation of the NLRP3 inflammasome requires co‐stimulation with LPS and nigericin.LPS activates the protein kinase IKKβ, while nigericin triggers dispersion of the trans‐Golgi network independent of IKKβ.IKKβ activity is needed to recruit NLRP3 to the dispersed TGN.TGN recruitment is followed by inflammasome formation, caspase 1 and gasdermin D activation and IL‐1/‐18 secretion. [ABSTRACT FROM AUTHOR]

The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat.

Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking.

Humans are usually exposed to nicotine through the use of tobacco products. Although it is generally believed that nicotine is relatively harmless in tobacco consumption, it is, in fact, a toxic substance that warrants careful consideration of its potential toxicity. However, the current understanding of the neurotoxicity of nicotine is still very limited. In this study, we aim to reveal the toxic risk of nicotine to key target neuronal cells and its potential toxic mechanisms. The results showed that nicotine induced cell death, ROS increase, mitochondrial membrane potential decrease, and DNA damage in SH-SY5Y human neuroblastoma cells at millimolar concentrations, but did not cause toxic effects at the physiological concentration. These toxic effects were accompanied by cytoplasmic vacuolation. The inhibition of cytoplasmic vacuolation by bafilomycin A1 greatly reduced nicotine-induced cell death, indicating that cytoplasmic vacuolation is the key driving factor of cell death. These cytoplasmic vacuoles originated from the trans-Golgi network (TGN) and expressed microtubule-associated protein 1 light chain 3-II (LC3-II) and lysosomal associated membrane protein 1(LAMP1). The presence of LC3-II and LAMP1 within these vacuoles serves as evidence of compromised TGN structure and function. These findings provide valuable new insights into the potential neurotoxic risk and mechanisms of nicotine. [ABSTRACT FROM AUTHOR]

The relation of organelle size to cellular function is a basic question in cell biology about which almost nothing is known. Reporting in this issue of Developmental Cell, Ferraro et al. (2014) show that the size and topology of the Golgi apparatus determines the size and functionality of a medically important secretory granule.

DNA is present in the nucleus and mitochondria of eukaryotic cells. There are, however, certain instances in which DNA emerges in the cytosol. The two major sources of cytosolic DNA are self DNA that is leaked out from the nucleus or mitochondria, and non-self DNA from DNA viruses. The cytosolic DNA triggers the host immune response. Recent studies have identified two key molecules, cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of interferon genes (STING) in this immune response. STING is an endoplasmic reticulum (ER) protein. After STING binding to cGAMP, STING exits the ER and translocates to the Golgi, where STING triggers the type I interferon- and proinflammatory responses through the activation of interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB). STING also activates other cellular responses including cell senescence, autophagy, and cell death. In this review, we focus on emerging issues regarding the regulation of STING by membrane traffic, with a particular focus on the retrograde membrane traffic from the Golgi to the ER. The retrograde membrane traffic is recently shown by us and others to be critical for silencing the STING signaling pathway and the defect in this traffic underlies the pathogenesis of the COPA syndrome, a monogenic autoinflammatory disease caused by missense mutations of coatomer protein complex subunit α (COP-α).

Objective: Hormone secretion from metabolically active tissues, such as pancreatic islets, is governed by specific and highly regulated signaling pathways. Defects in insulin secretion are among the major causes of diabetes. The molecular mechanisms underlying regulated insulin secretion are, however, not yet completely understood. In this work, we studied the role of the GTPase ARFRP1 on insulin secretion from pancreatic β-cells. Methods: A β-cell-specific Arfrp1 knockout mouse was phenotypically characterized. Pulldown experiments and mass spectrometry analysis were employed to screen for new ARFRP1-interacting proteins. Co-immunoprecipitation assays as well as super-resolution microscopy were applied for validation. Results: The GTPase ARFRP1 interacts with the Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). Both proteins are co-localized at the trans-Golgi network and regulate the first and second phase of insulin secretion by controlling the plasma membrane localization of the SNARE protein SNAP25. Downregulation of both GOPC and ARFRP1 in Min6 cells interferes with the plasma membrane localization of SNAP25 and enhances its degradation, thereby impairing glucose-stimulated insulin release from β-cells. In turn, overexpression of SNAP25 as well as GOPC restores insulin secretion in islets from β-cell-specific Arfrp1 knockout mice. Conclusion: Our results identify a hitherto unrecognized pathway required for insulin secretion at the level of trans-Golgi sorting.