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Vesicles bud from maturing Golgi cisternae in a programmed sequence. Budding is mediated by adaptors that recruit cargoes and facilitate vesicle biogenesis. In Saccharomyces cerevisiae, the AP-3 adaptor complex directs cargoes from the Golgi to the lysosomal vacuole. The AP-3 core consists of small and medium subunits complexed with two non-identical large subunits, β3 (Apl6) and δ (Apl5). The C-termini of β3 and δ were thought to be flexible hinges linking the core to ear domains that bind accessory proteins involved in vesicular transport. We found by computational modeling that the yeast β3 and δ hinges are intrinsically disordered and lack folded ear domains. When either hinge is truncated, AP-3 is recruited to the Golgi, but vesicle budding is impaired and cargoes normally sorted into the AP-3 pathway are mistargeted. This budding deficiency causes AP-3 to accumulate on ring-like Golgi structures adjacent to GGA adaptors that, in wild-type cells, bud vesicles downstream of AP-3 during Golgi maturation. Thus, each of the disordered hinges of yeast AP-3 has a crucial role in mediating transport vesicle formation at the Golgi., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The unprecedented research effort associated with the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) included several extensive proteomic studies that identified host proteins that interact with individual viral gene products. However, in most cases, the consequences of those virus-host interactions for virus replication were not experimentally pursued, which is a necessary step in determining whether the interactions represent pro- or anti-viral events. One putative interaction commonly identified in multiple studies was between the host adaptor protein complex 3 (AP-3) subunit B1 (AP3B1) and the SARS-CoV-2 envelope protein (E). AP3B1 is one subunit of AP-3 required for the biogenesis of lysosomal-related organelles (LROs), and its function impacts important disease processes including inflammation and vascular health. Thus, interactions between AP3B1 and SARS-CoV-2 might influence the clinical outcomes of infection. To determine if AP3B1 has a role in the SARS-CoV-2 replication cycle, we first confirmed the interaction in virus-infected cells using immunoprecipitation (IP) and immunofluorescence assays (IFA). AP3B1 is required by multiple viruses to aid in the replication cycle and therefore may be a therapeutic target. However, we found that the overexpression of AP3B1 suppressed SARS-CoV-2 replication, whereas the siRNA-mediated depletion of AP3B1 increased the release of infectious virus, suggesting an antiviral role for AP3B1. Together, our findings suggest that AP3B1 is an intrinsic barrier to SARS-CoV-2 replication through interactions with the viral E protein. Our work justifies further investigations of LRO trafficking in SARS-CoV-2 target cells and their role in viral pathogenesis.

Synaptic vesicle proteins (SVps) are transported by the motor UNC-104/KIF1A. We show that SVps travel in heterogeneous carriers in C. elegans neuronal processes, with some SVp carriers co-transporting lysosomal proteins (SV-lysosomes). LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 play a critical role in the sorting of SVps and lysosomal proteins away from each other at the SV-lysosomal intermediate trafficking compartment. Both SVp carriers lacking lysosomal proteins and SV-lysosomes are dependent on the motor UNC-104/KIF1A for their transport. In lrk-1 mutants, both SVp carriers and SV-lysosomes can travel in axons in the absence of UNC-104, suggesting that LRK-1 plays an important role to enable UNC-104 dependent transport of synaptic vesicle proteins. Additionally, LRK-1 acts upstream of the AP-3 complex and regulates its membrane localization. In the absence of the AP-3 complex, the SV-lysosomes become more dependent on the UNC-104-SYD-2/Liprin-α complex for their transport. Therefore, SYD-2 acts to link upstream trafficking events with the transport of SVps likely through its interaction with the motor UNC-104. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. SYD-2 acts in concert with AP complexes to ensure polarized trafficking & transport of SVps., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Neural systems encode information in the frequency of action potentials, which is then decoded by synaptic transmission. However, the rapid, synchronous release of neurotransmitters depletes synaptic vesicles (SVs), limiting release at high firing rates. How then do synapses convey information about frequency? Here, we show in mouse hippocampal neurons and slices that the adaptor protein AP-3 makes a subset of SVs that respond specifically to high-frequency stimulation. Neurotransmitter transporters slot onto these SVs in different proportions, contributing to the distinct properties of release observed at different excitatory synapses. Proteomics reveals that AP-3 targets the phospholipid flippase ATP8A1 to SVs; loss of ATP8A1 recapitulates the defect in SV mobilization at high frequency observed with loss of AP-3. The mechanism involves recruitment of synapsin by the cytoplasmically oriented phosphatidylserine translocated by ATP8A1. Thus, ATP8A1 enables the subset of SVs made by AP-3 to release at high frequency., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The time course of signaling by peptide hormones, neural peptides, and other neuromodulators depends on their storage inside dense core vesicles (DCVs). Adaptor protein 3 (AP-3) assembles the membrane proteins that confer regulated release of DCVs and is thought to promote their trafficking from endosomes directly to maturing DCVs. We now find that regulated monoamine release from DCVs requires sorting nexin 5 (SNX5). Loss of SNX5 disrupts trafficking of the vesicular monoamine transporter (VMAT) to DCVs. The mechanism involves a role for SNX5 in retrograde transport of VMAT from endosomes to the TGN. However, this role for SNX5 conflicts with the proposed function of AP-3 in trafficking from endosomes directly to DCVs. We now identify a transient role for AP-3 at the TGN, where it associates with DCV cargo. Thus, retrograde transport from endosomes by SNX5 enables DCV assembly at the TGN by AP-3, resolving the apparent antagonism. A novel role for AP-3 at the TGN has implications for other organelles that also depend on this adaptor., (© 2022 Xu et al.)

Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.

Chronic obstructive pulmonary disease (COPD) is a common, complex disease and a major cause of morbidity and mortality. Although multiple genetic determinants of COPD have been implicated by genome-wide association studies (GWASs), the pathophysiological significance of these associations remains largely unknown. From a COPD protein-protein interaction network module, we selected a network path between two COPD GWAS genes for validation studies: FAM13A (family with sequence similarity 13 member A)-AP3D1-CTGF- TGFβ2. We find that TGFβ2, FAM13A, and AP3D1 (but not CTGF) form a cellular protein complex. Functional characterization suggests that this complex mediates the secretion of TGFβ2 through an AP-3 (adaptor protein 3)-dependent pathway, with FAM13A acting as a negative regulator by targeting a late stage of this transport that involves the dissociation of coat-cargo interaction. Moreover, we find that TGFβ2 is a transmembrane protein that engages the AP-3 complex for delivery to the late endosomal compartments for subsequent secretion through exosomes. These results identify a pathophysiological context that unifies the biological network role of two COPD GWAS proteins and reveal novel mechanisms of cargo transport through an intracellular pathway.

Long-term potentiation (LTP) and long-term depression (LTD) of excitatory neurotransmission are believed to be the neuronal basis of learning and memory. Both processes are primarily mediated by neuronal activity-induced transport of postsynaptic AMPA-type glutamate receptors (AMPARs). While AMPAR subunits and their specific phosphorylation sites mediate differential AMPAR trafficking, LTP and LTD could also occur in a subunit-independent manner. Thus, it remains unclear whether and how certain AMPAR subunits with phosphorylation sites are preferentially recruited to or removed from synapses during LTP and LTD. Using immunoblot and immunocytochemical analysis, we show that phosphomimetic mutations of the membrane-proximal region (MPR) in GluA1 AMPAR subunits affect the subunit-dependent endosomal transport of AMPARs during chemical LTD. AP-2 and AP-3, adaptor protein complexes necessary for clathrin-mediated endocytosis and late endosomal/lysosomal trafficking, respectively, are reported to be recruited to AMPARs by binding to the AMPAR auxiliary subunit, stargazin (STG), in an AMPAR subunit-independent manner. However, the association of AP-3, but not AP-2, with STG was indirectly inhibited by the phosphomimetic mutation in the MPR of GluA1. Thus, although AMPARs containing the phosphomimetic mutation at the MPR of GluA1 were endocytosed by a chemical LTD-inducing stimulus, they were quickly recycled back to the cell surface in hippocampal neurons. These results could explain how the phosphorylation status of GluA1-MPR plays a dominant role in subunit-independent STG-mediated AMPAR trafficking during LTD., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Critically ill coronavirus disease 2019 (COVID-19) is characterized by severe cytokine storms, a hyperinflammatory condition intimately related to the development of fatal outcomes. Why some individuals seem particularly vulnerable to severe cytokine storms is still unknown. Primary immunodeficiency (PID)-related genes are inherited factors that dysregulate host inflammatory responses to infection, especially hemophagocytic lymphohistiocytosis (HLH)-related genes, established as contributors to the development of excessive cytokine storms. We analyzed the association between PID gene variants with severe cytokine storms in COVID-19. We conducted whole-exome sequencing in 233 hospitalized COVID-19 patients and identified four PID gene (UNC13D, AP3B1, RNF168, DHX58) variants were significantly enriched in COVID-19 patients experiencing severe cytokine storms. The total percentage of COVID-19 patients with variants in UNC13D or AP3B1, two typical HLH genes, was dramatically higher in high-level cytokine group than in low-level group (33.3 vs. 5.7%, P < 0.001). Germline variants in UNC13D and AP3B1 were associated with the development of severe cytokine storms, fatal outcomes in COVID-19. These findings advance the understanding of individual susceptibility to severe cytokine storms and help optimize the current management of COVID-19., (© 2021. The Author(s).)

Lamellar bodies (LBs) are lysosome-related organelles (LROs) of surfactant-producing alveolar type 2 (AT2) cells of the distal lung epithelium. Trafficking pathways to LBs have been understudied but are likely critical to AT2 cell homeostasis given associations between genetic defects of endosome to LRO trafficking and pulmonary fibrosis in Hermansky Pudlak syndrome (HPS). Our prior studies uncovered a role for AP-3, defective in HPS type 2, in trafficking Peroxiredoxin-6 to LBs. We now show that the P4-type ATPase ATP8A1 is sorted by AP-3 from early endosomes to LBs through recognition of a C-terminal dileucine-based signal. Disruption of the AP-3/ATP8A1 interaction causes ATP8A1 accumulation in early sorting and/or recycling endosomes, enhancing phosphatidylserine exposure on the cytosolic leaflet. This in turn promotes activation of Yes-activating protein, a transcriptional coactivator, augmenting cell migration and AT2 cell numbers. Together, these studies illuminate a mechanism whereby loss of AP-3-mediated trafficking contributes to a toxic gain-of-function that results in enhanced and sustained activation of a repair pathway associated with pulmonary fibrosis., Competing Interests: The authors declare no competing interest.

The common view of art and science as polar opposites along the educational spectrum can sometimes mask the degree to which they inform one another. In fact, art can also serve as a way to foster interest in querying the natural world, ultimately allowing us to recruit highly creative individuals to join the scientific community. We have experienced firsthand how cellular processes, such as autophagy, which are not usually highlighted or described in detail in foundational cell biology textbooks, have served as an on-ramp for artists at the undergraduate and high school levels in the context of scientific research and science outreach, respectively. We discuss our experiences in this article and highlight the ways in which art's many dimensions are well-suited, not only for forging connections between scientists and their communities but also for encouraging creativity in the way scientists engage with visually and conceptually complex phenomena, such as autophagy. Abbreviations: AP-3: adaptor protein complex 3; Atg27: autophagy related protein 27; STEAM: science, technology, engineering, arts, and mathematics; STEM: science, technology, engineering and math.

Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 β1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1 Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1 -/- endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2 +- mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 β1, also the μ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8 -/- endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8., (Copyright© 2019 Ferrata Storti Foundation.)

Objective: To describe phenotypes, treatment response, and outcomes of autoimmunity targeting a synaptic vesicle coat protein, the neuronal (B2) form of adaptor protein-3 (AP3)., Methods: Archived serum and CSF specimens (from 616,025 screened) harboring unclassified synaptic antibodies mimicking amphiphysin-immunoglobulin G (IgG) on tissue-based indirect immunofluorescence assay (IFA) were re-evaluated for novel IgG staining patterns. Autoantigens were identified by western blot and mass spectrometry. Recombinant western blot and cell-binding assay (CBA) were used to confirm antigen specificity. Clinical data were obtained retrospectively., Results: Serum (10) and CSF (6) specimens of 10 patients produced identical IFA staining patterns throughout mouse nervous system tissues, most prominently in cerebellum (Purkinje neuronal perikarya, granular layer synapses, and dentate regions), spinal cord gray matter, dorsal root ganglia, and sympathetic ganglia. The antigen revealed by mass spectrometry analysis and confirmed by recombinant assays (western blot and CBA) was AP3B2 in all. Of 10 seropositive patients, 6 were women; median symptom onset age was 42 years (range 24-58). Clinical information was available for 9 patients, all with subacute onset and rapidly progressive gait ataxia. Neurologic manifestations were myeloneuropathy (3), peripheral sensory neuropathy (2), cerebellar ataxia (2), and spinocerebellar ataxia (2). Five patients received immunotherapy; none improved, but they did not worsen over the follow-up period (median 36 months; range 3-94). Two patients (both with cancer) died. One of 50 control sera was positive by western blot only (but not by IFA or CBA)., Conclusion: AP3B2 (previously named β-neuronal adaptin-like protein) autoimmunity appears rare, is accompanied by ataxia (sensory or cerebellar), and is potentially treatable., (Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Stomatal movement is essential for plant growth. This process is precisely regulated by various cellular activities in guard cells. F-actin dynamics and vacuole morphology are both involved in stomatal movement. The sorting of cargoes by clathrin adaptor protein (AP) complexes from the Golgi to the vacuole is critical for establishing a normal vacuole morphology. In this study, we demonstrate that the medium subunit of the AP3 complex (AP3M) binds to and severs actin filaments in vitro and that it participates in the sorting of cargoes (such as the sucrose exporter SUC4) to the tonoplast, and thereby regulates stomatal closure in Arabidopsis thaliana Defects in AP3 or SUC4 led to more rapid water loss and delayed stomatal closure, as well as hypersensitivity to drought stress. In ap3m mutants, the F-actin status was altered compared to the wild type, and the sorted cargoes failed to localize to the tonoplast. AP3M contains a previously unidentified F-actin binding domain that is conserved in AP3M homologs in both plants and animals. Mutations in the F-actin binding domain of AP3M abolished its F-actin binding activity in vitro, leading to an aberrant vacuole morphology and reduced levels of SUC4 on the tonoplast in guard cells. Our findings indicate that the F-actin binding activity of AP3M is required for the precise localization of AP3-dependent cargoes to the tonoplast and for the regulation of vacuole morphology in guard cells during stomatal closure., Competing Interests: The authors declare no conflict of interest.

Planar cell polarity (PCP) regulates coordinated cellular polarity among neighboring cells to establish a polarity axis parallel to the plane of the tissue. Disruption in PCP results in a range of developmental anomalies and diseases. A key feature of PCP is the polarized and asymmetric localization of several membrane PCP proteins, which is essential to establish the polarity axis to orient cells coordinately. However, the machinery that regulates the asymmetric partition of PCP proteins remains largely unknown. In the present study, we show Van gogh-like 2 (Vangl2) in early and recycling endosomes as made evident by colocalization with diverse endosomal Rab proteins. Vangl2 biochemically interacts with adaptor protein-3 complex (AP-3). Using short hairpin RNA knockdown, we found that Vangl2 subcellular localization was modified in AP-3-depleted cells. Moreover, Vangl2 membrane localization within the cochlea is greatly reduced in AP-3-deficient mocha mice, which exhibit profound hearing loss. In inner ears from AP-3-deficient mocha mice, we observed PCP-dependent phenotypes, such as misorientation and deformation of hair cell stereociliary bundles and disorganization of hair cells characteristic of defects in convergent extension that is driven by PCP. These findings demonstrate a novel role of AP-3-mediated sorting mechanisms in regulating PCP proteins.

Adaptor proteins contribute to the selection, differentiation and activation of natural killer T (NKT) cells, an innate(-like) lymphocyte population endowed with powerful immunomodulatory properties. Distinct from conventional T lymphocytes NKT cells preferentially home to the liver, undergo a thymic maturation and differentiation process and recognize glycolipid antigens presented by the MHC class I-like molecule CD1d on antigen presenting cells. NKT cells express a semi-invariant T cell receptor (TCR), which combines the Vα14-Jα18 chain with a Vβ2, Vβ7, or Vβ8 chain in mice and the Vα24 chain with the Vβ11 chain in humans. The avidity of interactions between their TCR, the presented glycolipid antigen and CD1d govern the selection and differentiation of NKT cells. Compared to TCR ligation on conventional T cells engagement of the NKT cell TCR delivers substantially stronger signals, which trigger the unique NKT cell developmental program. Furthermore, NKT cells express a panoply of primarily inhibitory NK cell receptors (NKRs) that control their self-reactivity and avoid autoimmune activation. Adaptor proteins influence NKT cell biology through the integration of TCR, NKR and/or SLAM (signaling lymphocyte-activation molecule) receptor signals or the variation of CD1d-restricted antigen presentation. TCR and NKR ligation engage the SH2 domain-containing leukocyte protein of 76kDa slp-76 whereas the SLAM associated protein SAP serves as adaptor for the SLAM receptor family. Indeed, the selection and differentiation of NKT cells selectively requires co-stimulation via SLAM receptors. Furthermore, SAP deficiency causes X-linked lymphoproliferative disease with multiple immune defects including a lack of circulating NKT cells. While a deletion of slp-76 leads to a complete loss of all peripheral T cell populations, mutations in the SH2 domain of slp-76 selectively affect NKT cell biology. Furthermore, adaptor proteins influence the expression and trafficking of CD1d in antigen presenting cells and subsequently selection and activation of NKT cells. Adaptor protein complex 3 (AP-3), for example, is required for the efficient presentation of glycolipid antigens which require internalization and processing. Thus, our review will focus on the complex contribution of adaptor proteins to the delivery of TCR, NKR and SLAM receptor signals in the unique biology of NKT cells and CD1d-restricted antigen presentation.

In contrast to temporal coding by synaptically acting neurotransmitters such as glutamate, neuromodulators such as monoamines signal changes in firing rate. The two modes of signaling have been thought to reflect differences in release by different cells. We now find that midbrain dopamine neurons release glutamate and dopamine with different properties that reflect storage in different synaptic vesicles. The vesicles differ in release probability, coupling to presynaptic Ca 2+ channels and frequency dependence. Although previous work has attributed variation in these properties to differences in location or cytoskeletal association of synaptic vesicles, the release of different transmitters shows that intrinsic differences in vesicle identity drive different modes of release. Indeed, dopamine but not glutamate vesicles depend on the adaptor protein AP-3, revealing an unrecognized linkage between the pathway of synaptic vesicle recycling and the properties of exocytosis. Storage of the two transmitters in different vesicles enables the transmission of distinct signals., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder in humans and mice. Pale ear (ep) and pearl (pe) mice, bearing mutations in the biogenesis of lysosomal organelles complex 3 subunit 1 (Hps1) and adaptor-related protein complex 3, beta 1 subunit (Ap3b1) genes respectively, are mouse models of human HPS Type 1 (HPS1) and Type 2 (HPS2) respectively. In the present study we investigated and compared the reduced fertilities of ep and pe male mice. Both ep and pe males exhibited lower abilities to impregnate C57BL/6J (B6) females, and B6 females mated with ep males produced smaller litters than those mated with pe males. Delayed testis development, reduced sperm count and lower testosterone concentrations were observed in the pe but not ep male mice. However, the reduction in sperm motility was greater in ep than pe males, likely due to the mitochondrial and fibrous sheath abnormalities observed by electron microscopy in the sperm tails of ep males. Together, the results indicate that the Hps1 and Ap3b1 genes play distinct roles in male reproductive system development and spermatogenesis in mice, even though ep and pe males share common phenotypes, including reduced lysosomes in Sertoli cells and dislocated Zn2+ in sperm heads.

The regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in cellular physiology. However, the complete set of cellular membrane trafficking proteins that mediate the correct sorting of POMC to DCSGs remain unknown. Here, we report the roles of the phosphofurin acidic cluster sorting protein - 1 (PACS-1) and the clathrin adaptor protein 1 (AP-1) in the targeting of POMC to DCSGs. Upon knockdown of PACS-1 and AP-1, POMC is readily secreted into the extracellular milieu and fails to be targeted to DCSGs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Transport of late endosomes and lysosome-related organelles (LE/LROs) in axons is essential for supplying synaptic cargoes and for removing damaged macromolecules. Defects in this system are implicated in a range of human neurodegenerative and neurodevelopmental disorders. The findings reported here identify a novel mechanism regulating LE/LRO transport based on the coordinated coupling of microtubule motors and vesicle coat proteins to the neuron-enriched, transmembrane protein calcyon (Caly). We found that the cytoplasmic C-terminus of Caly pulled down proteins involved in microtubule-dependent transport (DIC, KIF5A, p150Glued, Lis1) and organelle biogenesis (AP-1 and AP-3) from the brain. In addition, RNA interference-mediated knockdown of Caly increased the percentage of static LE/LROs labeled by LysoTracker in cultured dorsal root ganglion axons. In contrast, overexpression of Caly stimulated movement of organelles positive for LysoTracker or the AP-3 cargo GFP-PI4KIIα. However, a Caly mutant (ATEA) that does not bind AP-3 was unable to pull down motor proteins from brain, and expression of the ATEA mutant failed to increase either LE/LRO flux or levels of associated dynein. Taken together, these data support the hypothesis that Caly is a multifunctional scaffolding protein that regulates axonal transport of LE/LROs by coordinately interacting with motor and vesicle coat proteins.

Pollen tube growth is an essential step for successful plant reproduction. Vacuolar trafficking and dynamic organization are important for pollen tube growth; however, the key proteins involved in these processes are not well understood. Here, we report that the ADAPTOR PROTEIN-3 (AP-3) complex and its tonoplast cargo PROTEIN S-ACYL TRANSFERASE10 (PAT10) are critical for pollen tube growth in Arabidopsis ( Arabidopsis thaliana ). AP-3 is a heterotetrameric protein complex consisting of four subunits, δ, β, µ, and σ. AP-3 regulates tonoplast targeting of several cargoes, such as PAT10. We show that functional loss of any of the four AP-3 subunits reduces plant fertility. In ap-3 mutants, pollen development was normal but pollen tube growth was compromised, leading to reduced male transmission. Functional loss of PAT10 caused a similar reduction in pollen tube growth, suggesting that the tonoplast association of PAT10 mediated by AP-3 is crucial for this process. Indeed, the Ca 2+ gradient during pollen tube growth was reduced significantly due to AP-3 loss of function, consistent with the abnormal targeting of CALCINUERIN B-LIKE2 (CBL2) and CBL3, whose tonoplast association depends on PAT10. Furthermore, we show that the pollen tubes of ap-3 mutants have vacuoles with simplified tubules and bulbous structures, indicating that AP-3 affects vacuolar organization. Our results demonstrate a role for AP-3 in plant reproduction and provide insights into the role of vacuoles in polarized cell growth., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

A deficit of GABA (γ-aminobutyric acid) transmission will lead to epilepsy and other cognitive disorders. Recent evidence has shown that neuronal miRNAs affect various synapses, including GABAergic synapses. However, the miRNAs that control GABAergic synapses remain not fully understood. Here, we identified miR-51, a member of Caenorhabditis elegans miR-99/100 family, as a key regulator of GABAergic synapses. Loss of mir-51 increased PTZ (Pentylenetetrazole) and aldicarb hypersensitivities, and decreased the number of GABAergic synapses and abundance of GABA A receptors. A Rab guaninenucleotide exchange factor (GEF) GLO-4, a well-known component in lysosomal trafficking-related GLO-4/GLO-1/AP-3 (GLO/AP-3) pathway, was discovered to be the direct target of miR-51. Rescue experiments showed that GLO-4 expressed in GABAergic motor neurons functioned as a suppressor of miR-51. Disruption of glo-1 or AP-3 gene apm-3 attenuated the defects of GABAergic synapse in mir-51 mutants, suggesting miR-51 regulated GABAergic synapses through GLO/AP-3 pathway. The present study implies the essential roles of miRNAs on the nervous pathologies characterized by mis-regulated GABA signaling, such as epilepsy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Human Respiratory Syncytial Virus (HRSV) is a leading cause of bronchopneumonia in infants and the elderly. To date, knowledge of viral and host protein interactions within HRSV is limited and are critical areas of research. Here, we show that HRSV Matrix (M) protein interacts with the cellular adaptor protein complex 3 specifically via its medium subunit (AP-3Mu3A). This novel protein-protein interaction was first detected via yeast-two hybrid screen and was further confirmed in a mammalian system by immunofluorescence colocalization and co-immunoprecipitation. This novel interaction is further substantiated by the presence of a known tyrosine-based adaptor protein MU subunit sorting signal sequence, YXXФ: where Ф is a bulky hydrophobic residue, which is conserved across the related RSV M proteins. Analysis of point-mutated HRSV M derivatives indicated that AP-3Mu3A- mediated trafficking is contingent on the presence of the tyrosine residue within the YXXL sorting sequence at amino acids 197-200 of the M protein. AP-3Mu3A is up regulated at 24 hours post-infection in infected cells versus mock-infected HEp2 cells. Together, our data suggests that the AP-3 complex plays a critical role in the trafficking of HRSV proteins specifically matrix in epithelial cells. The results of this study add new insights and targets that may lead to the development of potential antivirals and attenuating mutations suitable for candidate vaccines in the future.

Plant vacuoles are versatile organelles critical for plant growth and responses to environment. Vacuolar proteins are transported from the endoplasmic reticulum via multiple routes in plants. Two classic routes bear great similarity to other phyla with major regulators known, such as COPII and Rab5 GTPases. By contrast, vacuolar trafficking mediated by adaptor protein-3 (AP-3) or that independent of the Golgi has few recognized cargos and none of the regulators. In search of novel regulators for vacuolar trafficking routes and by using a fluorescence-based forward genetic screen, we demonstrated that the multispan transmembrane protein, Arabidopsis ( Arabidopsis thaliana ) PROTEIN S -ACYL TRANSFERASE10 (PAT10), is an AP-3-mediated vacuolar cargo. We show that the tonoplast targeting of PAT10 is mediated by the AP-3 complex but independent of the Rab5-mediated post-Golgi trafficking route. We also report that AP-3-mediated vacuolar trafficking involves a subpopulation of COPII and requires the vacuolar tethering complex HOPS. In addition, we have identified two novel mutant alleles of AP-3δ , whose point mutations interfered with the formation of the AP-3 complex as well as its membrane targeting. The results presented here shed new light on the vacuolar trafficking route mediated by AP-3 in plant cells., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Mast cell (MC) secretory granules are Lysosome-Related Organelles (LROs) whose biogenesis is associated with the post-Golgi secretory and endocytic pathways in which the sorting of proteins destined for a specific organelle relies on the recognition of sorting signals by adaptor proteins that direct their incorporation into transport vesicles. The adaptor protein 3 (AP-3) complex mediates protein trafficking between the trans-Golgi network (TGN) and late endosomes, lysosomes, and LROs. AP-3 has a recognized role in LROs biogenesis and regulated secretion in several cell types, including many immune cells such as neutrophils, natural killer cells, and cytotoxic T lymphocytes. However, the relevance of AP-3 for these processes in MCs has not been previously investigated. AP-3 was found to be expressed and distributed in a punctate fashion in rat peritoneal mast cells ex vivo. The rat MC line RBL-2H3 was used as a model system to investigate the role of AP-3 in mast cell secretory granule biogenesis and mediator release. By immunofluorescence and immunoelectron microscopy, AP-3 was localized both to the TGN and early endosomes indicating that AP-3 dependent sorting of proteins to MC secretory granules originates in these organelles. ShRNA mediated depletion of the AP-3 δ subunit was shown to destabilize the AP-3 complex in RBL-2H3 MCs. AP-3 knockdown significantly affected MC regulated secretion of β-hexosaminidase without affecting total cellular enzyme levels. Morphometric evaluation of MC secretory granules by electron microscopy revealed that the area of MC secretory granules in AP-3 knockdown MCs was significantly increased, indicating that AP-3 is involved in MC secretory granule biogenesis. Furthermore, AP-3 knockdown had a selective impact on the secretion of newly formed and newly synthesized mediators. These results show for the first time that AP-3 plays a critical role in secretory granule biogenesis and mediator release in MCs.

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit β3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.

Synaptic scaling is a form of homeostatic plasticity driven by transcription-dependent changes in AMPA-type glutamate receptor (AMPAR) trafficking. To uncover the pathways involved, we performed a cell-type-specific screen for transcripts persistently altered during scaling, which identified the μ subunit (μ3A) of the adaptor protein complex AP-3A. Synaptic scaling increased μ3A (but not other AP-3 subunits) in pyramidal neurons and redistributed dendritic μ3A and AMPAR to recycling endosomes (REs). Knockdown of μ3A prevented synaptic scaling and this redistribution, while overexpression (OE) of full-length μ3A or a truncated μ3A that cannot interact with the AP-3A complex was sufficient to drive AMPAR to REs. Finally, OE of μ3A acted synergistically with GRIP1 to recruit AMPAR to the dendritic membrane. These data suggest that excess μ3A acts independently of the AP-3A complex to reroute AMPAR to RE, generating a reservoir of receptors essential for the regulated recruitment to the synaptic membrane during scaling up., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3β and ap-4β knock-out mutants. In ap-3β mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4β mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3β and ap-4β compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity.

Unlabelled: Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a microtubule interacting and trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed, and characterized. Both were shown to interact strongly with the plasma membrane component, phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modeling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes., Database: Structural data are available in the Biological Magnetic Resonance Bank (BMRB) database under the accession number 19987 and the PDB database under the accession number 2MPK., (© 2016 Federation of European Biochemical Societies.)

Although human immunodeficiency virus (HIV) types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HIV-2 may help to shed light on this issue. In contrast to HIV-1, little is known about the assembly of HIV-2 particles, and the trafficking of HIV-2 Gag, the structural component of the virus, within cells. We have established that HIV-2 Gag accumulates in intracellular CD63 positive compartments, from which it may be delivered or recycled to the cell surface, or degraded. HIV-2 particle release was dependent on the adaptor protein complex AP-3 and the newly identified AP-5 complex, but much less so on AP-1. In contrast, HIV-1 particle release required AP-1 and AP-3, but not AP-5. AP-2, an essential component of clathrin-mediated endocytosis, which was previously shown to be inhibitory to HIV-1 particle release, had no effect on HIV-2. The differential requirement for adaptor protein complexes confirmed that HIV-1 and HIV-2 Gag have distinct cellular trafficking pathways, and that HIV-2 particles may be more susceptible to degradation prior to release.

The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The limiting membrane of lysosomes in animal cells and that of the vacuole in yeast include a wide variety of transporters, but little is known about how these proteins reach their destination membrane. The mammalian PQLC2 protein catalyzes efflux of basic amino acids from the lysosome, and the similar Ypq1, -2, and -3 proteins of yeast perform an equivalent function at the vacuole. We here show that the Ypq proteins are delivered to the vacuolar membrane via the alkaline phosphatase (ALP) trafficking pathway, which requires the AP-3 adaptor complex. When traffic via this pathway is deficient, the Ypq proteins pass through endosomes from where Ypq1 and Ypq2 properly reach the vacuolar membrane whereas Ypq3 is missorted to the vacuolar lumen via the multivesicular body pathway. When produced in yeast, PQLC2 also reaches the vacuolar membrane via the ALP pathway, but tends to sort to the vacuolar lumen if AP-3 is defective. Finally, in HeLa cells, inhibiting the synthesis of an AP-3 subunit also impairs sorting of PQLC2 to lysosomes. Our results suggest the existence of a conserved AP-3-dependent trafficking pathway for proper delivery of basic amino acid exporters to the yeast vacuole and to lysosomes of human cells.

Innate immune engagement results in the activation of host defenses that produce microbe-specific inflammatory responses. A long-standing interest in the field of innate immunity is to understand how varied host responses are generated through the signaling of just a limited number of receptors. Recently, intracellular trafficking and compartmental partitioning have been identified as mechanisms that provide signaling specificity for receptors by regulating signaling platform assembly. We show that cytokine activation as a result of TLR2 stimulation occurs at different intracellular locations and is mediated by the phagosomal trafficking molecule adaptor protein-3 (AP-3). AP-3 is required for trafficking TLR2 purified ligands or the Lyme disease causing bacterium, Borrelia burgdorferi, to LAMP-1 lysosomal compartments. The presence of AP-3 is necessary for the activation of cytokines such as IL-6 but not TNF-α or type I IFNs, suggesting induction of these cytokines occurs from a different compartment. Lack of AP-3 does not interfere with the recruitment of TLR signaling adaptors TRAM and MyD88 to the phagosome, indicating that the TLR-MyD88 signaling complex is assembled at a prelysosomal stage and that IL-6 activation depends on proper localization of signaling molecules downstream of MyD88. Finally, infection of AP-3-deficient mice with B. burgdorferi resulted in altered joint inflammation during murine Lyme arthritis. Our studies further elucidate the effects of phagosomal trafficking on tailoring immune responses in vitro and in vivo., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Mycobacterium avium subsp hominissuis (M. avium) is a pathogen that infects and survives in macrophages. Previously, we have identified the M. avium MAV&#95;2941 gene encoding a 73 amino acid protein exported by the oligopeptide transporter OppA to the macrophage cytoplasm. Mutations in MAV&#95;2941 were associated with significant impairment of M. avium growth in THP-1 macrophages. In this study, we investigated the molecular mechanism of MAV&#95;2941 action and demonstrated that MAV&#95;2941 interacts with the vesicle trafficking proteins syntaxin-8 (STX8), adaptor-related protein complex 3 (AP-3) complex subunit beta-1 (AP3B1) and Archain 1 (ARCN1) in mononuclear phagocytic cells. Sequencing analysis revealed that the binding site of MAV&#95;2941 is structurally homologous to the human phosphatidylinositol 3-kinase (PI3K) chiefly in the region recognized by vesicle trafficking proteins. The β3A subunit of AP-3, encoded by AP3B1, is essential for trafficking cargo proteins, including lysosomal-associated membrane protein 1 (LAMP-1), to the phagosome and lysosome-related organelles. Here, we show that while the heat-killed M. avium when ingested by macrophages co-localizes with LAMP-1 protein, transfection of MAV&#95;2941 in macrophages results in significant decrease of LAMP-1 co-localization with the heat-killed M. avium phagosomes. Mutated MAV&#95;2941, where the amino acids homologous to the binding region of PI3K were changed, failed to interact with trafficking proteins. Inactivation of the AP3B1 gene led to alteration in the trafficking of LAMP-1. These results suggest that M. avium MAV&#95;2941 interferes with the protein trafficking within macrophages altering the maturation of phagosome., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Cystinosin is a lysosomal cystine transporter defective in cystinosis, an autosomal recessive lysosomal storage disorder. It is composed of seven transmembrane (TM) domains and contains two lysosomal targeting motifs: a tyrosine-based signal (GYDQL) in its C-terminal tail and a non-classical motif in its fifth inter-TM loop. Using the yeast two-hybrid system, we showed that the GYDQL motif specifically interacted with the μ subunit of the adaptor protein complex 3 (AP-3). Moreover, cell surface biotinylation and total internal reflection fluorescence microscopy revealed that cystinosin was partially mislocalized to the plasma membrane (PM) in AP-3-depleted cells. We generated a chimeric CD63 protein to specifically analyze the function of the GYDQL motif. This chimeric protein was targeted to lysosomes in a manner similar to cystinosin and was partially mislocalized to the PM in AP-3 knockdown cells where it also accumulated in the trans-Golgi network and early endosomes. Together with the fact that the surface levels of cystinosin and of the CD63-GYDQL chimeric protein were not increased when clathrin-mediated endocytosis was impaired, our data show that the tyrosine-based motif of cystinosin is a 'strong' AP-3 interacting motif responsible for lysosomal targeting of cystinosin by a direct intracellular pathway., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The interferon-induced transmembrane proteins (IFITMs) broadly inhibit virus infections, particularly at the viral entry level. However, despite this shared ability to inhibit fusion, IFITMs differ in the potency and breadth of viruses restricted, an anomaly that is not fully understood. Here, we show that differences in the range of viruses restricted by IFITM1 are regulated by a C-terminal non-canonical dibasic sorting signal KRXX that suppresses restriction of some viruses by governing its intracellular distribution. Replacing the two basic residues with alanine (KR/AA) increased restriction of jaagsiekte sheep retrovirus and 10A1 amphotropic murine leukemia virus. Deconvolution microscopy revealed an altered subcellular distribution for KR/AA, with fewer molecules in LAMP1-positive lysosomes balanced by increased levels in CD63-positive multivesicular bodies, where jaagsiekte sheep retrovirus pseudovirions are colocalized. IFITM1 binds to cellular adaptor protein complex 3 (AP-3), an association that is lost when the dibasic motif is altered. Although knockdown of AP-3 itself decreases some virus entry, expression of parental IFITM1, but not its KR/AA mutant, potentiates inhibition of viral infections in AP-3 knockdown cells. By using the substituted cysteine accessibility method, we provide evidence that IFITM1 adopts more than one membrane topology co-existing in cellular membranes. Because the C-terminal dibasic sorting signal is unique to human IFITM1, our results provide novel insight into understanding the species- and virus-specific antiviral effect of IFITMs., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The position of lysosomes in the cytoplasm is important for their ability to fuse with the plasma membrane and release of proteases that are involved in tissue remodeling. Motor-directed bidirectional transport along microtubules is a critical process determining the distribution of lysosomes. How lysosomes are tethered to microtubules is incompletely understood, but a role for small GTPases of rab and arl families has been documented. We recently found that the rab5 and rab4 effector rabip4' interacts with the adaptor complex AP-3 in a rab4-dependent manner on tubular endosomes. We here describe the assays that led to the identification of AP-3 as a rabip4' partner and the role of the complex in regulating the spatial distribution of lysosomes.

Mutations in the large BEACH domain-containing protein LYST causes Chediak-Higashi syndrome. The diagnostic hallmark is enlarged lysosomes and lysosome-related organelles in various cell types. Dysfunctional secretion of enlarged lysosome-related organelles has been observed in cells with mutations in LYST, but the capacity of the enlarged lysosomes to degrade endogenous proteins has not been studied. Here, we show for the first time that small interfering RNA-depletion of LYST in human cell lines recapitulates the LYST mutant phenotype of enlarged lysosomes. We found no evidence for an effect of LYST depletion on autophagy or endocytic degradation. Autophagosomes are formed in normal size and quantities and are able to fuse to the enlarged lysosomes, leading to normal rates of degradation. Degradation of the epidermal growth factor receptor (EGFR) was similarly not affected, indicating that the enlarged lysosomes are fully functional in degrading endogenous proteins. Retrograde trafficking of toxins as well as the localization of transporters of lysosomal proteins, adaptor protein-3 (AP-3) and cation-independent mannose-6-phosphate receptor (CI-MPR), were all found to be unaffected by LYST. Quantitative analysis of the enlarged lysosomes shows that LYST depletion causes a reduction in vesicle quantity per cell, while the total enzymatic content and vesicular pH are unaffected, supporting a role for LYST in lysosomal fission and/or fusion events., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Action potentials trigger synchronous and asynchronous neurotransmitter release. Temporal properties of both types of release could be altered in an activity-dependent manner. While the effects of activity-dependent changes in synchronous release on postsynaptic signal integration have been studied, the contribution of asynchronous release to information transfer during natural stimulus patterns is unknown. Here we find that during trains of stimulations, asynchronous release contributes to the precision of action potential firing. Our data show that this form of release is selectively diminished in AP-3b2 KO animals, which lack functional neuronal AP-3, an adaptor protein regulating vesicle formation from endosomes generated during bulk endocytosis. We find that in the absence of neuronal AP-3, asynchronous release is attenuated and the activity-dependent increase in the precision of action potential timing is compromised. Lack of asynchronous release decreases the capacity of synaptic information transfer and renders synaptic communication less reliable in response to natural stimulus patterns.

Septins (SEPTs) form a family of GTP-binding proteins implicated in cytoskeleton and membrane organization, cell division and host/pathogen interactions. The precise function of many family members remains elusive. We show that SEPT6 and SEPT7 complexes bound to F-actin regulate protein sorting during multivesicular body (MVB) biogenesis. These complexes bind AP-3, an adapter complex sorting cargos destined to remain in outer membranes of maturing endosomes, modulate AP-3 membrane interactions and the motility of AP-3-positive endosomes. These SEPT-AP interactions also influence the membrane interaction of ESCRT (endosomal-sorting complex required for transport)-I, which selects ubiquitinated cargos for degradation inside MVBs. Whereas our findings demonstrate that SEPT6 and SEPT7 function in the spatial, temporal organization of AP-3- and ESCRT-coated membrane domains, they uncover an unsuspected coordination of these sorting machineries during MVB biogenesis. This requires the E3 ubiquitin ligase LRSAM1, an AP-3 interactor regulating ESCRT-I sorting activity and whose mutations are linked with Charcot-Marie-Tooth neuropathies.

Unlabelled: Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections., Importance: Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Extensive genetic and genomic studies of the relationship between alcohol drinking preference and withdrawal severity have been performed using animal models. Data from multiple such publications and public data resources have been incorporated in the GeneWeaver database with >60,000 gene sets including 285 alcohol withdrawal and preference-related gene sets. Among these are evidence for positional candidates regulating these behaviors in overlapping quantitative trait loci (QTL) mapped in distinct mouse populations. Combinatorial integration of functional genomics experimental results revealed a single QTL positional candidate gene in one of the loci common to both preference and withdrawal. Functional validation studies in Ap3m2 knockout mice confirmed these relationships. Genetic validation involves confirming the existence of segregating polymorphisms that could account for the phenotypic effect. By exploiting recent advances in mouse genotyping, sequence, epigenetics, and phylogeny resources, we confirmed that Ap3m2 resides in an appropriately segregating genomic region. We have demonstrated genetic and alcohol-induced regulation of Ap3m2 expression. Although sequence analysis revealed no polymorphisms in the Ap3m2-coding region that could account for all phenotypic differences, there are several upstream SNPs that could. We have identified one of these to be an H3K4me3 site that exhibits strain differences in methylation. Thus, by making cross-species functional genomics readily computable we identified a common QTL candidate for two related bio-behavioral processes via functional evidence and demonstrate sufficiency of the genetic locus as a source of variation underlying two traits., (Copyright © 2014 by the Genetics Society of America.)

Background: Alzheimer's disease (AD) is characterized by cerebral deposition of β-amyloid peptide (Aβ). Aβ is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate Aβ production, although the exact organelle in which Aβ is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating Aβ production, but these pathways are relatively under-studied. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing., Results: We have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein (paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the Golgi to downstream compartments identified with compartment markers tagged with red fluorescent protein (mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because γ-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of fluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that APP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the highly selective γ-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is being cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal APP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3 (AP-3; a heterotetrameric protein complex required for trafficking many proteins to the lysosome) using siRNA, we are able to reduce this lysosomal transport. Blocking lysosomal transport of APP reduces Aβ production by more than a third., Conclusion: These data suggests that AP-3 mediates rapid delivery of APP to lysosomes, and that the lysosome is a likely site of Aβ production.

The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes.

BST2/tetherin, an antiviral restriction factor, inhibits the release of enveloped viruses from the cell surface. Human immunodeficiency virus-1 (HIV-1) antagonizes BST2 through viral protein u (Vpu), which downregulates BST2 from the cell surface. We report the crystal structure of a protein complex containing Vpu and BST2 cytoplasmic domains and the core of the clathrin adaptor protein complex 1 (AP1). This, together with our biochemical and functional validations, reveals how Vpu hijacks the AP1-dependent membrane trafficking pathways to mistraffick BST2. Vpu mimics a canonical acidic dileucine-sorting motif to bind AP1 in the cytosol, while simultaneously interacting with BST2 in the membrane. These interactions enable Vpu to build on an intrinsic interaction between BST2 and AP1, presumably causing the observed retention of BST2 in juxtanuclear endosomes and stimulating its degradation in lysosomes. The ability of Vpu to hijack AP-dependent trafficking pathways suggests a potential common theme for Vpu-mediated downregulation of host proteins.DOI: http://dx.doi.org/10.7554/eLife.02362.001., (Copyright © 2014, Jia et al.)

The yeast Rab5 homologue, Vps21p, is known to be involved both in the vacuolar protein sorting (VPS) pathway from the trans-Golgi network to the vacuole, and in the endocytic pathway from the plasma membrane to the vacuole. However, the intracellular location at which these two pathways converge remains unclear. In addition, the endocytic pathway is not completely blocked in yeast cells lacking all Rab5 genes, suggesting the existence of an unidentified route that bypasses the Rab5-dependent endocytic pathway. Here we show that convergence of the endocytic and VPS pathways occurs upstream of the requirement for Vps21p in these pathways. We also identify a previously unidentified endocytic pathway mediated by the AP-3 complex. Importantly, the AP-3-mediated pathway appears mostly intact in Rab5-disrupted cells, and thus works as an alternative route to the vacuole/lysosome. We propose that the endocytic traffic branches into two routes to reach the vacuole: a Rab5-dependent VPS pathway and a Rab5-independent AP-3-mediated pathway.

Intracellular protein trafficking through secretory and endocytic pathways depends on the function of adaptor proteins that bind motifs on cargo proteins. The adaptor proteins then recruit coat proteins such as clathrin, enabling the formation of a transport vesicle. While studying the role of the clathrin adaptor proteins, AP-1, AP-2 and AP-3 in viral protein trafficking, we discovered that AP-1 and AP-3 potentially have a role in successful transfection of mammalian cells with DNA-liposome complexes (lipoplexes). We showed that AP-1, -2 and -3 are not required for lipoplexes to enter cells, but that lipoplexes and/or released DNA are unable to reach the nucleus in the absence of AP-1 or AP-3, leading to minimal exogenous gene expression. In contrast, gene expression from liposome-delivered mRNA, which does not require nuclear entry, was not impaired by the absence of AP-1 or AP-3. Despite the use of lipoplexes to mediate gene delivery being so widely used in cell biology and, more recently, gene therapy, the mechanism by which lipoplexes or DNA reach the nucleus is poorly characterised. This work sheds light on the components involved in this process, and demonstrates a novel role for AP-1 and AP-3 in trafficking lipoplexes.

Viroporins, which are encoded by a wide range of animal viruses, oligomerize in host cell membranes and form hydrophilic pores that can disrupt a number of physiological properties of the cell. Little is known about the relationship between host cell proteins and viroporin activity. The human JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy. The JCV-encoded agnoprotein, which is essential for viral replication, has been shown to act as a viroporin. Here we demonstrate that the JCV agnoprotein specifically interacts with adaptor protein complex 3 through its δ subunit. This interaction interrupts adaptor protein complex 3-mediated vesicular trafficking with suppression of the targeting of the protein to the lysosomal degradation pathway and instead permits the transport of agnoprotein to the cell surface with resulting membrane permeabilization. The findings demonstrate a previously undescribed paradigm in virus-host interactions allowing the host to regulate viroporin activity and suggest that the viroporins of other viruses may also be highly regulated by specific interactions with host cell proteins.

Newly formed synapses undergo maturation during ontogenetic development via mechanisms that remain poorly understood. We show that maturation of the presynaptic endocytotic machinery in CNS neurons requires substitution of the adaptor protein 3 (AP-3) with AP-2 at the presynaptic plasma membrane. In mature synapses, AP-2 associates with the intracellular domain of the neural cell adhesion molecule (NCAM). NCAM promotes binding of AP-2 over binding of AP-3 to presynaptic membranes, thus favoring the substitution of AP-3 for AP-2 during formation of mature synapses. The presynaptic endocytotic machinery remains immature in adult NCAM-deficient (NCAM-/-) mice accumulating AP-3 instead of AP-2 and its partner protein AP180 in synaptic membranes and vesicles. NCAM deficiency or disruption of the NCAM/AP-2 complex in wild-type (NCAM+/+) neurons by overexpression of AP-2 binding-defective mutant NCAM interferes with efficient retrieval of the synaptic vesicle v-SNARE synaptobrevin 2. Abnormalities in synaptic vesicle endocytosis and recycling may thus contribute to neurological disorders associated with mutations in NCAM.