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Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s -1 ) of the Dab2 MIR-myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 μM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and "stop-and-go" motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR-myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor-cargo association in enabling cargo transportation without disrupting cytoskeletal organization., Competing Interests: Conflict of interests 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.)

Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 "uncoating ATPase." The J- and PTEN-like domain-containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin-mediated membrane scission has released the vesicle from the plasma membrane. These bursts contain a very small number of auxilins, and even four to six molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated pit assembly. We previously showed that clathrin-coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. The differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism., (© 2020 He et al.)

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Chlamydial infection requires the formation of a membrane-bound vacuole, termed the inclusion, that undergoes extensive interactions with select host organelles. The importance of the Inc protein CT229 in the formation and maintenance of the chlamydial inclusion was recently highlighted by studies demonstrating that its absence during infection results in reduced bacterial replication, premature inclusion lysis, and host cell death. Previous reports have indicated that CT229 binds Rab GTPases; however, the physiological implications of this interaction are unknown. Here, we show that CT229 regulates host multivesicular trafficking by recruiting multiple Rab GTPases and their cognate effectors to the inclusion. We demonstrate that CT229 specifically modulates clathrin-coated vesicle trafficking and regulates the trafficking of transferrin and the mannose-6-phosphate receptor, both of which are crucial for proper chlamydial development. This study highlights CT229 as a master regulator of multiple host vesicular trafficking pathways essential for chlamydial infection., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life-threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non-iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C. neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin-mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C. neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin-containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C. neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C. neoformans and reveals a role for CME in fungal pathogenesis., (© 2018 John Wiley & Sons Ltd.)

ORF9p (homologous to herpes simplex virus 1 [HSV-1] VP22) is a varicella-zoster virus (VZV) tegument protein essential for viral replication. Even though its precise functions are far from being fully described, a role in the secondary envelopment of the virus has long been suggested. We performed a yeast two-hybrid screen to identify cellular proteins interacting with ORF9p that might be important for this function. We found 31 ORF9p interaction partners, among which was AP1M1, the μ subunit of the adaptor protein complex 1 (AP-1). AP-1 is a heterotetramer involved in intracellular vesicle-mediated transport and regulates the shuttling of cargo proteins between endosomes and the trans -Golgi network via clathrin-coated vesicles. We confirmed that AP-1 interacts with ORF9p in infected cells and mapped potential interaction motifs within ORF9p. We generated VZV mutants in which each of these motifs was individually impaired and identified leucine 231 in ORF9p to be critical for the interaction with AP-1. Disrupting ORF9p binding to AP-1 by mutating leucine 231 to alanine in ORF9p strongly impaired viral growth, most likely by preventing efficient secondary envelopment of the virus. Leucine 231 is part of a dileucine motif conserved among alphaherpesviruses, and we showed that VP22 of Marek's disease virus and HSV-2 also interacts with AP-1. This indicates that the function of this interaction in secondary envelopment might be conserved as well. IMPORTANCE Herpesviruses are responsible for infections that, especially in immunocompromised patients, can lead to severe complications, including neurological symptoms and strokes. The constant emergence of viral strains resistant to classical antivirals (mainly acyclovir and its derivatives) pleads for the identification of new targets for future antiviral treatments. Cellular adaptor protein (AP) complexes have been implicated in the correct addressing of herpesvirus glycoproteins in infected cells, and the discovery that a major constituent of the varicella-zoster virus tegument interacts with AP-1 reveals a previously unsuspected role of this tegument protein. Unraveling the complex mechanisms leading to virion production will certainly be an important step in the discovery of future therapeutic targets., (Copyright © 2018 American Society for Microbiology.)

Endocytic recycling returns receptors to the plasma membrane following internalization and is essential to maintain receptor levels on the cell surface, re-sensitize cells to extracellular ligands and for continued nutrient uptake. Yet, the protein machineries and mechanisms that drive endocytic recycling remain ill-defined. Here, we establish that NECAP2 regulates the endocytic recycling of EGFR and transferrin receptor. Our analysis of the recycling dynamics revealed that NECAP2 functions in the fast recycling pathway that directly returns cargo from early endosomes to the cell surface. In contrast, NECAP2 does not regulate the clathrin-mediated endocytosis of these cargos, the degradation of EGFR or the recycling of transferrin along the slow, Rab11-dependent recycling pathway. We show that protein knockdown of NECAP2 leads to enlarged early endosomes and causes the loss of the clathrin adapter AP-1 from the organelle. Through structure-function analysis, we define the protein-binding interfaces in NECAP2 that are crucial for AP-1 recruitment to early endosomes. Together, our data identify NECAP2 as a pathway-specific regulator of clathrin coat formation on early endosomes for fast endocytic recycling., (© 2016. Published by The Company of Biologists Ltd.)

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER-Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway., (© 2016 Ding, Segarra, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The AP-2 clathrin adaptor complex oversees endocytic cargo selection in two parallel but independent manners. First, by physically engaging peptide-based endocytic sorting signals, a subset of clathrin-dependent transmembrane cargo is directly collected into assembling buds. Synchronously, by interacting with an assortment of clathrin-associated sorting proteins (CLASPs) that independently select different integral membrane cargo for inclusion within the incipient bud, AP-2 handles additional cargo capture indirectly. The distal platform subdomain of the AP-2 β2 subunit appendage is a privileged CLASP-binding surface that recognizes a cognate, short α-helical interaction motif. This signal, found in the CLASPs β-arrestin and the autosomal recessive hypercholesterolemia (ARH) protein, docks into an elongated groove on the β2 appendage platform. Tyr-888 is a critical constituent of this spatially confined β2 appendage contact interface and is phosphorylated in numerous high-throughput proteomic studies. We find that a phosphomimetic Y888E substitution does not interfere with incorporation of expressed β2-YFP subunit into AP-2 or alter AP-2 deposition at surface clathrin-coated structures. The Y888E mutation does not affect interactions involving the sandwich subdomain of the β2 appendage, indicating that the mutated appendage is folded and operational. However, the Y888E, but not Y888F, switch selectively uncouples interactions with ARH and β-arrestin. Phyogenetic conservation of Tyr-888 suggests that this residue can reversibly control occupancy of the β2 platform-binding site and, hence, cargo sorting., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

ADP ribosylation factors (Arfs) are small GTP-binding proteins known for their role in vesicular transport, where they nucleate the assembly of coat protein complexes at sites of carrier vesicle formation. Similar to other GTPases, Arfs require guanine nucleotide exchange factors to catalyze GTP loading and activation. One subfamily of ArfGEFs, the BRAGs, has been shown to activate Arf6, which acts in the endocytic pathway to control the trafficking of a subset of cargo proteins including integrins. We have previously shown that BRAG2 modulates cell adhesion by regulating integrin surface expression. Here, we show that, in addition to Arf6, endogenous BRAG2 also activates the class II Arfs, Arf4 and Arf5, and that surprisingly, it is Arf5 that mediates integrin internalization. We observed that cell spreading on fibronectin is enhanced upon inhibition of BRAG2 or Arf5 but not Arf6. Similarly, spreading in BRAG2-depleted cells is reverted by expression of a rapid cycling Arf5 mutant (T161A) but not by a corresponding Arf6 construct (T157A). We also show that BRAG2 binds clathrin and the AP-2 adaptor complex and that both BRAG2 and Arf5 localize to clathrin-coated pits at the plasma membrane. Consistent with these observations, depletion of Arf5, but not Arf6 or Arf4, slows internalization of β1 integrins without affecting transferrin receptor uptake. Together, these findings indicate that BRAG2 acts at clathrin-coated pits to promote integrin internalization by activating Arf5 and suggest a previously unrecognized role for Arf5 in clathrin-mediated endocytosis of specific cargoes.

We show that a single gene locus gives rise to two fully processed and functional miRNAs, i.e. that due to imperfect base pairing, two distinct microRNAs (miRNAs) can be produced from the fully complementary DNA strands. The antisense strand encodes miR-214, which is transcribed by its own promoter, whereas a novel miRNA, miR-3120, is co-expressed with its host gene mRNA. We also found that miR-3120 regulates important aspects of cellular function that are similar to that of its host gene, dynamin-3. miR-3120 was found to be located in neuronal cell bodies and to target Hsc70 and auxilin, and its lentivirus-mediated expression inhibited the uncoating of clathrin-coated vesicles. Finally, mirror miRNAs are likely to represent a new group of miRNAs with complex roles in coordinating gene expression.

The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca(2+)-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance.

Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b(558)) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91(phox) and CeCl(3) cytochemistry showed the presence of gp91(phox) and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b(558) is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b(558)-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5'-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b(558) under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNFα, and CD40L as physiological inducers of cyt b(558) exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b(558), which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b(558) did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b(558) depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.

Hypoxia in solid tumors contributes to decreased immunosurveillance via down-regulation of Kv1.3 channels in T lymphocytes and associated T cell function inhibition. However, the mechanisms responsible for Kv1.3 down-regulation are not understood. We hypothesized that chronic hypoxia reduces Kv1.3 surface expression via alterations in membrane trafficking. Chronic hypoxia decreased Kv1.3 surface expression and current density in Jurkat T cells. Inhibition of either protein synthesis or degradation and endocytosis did not prevent this effect. Instead, blockade of clathrin-coated vesicle formation and forward trafficking prevented the Kv1.3 surface expression decrease in hypoxia. Confocal microscopy revealed an increased retention of Kv1.3 in the trans-Golgi during hypoxia. Expression of adaptor protein-1 (AP1), responsible for clathrin-coated vesicle formation at the trans-Golgi, was selectively down-regulated by hypoxia. Furthermore, AP1 down-regulation increased Kv1.3 retention in the trans-Golgi and reduced Kv1.3 currents. Our results indicate that hypoxia disrupts AP1/clathrin-mediated forward trafficking of Kv1.3 from the trans-Golgi to the plasma membrane thus contributing to decreased Kv1.3 surface expression in T lymphocytes.

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation., Competing Interests: The authors have declared that no competing interests exist.

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.

Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-coated pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP(2)), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP(2) synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5'-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP(2) synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.

Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to down-regulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC.

Heat shock cognate protein-70 (Hsc70) supports remodeling of protein complexes, such as disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP-driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by cryo-EM, in the vicinity of each occupied site and that accumulation of local strains destabilizes the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.

The Ca(2+)-sensing receptor (CaR) regulates salt and water transport in the kidney as demonstrated by the association of gain of function CaR mutations with a Bartter syndrome-like, salt-wasting phenotype, but the precise mechanism for this effect is not fully established. We found previously that the CaR interacts with and inactivates an inwardly rectifying K(+) channel, Kir4.1, which is expressed in the distal nephron that contributes to the basolateral K(+) conductance, and in which loss of function mutations are associated with a complex phenotype that includes renal salt wasting. We now find that CaR inactivates Kir4.1 by reducing its cell surface expression. Mutant CaRs reduced Kir4.1 cell surface expression and current density in HEK-293 cells in proportion to their signaling activity. Mutant, activated Gα(q) reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via Gα(i) and Gα(q). Other α subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of Gα(q) on Kir4.1, whereas knockdown of the clathrin heavy chain had no effect. CaR had no comparable effect on the renal outer medullary K(+) channel, an apical membrane distal nephron K(+) channel that is internalized by clathrin-coated vesicles. Co-immunoprecipitation studies showed that the CaR and Kir4.1 physically associate with caveolin-1 in HEK cells and in kidney extracts. Thus, the CaR decreases cell surface expression of Kir4.1 channels via a mechanism that involves Gα(q) and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.

Clathrin-mediated vesicle recycling in synapses is maintained by a unique set of endocytic proteins and interactions. We show that endophilin localizes in the vesicle pool at rest and in spirals at the necks of clathrin-coated pits (CCPs) during activity in lamprey synapses. Endophilin and dynamin colocalize at the base of the clathrin coat. Protein spirals composed of these proteins on lipid tubes in vitro have a pitch similar to the one observed at necks of CCPs in living synapses, and lipid tubules are thinner than those formed by dynamin alone. Tubulation efficiency and the amount of dynamin recruited to lipid tubes are dramatically increased in the presence of endophilin. Blocking the interactions of the endophilin SH3 domain in situ reduces dynamin accumulation at the neck and prevents the formation of elongated necks observed in the presence of GTPγS. Therefore, endophilin recruits dynamin to a restricted part of the CCP neck, forming a complex, which promotes budding of new synaptic vesicles.

Background: Dengue virus (DENV) is the causative agent of Dengue fever and the life-threatening Dengue Haemorrhagic fever or Dengue shock syndrome. In the absence of anti-viral agents or vaccine, there is an urgent need to develop an effective anti-viral strategy against this medically important viral pathogen. The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites. Currently the involvements of human membrane trafficking host genes or factors that mediate the infectious cellular entry of dengue virus are not well defined., Results: In this study, we have used a targeted small interfering RNA (siRNA) library to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics and endosome trafficking that are important and essential for DENV infection. The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. Furthermore, DENV infection was shown to be sensitive to the disruption of human genes in regulating the early to late endosomal trafficking as well as the endosomal acidic pH. The importance and involvement of both actin and microtubule dynamics in mediating the infectious entry of DENV was also revealed in this study., Conclusions: Together, the findings from this study have provided a detail profiling of the human membrane trafficking cellular genes and the mechanistic insight into the interplay of these host genes with DENV to initiate an infection, hence broadening our understanding on the entry pathway of this medically important viral pathogen. These data may also provide a new potential avenue for development of anti-viral strategies and treatment of DENV infection.

Antigen binding to the B cell antigen receptor (BCR) initiates an array of signaling events. These include endocytosis of ligand-receptor complexes via clathrin-coated pits, trafficking of the internalized ligand to lysosomes, degradation of the associated proteins to peptides, and peptide presentation on nascent major histocompatibility complex class II to T cells. The signal transduction events supporting BCR internalization are not well understood. We have identified a pathway supporting BCR internalization that includes the Vav1 and/or Vav3 isoforms and the GTPase dynamin. Vav1 and -3 are not required for B cell development and maturation, nor for a variety of BCR-induced signaling events nor for BCR signaling leading to major histocompatibility complex class II and CD80 expression, but Vav1 and/or -3 are absolutely required for BCR endocytosis and BCR-induced Rac-GTP loading. This is the first demonstration of a link between Vav and Rac in BCR internalization leading to antigen presentation to T cells.

The AP-2 clathrin adaptor differs fundamentally from the related AP-1, AP-3, and AP-4 sorting complexes because membrane deposition does not depend directly on an Arf family GTPase. Instead phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) appears to act as the principal compartmental cue for AP-2 placement at the plasma membrane as well as for the docking of numerous other important clathrin coat components at the nascent bud site. This PtdIns(4,5)P(2) dependence makes type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIs) lynchpin enzymes in the assembly of clathrin-coated structures at the cell surface. PIPKIgamma is the chief 5-kinase at nerve terminals, and here we show that the 26-amino acid, alternatively spliced C terminus of PIPKIgamma661 is an intrinsically unstructured polypeptide that binds directly to the sandwich subdomain of the AP-2 beta2 subunit appendage. An aromatic side chain-based, extended interaction motif that also includes the two bulky C-terminal residues of the short PIPKIgamma635 variant is necessary for beta2 appendage engagement. The clathrin heavy chain accesses the same contact surface on the AP-2 beta2 appendage, but because of additional clathrin binding sites located within the unstructured hinge segment of the beta2 subunit, clathrin binds the beta2 chain with a higher apparent affinity than PIPKIgamma661. A clathrin-regulated interaction with AP-2 could allow PIPKIgamma661 to be strategically positioned for regional PtdIns(4,5)P(2) generation during clathrin-coated vesicle assembly at the synapse.

ACK1 (activated Cdc42-associated kinase 1) is a cytoplasmic tyrosine kinase implicated in trafficking through binding to epidermal growth factor (EGF) receptor and clathrin. Here, we have identified a new ACK1-binding partner, the E3 ubiquitin ligase Nedd4-2, which binds ACK1 via a conserved PPXY-containing region. We show that this motif also binds Nedd4-related proteins and several other WW domain-containing proteins, including the tumor suppressor oxidoreductase Wwox. In HeLa cells ACK1 colocalizes with Nedd4-2 in clathrin-rich vesicles, requiring this PPXY motif. Nedd4-2 strongly down-regulates ACK1 levels when coexpressed, and this process can be blocked by proteasome inhibitor MG132. ACK1 degradation via Nedd4 requires their mutual interaction and a functional E3 ligase; it is also driven by ACK1 activity. ACK1 is polyubiquitinated in vivo, and dominant inhibitory Nedd4 blocks endogenous ACK1 turnover in response to acute EGF treatment. Because EGF stimulation activates ACK1 ( Galisteo, M., Y., Y., Urena, J., and Schlessinger, J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 9796-9801 ), our result suggest that EGF receptor-mediated ACK1 activation allows Nedd4-2 to drive kinase degradation. Thus the interplay between Nedd4-2-related E3 ligases that regulate ACK1 levels and Cbl that modifies EGF receptor impinges on cell receptor dynamics. These processes are particularly pertinent given the report of genomic amplification of the ACK1 locus in metastatic tumors.

Epsin contains a phospholipid-binding ENTH domain coupled to C-terminal domain motifs that bind coated pit proteins. We examined how these domains interact to influence epsin function and localization in Dictyostelium. Although not required for global clathrin function, epsin was essential for constructing oval spores during development. Within the epsin protein, we found that features important for essential function were distinct from features targeting epsin to clathrin-coated pits. On its own, the phospholipid-binding ENTH domain could rescue the epsin-null phenotype. Although necessary and sufficient for function, the isolated ENTH domain was not targeted within clathrin-coated pits. The C-terminal domain containing the coated-pit motif was also insufficient, highlighting a requirement for both domains for targeting to coated pits. Replacement of the ENTH domain by an alternative membrane-binding domain resulted in epsin that sequestered clathrin and AP2 and ablated clathrin function, supporting a modulatory role for the ENTH domain. Within the ENTH domain, residues important for PtdIns(4,5)P2 binding were essential for both epsin localization and function, whereas residue T107 was essential for function but not coated pit localization. Our results support a model where the ENTH domain coordinates with the clathrin-binding C-terminal domain to allow a dynamic interaction of epsin with coated pits.

Background: Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux), a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood., Results: To understand the role of auxilin in Notch ligand endocytosis, we have analyzed several point mutations affecting specific domains of dAux. In agreement with previous work, analysis using these stronger dAux alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in dAux deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing dAux phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in dAux mutant cells, Delta accumulates at the cell surface. In dAux mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures., Conclusion: Our data suggest that dAux mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in dAux mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, our observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization.

The AP-2 complex is a key factor in the formation of endocytic clathrin-coated vesicles (CCVs). AP-2 sorts and packages cargo membrane proteins into CCVs, binds the coat protein clathrin, and recruits numerous other factors to the site of vesicle formation. Structural information on the AP-2 complex and biochemical work have allowed understanding its function on the molecular level, and recent studies showed that cycles of phosphorylation are key steps in the regulation of AP-2 function. The complex is phosphorylated on both large subunits (alpha- and beta2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit mu2. Phosphorylation of mu2 is necessary for efficient cargo recruitment, whereas the functional context of the large subunit phosphorylation is unknown. Here, we show that the subunit phosphorylation of AP-2 exhibits striking differences, with calculated half-lives of <1 min for mu2, approximately 25 min for beta2, and approximately 70 min for alpha. We were also able to purify a phosphatase that dephosphorylates the mu2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic Cbeta subunit, a scaffolding Abeta subunit, and a regulatory Balpha subunit. RNA interference knock down of the latter subunit in HeLa cells resulted in increased levels of phosphorylated adaptors and altered endocytosis, showing that a specific PP2A holoenzyme is an important regulatory enzyme in CCV-mediated transport.

The B cell antigen receptor (BCR) delivers antigen to the endocytic compartment and transduces signals that regulate the stability of the receptor complex. Previous studies showed that BCR-mediated signal transduction dissociates micro-heavy chain (microm) from Ig-alpha/Ig-beta, facilitating the delivery of antigen to clathrin-coated vesicles (CCVs). Herein, we demonstrate that the dissociation of Ig-alpha/Ig-beta from microm requires tyrosine-587 of the microm transmembrane domain. Receptors expressing a mutation at tyrosine-587 (Y587F) transduced signals that were comparable to wild type, yet they failed to dissociate microm from Ig-alpha/Ig-beta. Further, receptors harboring the Y587F mutation failed to associate with CCVs, resulting in diminished antigen in the lysosome-associated membrane protein-1 (LAMP-1(+)) compartment and severely impaired antigen presentation, indicating that endocytosis through CCVs is required for antigen presentation. Thus, the transmembrane tyrosine of mum mediates destabilization of the BCR complex, facilitating antigen processing by promoting the association of antigen with CCVs.

SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1alpha. SGIP1alpha bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1alpha furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1alpha was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1alpha was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1alpha at clathrin-coated pits/vesicles. SGIP1alpha overexpression reduced transferrin and EGF endocytosis. SGIP1alpha knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1alpha plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15.

Clathrin-coated vesicles (CCVs) are responsible for the endocytosis of multiple cargo, including synaptic vesicle membranes. We now describe a new CCV protein, termed connecdenn, that contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a poorly characterized protein module found in multiple proteins of unrelated function and a C-terminal peptide motif domain harboring three distinct motifs for binding the alpha-ear of the clathrin adaptor protein 2 (AP-2). Connecdenn coimmunoprecipitates and partially colocalizes with AP-2, and nuclear magnetic resonance and peptide competition studies reveal that all three alpha-ear-binding motifs contribute to AP-2 interactions. In addition, connecdenn contains multiple Src homology 3 (SH3) domain-binding motifs and coimmunoprecipitates with the synaptic SH3 domain proteins intersectin and endophilin A1. Interestingly, connecdenn is enriched on neuronal CCVs and is present in the presynaptic compartment of neurons. Moreover, connecdenn has a uniquely stable association with CCV membranes because it resists extraction with Tris and high-salt buffers, unlike most other CCV proteins, but it is not detected on purified synaptic vesicles. Together, these observations suggest that connecdenn functions on the endocytic limb of the synaptic vesicle cycle. Accordingly, disruption of connecdenn interactions with its binding partners through overexpression of the C-terminal peptide motif domain or knock down of connecdenn through lentiviral delivery of small hairpin RNA both lead to defects in synaptic vesicle endocytosis in cultured hippocampal neurons. Thus, we identified connecdenn as a component of the endocytic machinery functioning in synaptic vesicle endocytosis, providing the first evidence of a role for a DENN domain-containing protein in endocytosis.

Rab14 localizes to the Golgi/TGN and to early endosomes, but its biological function remains unclear. By structural modeling, we identified Rab14-specific residues and established a close relationship between the Rab2/Rab4/Rab14, Rab11/25 and Rab39 sub-groups within the Rab protein family. By quantitative confocal microscopy and by density centrifugation we show that Rab14 is part of the early endosomal AP-1 microdomain. Overexpression of a dominant-negative Rab14 GTP-binding mutant that solely localizes to the Golgi donor compartment accelerated EGF degradation. We suggest that the AP-1 microdomain represents the interconnecting compartment in which Rab14 vesicles cycle between early endosomes and the Golgi cisternae.

The AP-1 and AP-2 complexes are the most abundant adaptors in clathrin-coated vesicles (CCVs), but clathrin-mediated trafficking can still occur in the absence of any detectable AP-1 or AP-2. To find out whether adaptor abundance reflects cargo abundance, we used lectin pulldowns to identify the major membrane glycoproteins in CCVs from human placenta and rat liver. Both preparations contained three prominent high molecular-weight proteins: the cation-independent mannose 6-phosphate receptor (CIMPR), carboxypeptidase D (CPD) and low-density lipoprotein receptor-related protein 1 (LRP1). To investigate how these proteins are sorted, we constructed and stably transfected CD8 chimeras into HeLa cells. CD8-CIMPR localized mainly to early/tubular endosomes, CD8-CPD to the trans Golgi network and CD8-LRP1 to late/multivesicular endosomes. All three constructs redistributed to the plasma membrane when clathrin was depleted by siRNA. CD8-CIMPR was also strongly affected by AP-2 depletion. CD8-CPD was moderately affected by AP-2 depletion but strongly affected by depleting AP-1 and AP-2 together. CD8-LRP1 was only slightly affected by AP-2 depletion; however, mutating an NPXY motif in the LRP1 tail caused it to become AP-2 dependent. These results indicate that all three proteins have AP-dependent sorting signals, which may help to explain the relative abundance of AP complexes in CCVs. However, the relatively low abundance of cargo proteins in CCV preparations suggests either that some of the APs may be empty or that the preparations may be dominated by empty coats.

Endocytosis by clathrin-coated vesicles (CCVs) is an important mechanism mediating protein internalization. Here, we show that two proteins identified through a proteomics analysis of CCVs are new components of the endocytic machinery. The proteins, named NECAP (adaptin-ear-binding coat-associated protein) 1 and 2, are paralogues that display no sequence similarity or common domains with any known protein. Both are enriched in CCV coats, and further analysis of the brain-enriched isoform, NECAP 1, shows its partial localization to clathrin-coated pits and direct binding to the globular ear domain of the alpha-adaptin subunit (alpha-ear) of the adaptor protein 2 (AP-2) complex. Intriguingly, this interaction is mediated by a new motif, WVQF, that uses a distinct alpha-ear interface relative to known alpha-ear-binding partners. Disruption of this interaction blocks clathrin-mediated endocytosis. Together, our studies identify a new family of endocytic proteins that define a unique AP-2-binding motif.

EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

Clathrin-adaptor complexes (APs) are vesicle coat components that participate in cargo selectivity and transport vesicle formation. Here we cloned and characterized apm1, apm3 and apm4 cDNAs encoding AP medium chains (mu) in D. discoideum. Amino acid comparison suggested that predicted proteins were homologous to known mu1, mu3 and mu4 subunits of mammalian APs as they shared 69, 51, and 26% identity with mouse mu1A, human mu3A and human mu4, respectively. In all chains, amino acid residues predicted to interact with tyrosine based sorting signals were conserved. Southern blot analysis indicated only one copy of each gene in D. discoideum genome. Expression of apm1 and apm3 mRNAs stayed relatively constant during vegetative growth and throughout development. In contrast, apm4 was poorly expressed in amoebae but became well detectable by RT-PCR upon cell differentiation. This regulated expression of coat proteins enlightens the importance of intracellular membrane transport vesicles during development in D. discoideum and strengthens this attractive model organism for studying the function of coat complexes in vivo.