Eps15 homology domain-NPF motif interactions regulate clathrin coat assembly during synaptic vesicle recycling

Although genetic and biochemical studies suggest a role for Eps15 homology domain containing proteins in clathrin-mediated endocytosis, the specific functions of these proteins have been elusive. Eps15 is found at the growing edges of clathrin-coated pits, leading to the hypothesis that it participates in the formation of coated vesicles. We have evaluated this hypothesis by examining the effect of Eps15 on clathrin assembly. We found that although Eps15 has no intrinsic ability to assemble clathrin, it potently stimulates the ability of the clathrin adaptor protein, AP180, to assemble clathrin at physiological pH. We have also defined the binding sites for Eps15 on squid AP180. These sites contain an NPF motif, and peptides derived from these binding sites inhibit the ability of Eps15 to stimulate clathrin assembly in vitro. Furthermore, when injected into squid giant presynaptic nerve terminals, these peptides inhibit the formation of clathrin-coated pits and coated vesicles during synaptic vesicle endocytosis. This is consistent with the hypothesis that Eps15 regulates clathrin coat assembly in vivo, and indicates that interactions between Eps15 homology domains and NPF motifs are involved in clathrin-coated vesicle formation during synaptic vesicle recycling.     

Two WXXF-based motifs in NECAPs define the specificity of accessory protein binding to AP-1 and AP-2

The adaptor proteins AP-2 and AP-1/GGAs are essential components of clathrin coats at the plasma membrane and trans-Golgi network, respectively. The adaptors recruit accessory proteins to clathrin-coated pits, which is dependent on the adaptor ear domains engaging short peptide motifs in the accessory proteins. Here, we perform an extensive mutational analysis of a novel WXXF-based motif that functions to mediate the binding of an array of accessory proteins to the alpha-adaptin ear domain of AP-2. Using nuclear magnetic resonance and mutational studies, we identified WXXF-based motifs as major ligands for a site on the alpha-ear previously shown to bind the DPW-bearing proteins epsin 1/2. We also defined the determinants that allow for specific binding of the alpha-ear motif to AP-2 as compared to those that allow a highly related WXXF-based motif to bind to the ear domains of AP-1/GGAs. Intriguingly, placement of acidic residues around the WXXF cores is critical for binding specificity. These studies provide a structural basis for the specific recruitment of accessory proteins to appropriate sites of clathrin-coated vesicle formation.     

Tyrosine-based endocytic motifs stimulate oligomerization of AP-2 adaptor complexes

The clathrin adaptor complex AP-2 functions in the assembly of clathrin-coated vesicles at the plasma membrane where it serves to couple endocytic vesicle formation to the selection of membrane cargo proteins. Recent evidence suggests that binding of tyrosine-based endocytic sorting motifs may induce a conformational change within the AP-2 adaptor complex that could enhance its interaction with other cargo molecules and with the membrane. We report here that soluble tyrosine-based endocytic sorting motif peptides facilitate clathrin/AP-2 recruitment to liposomal membranes and induce adaptor oligomerization even in the absence of a lipid bilayer. These effects are specific for endocytic motifs of the type Yxxphi whereas peptides corresponding to NPxY- or di-leucine-containing sorting signals are ineffective. Our data may help to explain how the highly cooperative assembly of clathrin and adaptors could be linked to the selection of membrane cargo proteins.     

P4-ATPase requirement for AP-1/clathrin function in protein transport from the trans-Golgi network and early endosomes

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Delta cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.     

Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site.

In previous work, we showed that peptides from endocytosed proteins containing the tyrosine YXXphi sorting motif are recognized by the mu 2 subunit of AP-2, the plasma membrane clathrin adaptor protein complex. This interaction is activated by phosphoinositide lipids that are phosphorylated at the D-3 position of the inositol ring, and is also enhanced by the formation of clathrin-AP-2 coats. Here, we describe the detection of a specific interaction between peptides containing a second sorting motif, the dileucine motif, and AP-1, the clathrin adaptor complex responsible for sorting proteins at the trans-Golgi network (TGN). Surprisingly, the site of dileucine binding is the beta1 subunit, not mu 1. A YXXphi-containing peptide from a protein trafficked within the TGN does bind to mu 1, however. Phosphatidylinositol 3,4-diphosphate and 3,4, 5-triphosphate did not activate the interaction between dileucine-containing peptides and AP-1 but instead inhibited it, and clathrin-AP-1 coat formation did not alter the interaction. Thus, there are at least two physically separate binding sites for sorting signals on APs, which are also regulated independently.     

The tyrosine-based lysosomal targeting signal in lamp-1 mediates sorting into Golgi-derived clathrin-coated vesicles.

Diversion of membrane proteins from the trans-Golgi network (TGN) or the plasma membrane into the endosomal system occurs via clathrin-coated vesicles (CCVs). These sorting events may require the interaction of cytosolic domain signals with clathrin adaptor proteins (APs) at the TGN (AP-1) or the plasma membrane (AP-2). While tyrosine- and di-leucine-based signals in several proteins mediate endocytosis via cell surface CCVs, segregation into Golgi-derived CCVs has so far only been documented for the mannose 6-phosphate receptors, where it is thought to require a casein kinase II phosphorylation site adjacent to a di-leucine motif. Although recently tyrosine-based signals have also been shown to interact with the mu chain of AP-1 in vitro, it is not clear if these signals also bind intact AP-1 adaptors, nor if they can mediate sorting of proteins into AP-1 CCVs. Here we show that the cytosolic domain of the lysosomal membrane glycoprotein lamp-1 binds AP-1 and AP-2. Furthermore, lamp-1 is present in AP-1-positive vesicles and tubules in the trans-region on the Golgi complex. AP-1 binding as well as localization to AP-1 CCVs require the presence of the functional tyrosine-based lysosomal targeting signal of lamp-1. These results indicate that lamp-1 can exit the TGN in CCVs and that tyrosine signals can mediate these sorting events.     

AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways

The adaptor complexes AP-1 and AP-3 are localized to endosomes and/or the trans Golgi network (TGN). Because of limitations in analysing intracellular adaptor function directly, their site of function is a matter of ongoing uncertainty. To overcome this problem and to analyse adaptor sorting at the TGN, we reconstituted vesicle formation from Golgi/TGN-enriched membranes in a novel in vitro budding assay. Melanocytes were metabolically labelled followed by a 19°C temperature block to accumulate newly synthesized proteins in Golgi membranes, which were then enriched by subcellular fractionation and used as donor membranes for vesicle formation in vitro . The incorporation of the melanosomal proteins tyrosinase and tyrosinase-related protein 1 (TRP-1) as well as Lamp-1 and 46 kDa mannose-6-phosphate receptor (MPR46) into Golgi/TGN-derived vesicles was temperature, nucleotide, cytosol, ADP ribosylation factor 1 and adaptor dependent. We show that sorting of TRP-1 and MPR46 was AP-1 dependent, while budding of tyrosinase and Lamp-1 required AP-3. Depletion of clathrin inhibited sorting of all four cargo proteins, suggesting that AP-1 and AP-3 are involved in the formation of distinct types of clathrin-coated vesicles, each of which is characterized by the incorporation of specific cargo membrane proteins.     

γ Subunit of the AP-1 Adaptor Complex Binds Clathrin: Implications for Cooperative Binding in Coated Vesicle Assembly

The heterotetrameric AP-1 adaptor complex is involved in the assembly of clathrin-coated vesicles originating from the trans-Golgi network (TGN). The beta 1 subunit of AP-1 is known to contain a consensus clathrin binding sequence, LLNLD (the so-called clathrin box motif), in its hinge segment through which the beta chain interacts with the N-terminal domains of clathrin trimers. Here, we report that the hinge region of the gamma subunit of human and mouse AP-1 contains two copies of a new variant, LLDLL, of the clathrin box motif that also bind to the terminal domain of the clathrin heavy chain. High-affinity binding of the gamma hinge to clathrin trimers requires both LLDLL sequences to be present and the spacing between them to be maintained. We also identify an independent clathrin-binding site within the appendage domain of the gamma subunit that interacts with a region of clathrin other than the N-terminal domain. Clathrin polymerization is promoted by glutathione S-transferase (GST)-gamma hinge, but not by GST-gamma appendage. However, the hinge and appendage domains of gamma function in a cooperative manner to recruit and polymerize clathrin, suggesting that clathrin lattice assembly at the TGN involves multivalent binding of clathrin by the gamma and beta1 subunits of AP-1.     

The interaction of beta-arrestin with the AP-2 adaptor is required for the clustering of beta 2-adrenergic receptor into clathrin-coated pits

Beta-arrestins are cytosolic proteins that regulate the signaling and the internalization of G protein-coupled receptors (GPCRs). Although termination of receptor coupling requires beta-arrestin binding to agonist-activated receptors, GPCR endocytosis involves the coordinate interactions between receptor-beta-arrestin complexes and other endocytic proteins such as adaptor protein 2 (AP-2) and clathrin. Clathrin interacts with a conserved motif in the beta-arrestin C-terminal tail; however, the specific molecular determinants in beta-arrestin that bind AP-2 have not been identified. Moreover, the respective contributions of the interactions of beta-arrestin with AP-2 and clathrin toward the targeting of GPCRs to clathrin-coated vesicles have not been established. Here, we identify specific arginine residues (Arg(394) and Arg(396)) in the beta-arrestin 2 C terminus that mediate beta-arrestin binding to AP-2 and show, in vitro, that these domains in beta-arrestin 1 and 2 interact equally well with AP-2 independently of clathrin binding. We demonstrate in HEK 293 cells by fluorescence microscopy that beta(2)-adrenergic receptor-beta-arrestin complexes lacking the beta-arrestin-clathrin binding motif are still targeted to clathrin-coated pits. In marked contrast, receptor-beta-arrestin complexes lacking the beta-arrestin/AP-2 interactions are not effectively compartmentalized in punctated areas of the plasma membrane. These results reveal that the binding of a receptor-beta-arrestin complex to AP-2, not to clathrin, is necessary for the initial targeting of beta(2)-adrenergic receptor to clathrin-coated pits.     

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane.     

Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes.

Many plasma membrane proteins destined for endocytosis are concentrated into clathrin-coated pits through the recognition of a tyrosine-based motif in their cytosolic domains by an adaptor (AP-2) complex. The mu2 subunit of isolated AP-2 complexes binds specifically, but rather weakly, to proteins bearing the tyrosine-based signal. We now demonstrate, using peptides with a photoreactive probe, that this binding is strengthened significantly when the AP-2 complex is present in clathrin coats, indicating that there is cooperativity between receptor-AP-2 interactions and coat formation. Phosphoinositides with a phosphate at the D-3 position of the inositol ring, but not other isomers, also increase the affinity of the AP-2 complex for the tyrosine-based motif. AP-2 is the first protein known (in any context) to interact with phosphatidylinositol 3-phosphate. Our findings indicate that receptor recruitment can be coupled to clathrin coat assembly and suggest a mechanism for regulation of membrane traffic by lipid products of phosphoinositide 3-kinases.     

Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells

Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.     

High-affinity binding of the AP-1 adaptor complex to trans-golgi network membranes devoid of mannose 6-phosphate receptors

The GTP-binding protein ADP-ribosylation factor (ARF) initiates clathrin-coat assembly at the trans-Goli network (TGN) by generating high-affinity membrane-binding sites for the AP-1 adaptor complex. Both transmembrane proteins, which are sorted into the assembling coated bud, and novel docking proteins have been suggested to be partners with GTP-bound ARF in generating the AP-1-docking sites. The best characterized, and probably the major transmembrane molecules sorted into the clathrin-coated vesicles that form on the TGN, are the mannose 6-phosphate receptors (MPRs). Here, we have examined the role of the MPRs in the AP-1 recruitment process by comparing fibroblasts derived from embryos of either normal or MPR-negative animals. Despite major alterations to the lysosome compartment in the MPR-deficient cells, the steady-state distribution of AP-1 at the TGN is comparable to that of normal cells. Golgi-enriched membranes prepared from the receptor-negative cells also display an apparently normal capacity to recruit AP-1 in vitro in the presence of ARF and either GTP or GTPgammaS. The AP-1 adaptor is recruited specifically onto the TGN and not onto the numerous abnormal membrane elements that accumulate within the MPR-negative fibroblasts. AP-1 bound to TGN membranes from either normal or MPR-negative fibroblasts is fully resistant to chemical extraction with 1 M Tris-HCl, pH 7, indicating that the adaptor binds to both membrane types with high affinity. The only difference we do note between the Golgi prepared from the MPR-deficient cells and the normal cells is that AP-1 recruited onto the receptor-lacking membranes in the presence of ARF1.GTP is consistently more resistant to extraction with Tris. Because sensitivity to Tris extraction correlates well with nucleotide hydrolysis, this finding might suggest a possible link between MPR sorting and ARF GAP regulation. We conclude that the MPRs are not essential determinants in the initial steps of AP-1 binding to the TGN but, instead, they may play a regulatory role in clathrin-coated vesicle formation by affecting ARF.GTP hydrolysis.     

Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation.

The composition of secretory granules in neuroendocrine and endocrine cells is determined by two sorting events; the first in the trans-Golgi complex (TGN), the second in the immature secretory granule (ISG). Sorting from the ISG, which may be mediated by the AP-1 type adaptor complex and clathrin-coated vesicles, occurs during ISG maturation. Here we show that furin, a ubiquitously expressed, TGN/endosomal membrane endoprotease, is present in the regulated pathway of neuroendocrine cells where it is found in ISGs. By contrast, TGN38, a membrane protein that is also routed through the TGN/endosomal system does not enter ISGs. Furin, however, is excluded from mature secretory granules, suggesting that the endoprotease is retrieved from the clathrin-coated ISGs. Consistent with this, we show that the furin cytoplasmic domain interacts with AP-1, a component of the TGN/ISG-localized clathrin sorting machinery. Interaction between AP-1 and furin is dependent on phosphorylation of the enzyme's cytoplasmic domain by casein kinase II. Finally, in support of a requirement for the phosphorylation-dependent association of furin with AP-1, expression of furin mutants that mimic either the phosphorylated or unphosphorylated forms of the endoprotease in AtT-20 cells demonstrates that the integrity of the CKII sites is necessary for removal of furin from the regulated pathway.     

AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.     

Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification

The effects of methods known to perturb endocytosis from clathrin- coated pits on the localization of clathrin and HA2 adaptors in HEp-2 carcinoma cells have been studied by immunofluorescence and ultrastructural immunogold microscopy, using internalization of transferrin as a functional assay. Potassium depletion, as well as incubation in hypertonic medium, remove membrane-associated clathrin lattices: flat clathrin lattices and coated pits from the plasma membrane, and clathrin-coated vesicles from the cytoplasm, as well as those budding from the TGN. In contrast, immunofluorescence microscopy using antibodies specific for the alpha- and beta-adaptins, respectively, and immunogold labeling of cryosections with anti-alpha- adaptin antibodies shows that under these conditions HA2 adaptors are aggregated at the plasma membrane to the same extent as in control cells. After reconstitution with isotonic K(+)-containing medium, adaptor aggregates and clathrin lattices colocalize at the plasma membrane as normally and internalization of transferrin resumes. Acidification of the cytosol affects neither clathrin nor HA2 adaptors as studied by immunofluorescence microscopy. However, quantitative ultrastructural observations reveal that acidification of the cytosol results in formation of heterogeneously sized and in average smaller clathrin-coated pits at the plasma membrane and buds on the TGN. Collectively, our observations indicate that the methods to perturb formation of clathrin-coated vesicles act by different mechanisms: acidification of the cytosol by affecting clathrin-coated membrane domains in a way that interferes with budding of clathrin-coated vesicles from the plasma membrane as well as from the TGN; potassium depletion and incubation in hypertonic medium by preventing clathrin and adaptors from interacting. Furthermore our observations show that adaptor aggregates can exist at the plasma membrane independent of clathrin lattices and raise the possibility that adaptor aggregates can form nucleation sites for clathrin lattices.     

SGIP1alpha is an endocytic protein that directly interacts with phospholipids and Eps15

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.     

Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-α results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both α-adaptin and clathrin. Upon EGF stimulation, Eps15 and α-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.     

Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites

Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin.     

ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma

Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.