Self-association of the plasma membrane-associated clathrin assembly protein AP-2

A self-association reaction involving the plasma membrane-associated clathrin assembly protein AP-2 has been detected by incubating AP-2 alone under solution conditions that would favor the assembly of complete coat structures if clathrin were present. Self-association was rapid, unaffected by nonionic detergents, readily reversible, and gave rise to sedimentable aggregates. Only the AP subtype AP-2 exhibited self-association: the structurally or functionally related assembly proteins AP-1 and AP-3 and unrelated proteins neither self-associated nor were incorporated into the AP-2 aggregate. AP-2 interactions responsible for self-association were of high affinity, with an apparent Kd of approximately 10(-8)M. By proteolytic dissection, the self-association domain was localized to the core of the molecule containing the intact 50- and 16-kDa polypeptides in association with the truncated 60-66-kDa moieties of the parent alpha/beta polypeptides. Self-association of the intact AP-2 molecule was pH-dependent, exhibiting an apparent pKa approximately 7.4. While it is unlikely that the large AP-2 aggregates formed in solution are themselves biologically relevant structures, the AP-2 interactions involved in their formation have properties consistent with their occurrence in intact cells and thus may be important in cellular functions of the plasma membrane-localized assembly protein.     

Interaction of assembly protein AP-2 and its isolated subunits with clathrin

The clathrin assembly protein complex AP-2 is a multimeric subunit complex consisting of two 100-115-kDa subunits known as alpha and beta and 50- and 16-kDa subunits. The subunits have been dissociated and separated by ion-exchange chromatography in 7.5 M urea. Fractions highly enriched in either the alpha or beta subunit were obtained. The alpha fraction interacted with clathrin as evidenced by its ability to bind to preassembled clathrin cages. It also reacted with dissociated clathrin trimers under conditions that favor assembly of coat structures, but did not yield discrete clathrin polygonal lattices. The enriched beta fraction (containing small amounts of alpha) reacted with clathrin to yield intact coats with the incorporation of approximately equivalent amounts of alpha and beta subunits into the polymerized species; excess free beta subunit was unreactive. The AP-2 complex was also completely dissociated in a highly denaturing solvent, 6 M Gdn.HCl, and the constituent subunits of 100-115, 50, and 16 kDa were separated by gel filtration. In a coassembly assay with clathrin, the clathrin polymerizing activity was exclusively associated with the 100-kDa subunit fraction with stoichiometric incorporation of both alpha and beta subunits of 100 kDa into the polymerized coats, and with no requirement for 50- or 16-kDa subunits. These observations demonstrate that the assembly activity of the complex is associated with the alpha and beta subunits and suggest that both subunits, through independent interactions with clathrin, are required for expression of complete lattice assembly activity.     

Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2.

To clarify the function of the receptor binding protein for inositol hexakisphosphate (IP6), we obtained a partial amino acid sequence from the purified protein and a partial nucleotide sequence from a cDNA clone of the gene. The sequences are essentially identical to those of the alpha-subunit of the clathrin assembly protein AP-2. The IP6 receptor protein analyzed by SDS-PAGE contains a series of subunits which are the same as those of AP-2. Antibodies to AP-2 react with the IP6 receptor protein in immunoblot analysis.     

The AP-2 complex is excluded from the dynamic population of plasma membrane-associated clathrin

Numerous biologically relevant substrates are selectively internalized via clathrin-mediated endocytosis. At the plasma membrane the AP-2 complex plays a major role in clathrin coat formation, interacting with both cargo and clathrin. Utilizing simultaneous dual-channel total internal reflection fluorescence microscopy we have analyzed components of the AP-2 complex (alpha- and beta 2-adaptin) during clathrin-mediated endocytosis. Although in static images enhanced green fluorescent protein-tagged AP-2 markers significantly co-localized with clathrin and other components of clathrin-coated pits, AP-2 did not seem to be present in clathrin spots that appeared to undergo internalization or motility parallel to the plane of the plasma membrane. Two populations of clathrin at the plasma membrane seem to exist, the dynamic and the static, and AP-2 appears to be only found within the latter. These results suggest that colocalized clathrin/AP-2 puncta may represent loci for coated pit production and that previous models that assumed AP-2 was retained within clathrin coats during endocytosis may need to be re-evaluated.     

SMAP2, a novel ARF GTPase-activating protein, interacts with clathrin and clathrin assembly protein and functions on the AP-1-positive early endosome/trans-Golgi network

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1-dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.     

Characterization of a protein phosphatase 2A holoenzyme that dephosphorylates the clathrin adaptors AP-1 and AP-2

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.     

Stabilization of clathrin coats by the core of the clathrin-associated protein complex AP-2

AP-2 is the class of clathrin-associated protein complex found in coated vesicles derived from the plasma membrane of eukaryotic cells. We demonstrate here, using a chemical method, that an AP-2 complex is an asymmetric structure consisting of one large alpha chain, one large beta chain, one medium AP50 chain, and one small AP17 chain. The complex has been shown to contain a core and two appendages. The AP core includes the small AP17 and the medium AP50 chains together with the amino-terminal domains of the large alpha and beta chains. One appendage corresponds to the carboxy-terminal domain of the beta chain. We find that as in the case of the beta chains, the carboxy-terminal portion of the alpha chains is an independently folded domain corresponding to the second appendage. We use limited tryptic proteolysis of clathrin/AP-2 coats to show the release of the appendages from the interior of the coats and the retention of the AP core by the remaining clathrin lattice. In addition, we find that the AP core stabilizes the coat and prevents its depolymerization. These results are consistent with the proposal that the AP core contains the binding site(s) for clathrin, while the alpha- and beta-chain appendages interact with membrane components of coated pits and coated vesicles.     

Molecular switches involving the AP-2 beta2 appendage regulate endocytic cargo selection and clathrin coat assembly

Clathrin-associated sorting proteins (CLASPs) expand the repertoire of endocytic cargo sorted into clathrin-coated vesicles beyond the transmembrane proteins that bind physically to the AP-2 adaptor. LDL and GPCRs are internalized by ARH and beta-arrestin, respectively. We show that these two CLASPs bind selectively to the AP-2 beta2 appendage platform via an alpha-helical [DE](n)X(1-2)FXX[FL]XXXR motif, and that this motif also occurs and is functional in the epsins. In beta-arrestin, this motif maintains the endocytosis-incompetent state by binding back on the folded core of the protein in a beta strand conformation. Triggered via a beta-arrestin/GPCR interaction, the motif must be displaced and must undergo a strand to helix transition to enable the beta2 appendage binding that drives GPCR-beta-arrestin complexes into clathrin coats. Another interaction surface on the beta2 appendage sandwich is identified for proteins such as eps15 and clathrin, suggesting a mechanism by which clathrin displaces eps15 to lattice edges during assembly.     

Endocytosis: an assembly protein for clathrin cages.

The protein AP180 is known to have clathrin-assembly activity in vitro. AP180 has now been found to be crucial for synaptic vesicle endocytosis and the maintenance of a uniform-size vesicle population in vivo. These results significantly advance our understanding of clathrin-mediated endocytosis in the synapse and elsewhere.     

Direct interaction of the 170 kDa isoform of synaptojanin 1 with clathrin and with the clathrin adaptor AP-2

Synaptojanin 1, a polyphosphoinositide phosphatase, is expressed as two major alternatively spliced isoforms of 145 kDa (SJ145) and 170 kDa (SJ170) [1] [2], which are thought to have pleiotropic roles in endocytosis, signaling and actin function [3] [4] [5]. SJ145 is highly enriched in nerve terminals where it participates in clathrin-dependent synaptic vesicle recycling [1] [5]. SJ170, which differs from SJ145 by the presence of a carboxy-terminal extension, is the predominant isoform in developing neurons and is expressed in a variety of tissues [2]. The carboxy-terminal domain unique to SJ170 was previously shown to bind Eps15 [6], a protein involved in receptor-mediated endocytosis. Here, we show that the same domain also binds clathrin and the clathrin adaptor AP-2. These interactions occur both in vitro and in vivo and are direct. Binding of AP-2 is mediated by the ear domain of its alpha-adaptin subunit and binding of clathrin by the amino-terminal domain of its heavy chain. Overexpression in chinese hamster ovary (CHO) cells of full-length SJ170 or its unique carboxy-terminal region caused mislocalization of Eps15, AP-2 and clathrin, as well as inhibition of clathrin-dependent transferrin uptake. These findings suggest a close association of SJ170 with the clathrin coat and provide new evidence for its physiological role in the regulation of clathrin coat dynamics.     

Recognition sites for clathrin-associated proteins AP-2 and AP-3 on clathrin triskelia.

AP-2 and AP-3 are cellular proteins that drive the in vitro polymerization of clathrin triskelia into cage structures. The interaction of these two types of assembly proteins (APs) with preassembled clathrin cages has been studied in order to identify the sites on the triskelia required for binding. Comparing binding of the APs to intact or to proteolytically clipped cages, we attempted to distinguish between binding to the terminal domain, the globular end of the heavy chain, and binding to the hub of the clathrin triskelia, the portion that remains assembled after trypsin treatment. AP-3 binds to intact clathrin cages but not to those that were treated with trypsin. AP-3 also bound to cages consisting solely of clathrin heavy chains; proteolysis of these cages also eliminated AP-3 binding. In addition, AP-3 did not bind to either isolated hubs or terminal domains that had been immobilized on Sepharose. These data indicate that clathrin light chains are not required for binding of AP-3, and that neither terminal domain nor hubs alone will suffice. However, an intact heavy chain is both necessary and sufficient for the binding of AP-3. Previous work has demonstrated one binding site for AP-2 on proteolyzed cages containing only clathrin hubs; the existence of a second binding site associated with the terminal domain was hypothesized. Here we provide direct evidence for recognition by AP-2 of isolated terminal domains immobilized on Sepharose and show that the core of the AP-2 molecule is responsible for this interaction. These results provide the first demonstration of a functional role for the conserved terminal domain of the clathrin heavy chain.     

Purification and properties of a new clathrin assembly protein.

A clathrin assembly protein (AP180) has been purified and characterized from coated vesicles of bovine brain. This protein has hitherto escaped detection because in SDS-gel electrophoresis it is obscured by the 180 kd heavy chain of clathrin. Despite the similarity in electrophoretic mobility, AP180 differs from clathrin in both its subunit and native mol. wt, as well as hydrodynamic properties, surface charge and tryptic peptide composition. It also appears immunologically distinct from clathrin, since neither a polyclonal antiserum nor a monoclonal antibody, that have been shown to be specific for AP180, cross-react with the heavy chain of clathrin. AP180 binds to clathrin triskelia and thereby promotes clathrin assembly into regular polyhedral structures of narrow size-distribution (60-90 nm), reminiscent of the surface coat of coated vesicles. In this respect AP180 bears a functional resemblance to the 100-110 kd clathrin assembly polypeptides that have been previously described.     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL₂ and HDL₃, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes.

When incubated with intact erythrocytes, low density lipoproteins (LDL) decrease the phosphate content of erythrocyte spectrin allowing the cells to undergo morphological transformation. The phosphate content of spectrin depends on the balance between the activity of membrane-associated cyclic AMP-independent protein kinases and phosphoprotein phosphates. LDL do not influence the activity of membrane-associated cyclic AMP-independent protein kinases; these lipoproteins activate by 2-fold and greater membrane-associated phosphatases as determined by hydrolysis of p-nitrophenyl phosphate and by phosphate hydrolysis of phosphorylated erythrocyte membrane proteins. We conclude that LDL interact at the exterior surface of the erythrocyte to stimulate dephosphorylation of spectrin. The significance of this conclusion is augmented by the fact that spectrin, the target for LDL-induced dephosphorylation, specifies cell morphology and modulates the distribution of cell-surface receptors. LDL also render erythrocyte acetylcholinesterase less susceptible to inhition by F-. Lipoproteins in the high density class (HDL) do not stimulate dephosphorylation of spectrin, and they are consequently unable to alter erythrocyte morphology. HDL do prevent the LDL-induced activation of membrane phosphatase. The inhibitory capacity of HDL is observed over the range of LDL:HDL (w/w) which exists in the plasma of normolipemic humans.     

Multiple interactions of auxilin 1 with clathrin and the AP-2 adaptor complex

The removal of the clathrin coat is essential for vesicle fusion with acceptor membranes. Disassembly of the coat involves hsc70, which is specifically recruited by members of the auxilin protein family to clathrin lattices. In vitro, this function of auxilin does not require the globular amino-terminal domain of the clathrin heavy chain, which is known to play a prominent role in the interaction of clathrin with adaptors and numerous endocytic accessory proteins. Here we report the unexpected finding that the neuron-specific form of auxilin (auxilin 1) can also associate with the clathrin amino-terminal domain. This interaction is mediated through tandemly arranged sites within the auxilin 1 carboxyl-terminal segment 547-910. The overlapping auxilin 1 fragments 547-714 and 619-738 bind the clathrin terminal domain with high affinity, whereas auxilin 1-(715-901) interacts only poorly with it. All three fragments also associate with the clathrin distal domain and the alpha-appendage domain of AP-2. Moreover, they support efficient assembly of clathrin triskelia into regular cages. A novel uncoating assay was developed to demonstrate that auxilin 1-(715-901) functions efficiently as a cofactor for hsc70 in the uncoating of clathrin-coated vesicles. The multiple protein-protein interactions of auxilin 1 suggest that its function in endocytic trafficking may be more complex than previously anticipated.     

ARH is a modular adaptor protein that interacts with the LDL receptor,clathrin, and AP-2

Mutations in the phosphotyrosine binding domain protein ARH cause autosomal recessive hypercholesterolemia, a disorder caused by defective internalization of low density lipoprotein receptors (LDLR) in the liver. To examine the function of ARH, we used pull-down experiments to test for interactions between ARH, the LDLR, and proteins involved in clathrin-mediated endocytosis. The phosphotyrosine binding domain of ARH interacted with the internalization sequence (NPVY) in the cytoplasmic tail of LDLR in a sequence-specific manner. Mutations in the NPVY sequence that were previously shown to decrease LDLR internalization abolished in vitro binding to ARH. Recombinant ARH bound purified bovine clathrin with high affinity (K(D), approximately 44 nm). The interaction between ARH and clathrin was mapped to a canonical clathrin box sequence (LLDLE) in ARH and to the N-terminal domain of the clathrin heavy chain. A highly conserved 20-amino acid sequence in the C-terminal region of ARH bound the beta(2)-adaptin subunit of AP-2. Mutation of a glutamic acid residue in the appendage domain of beta(2)-adaptin that is required for interaction with the adapter protein beta-arrestin markedly reduced binding to ARH. These data are consistent with the hypothesis that ARH functions as an adaptor protein that couples LDLR to the endocytic machinery.     

Effect of clathrin assembly lymphoid myeloid leukemia protein depletion on clathrin coat formation

The endocytic accessory clathrin assembly lymphoid myeloid leukemia protein (CALM) is the ubiquitously expressed homolog of the neuron-specific protein AP180 that has been implicated in the retrieval of synaptic vesicle. Here, we show that CALM associates with the alpha-appendage domain of the AP2 adaptor via the three peptide motifs 420DPF, 375DIF and 489FESVF and to a lesser extent with the amino-terminal domain of the clathrin heavy chain. Reducing clathrin levels by RNA interference did not significantly affect CALM localization, but depletion of AP2 weakens its association with the plasma membrane. In cells, where CALM levels were reduced by RNA interference, AP2 and clathrin remained organized in somewhat enlarged bright fluorescent puncta. Electron microscopy showed that the depletion of CALM drastically affected the clathrin lattice structure. Round-coated buds, which are the predominant features in control cells, were replaced by irregularly shaped buds and long clathrin-coated tubules. Moreover, we noted an increase in the number of very small cages that formed on flat lattices. Furthermore, we noticed a redistribution of endosomal markers and AP1 in cells that were CALM depleted. Taken together, our findings indicate a critical role for CALM in the regulation and orderly progression of coated bud formation at the plasma membrane.     

Clathrin domains involved in recognition by assembly protein AP-2

The domains on clathrin responsible for interaction with the plasma membrane-associated assembly protein AP-2 have been studied using a novel cage binding assay. AP-2 bound to pure clathrin cages but not to coat structures already containing AP that had been prepared by coassembly. Binding to preassembled cages also occurred in the presence of elevated Tris-HCl concentrations (greater than or equal to 200 mM) which block AP-2 interactions with free clathrin. AP-2 interactions with assembled cages could also be distinguished from AP-2 binding to clathrin trimers by sodium tripolyphosphate (NaPPPi), which binds to the alpha subunit of AP-2 (Beck, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4442-4447). At concentrations of 1-5 mM, NaPPPi blocked clathrin-triskelion binding; in contrast, interactions with cages persisted in the presence of 25 mM NaPPPi. To begin to identify the region(s) of the clathrin molecule important in recognition by AP-2, clathrin cages were proteolyzed to remove heavy chain terminal domains and portions of the distal leg as well as all of the light chains. AP-2 bound to these "clipped cages"; however, unlike the interaction with native cages, binding of AP-2 to clipped cages was sensitive to the lower concentrations of both Tris-HCl and NaPPPi which disrupt interactions of AP-2 with clathrin trimers. Reconstitution of the clipped cages with clathrin light chains did not restore resistance of AP-2 binding to Tris-HCl. We conclude that one binding site for AP-2 resides on the hub and/or proximal part of the clathrin triskelion whereas a second site is likely to involve the terminal domain and/or distal leg; the second site is manifested only in the assembled lattice structure. We suggest that these two distinct binding interactions may be mediated by the two unique large subunits within the AP-2 complex, acting sequentially during assembly.     

Insulin stimulates the assembly of cytosolic clathrin onto adipocyte plasma membranes

The effects of insulin on the subcellular distribution of the heavy chain of clathrin and on the insulin-like growth factor II (IGF-II) mannose 6-phosphate receptor were investigated in isolated rat adipocytes. Plasma membranes, intracellular membranes, and cytosol were separated by differential centrifugation, and the concentration of clathrin and receptor in each fraction was quantified by sequential immunoblotting with monoclonal and polyclonal antibodies against these proteins. A 3-fold increase in the amount of clathrin heavy chain associated with isolated plasma membranes was found after treatment of cells with low concentrations of insulin. This effect was complete within 2 min of stimulation at 37 degrees C and was abolished at 5-10 degrees C. The insulin-mediated increase in the cell surface concentration of receptors for IGF-II/mannose 6-phosphate displayed a similar time course and temperature dependence. A concomitant decrease in the concentration of IGF-II/mannose 6-phosphate receptors in intracellular membranes was observed. In contrast, no significant changes in the concentration of clathrin in this fraction could be detected. Instead, a marked decrease in the level of unassembled cytosolic clathrin was observed in insulin-treated cells compared with controls. These results suggest that insulin induces an increase in the assembly of cytosolic clathrin onto the plasma membrane in conjunction with its ability to increase the concentration of receptors on the cell surface.