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.     

Adaptable adaptors for coated vesicles

Adaptors select cargo for inclusion into coated vesicles in the late secretory and endocytic pathways. Although originally there were thought to be just two adaptors, AP-1 and AP-2, it is now clear that there are many more: two additional adaptor complexes, AP-3 and AP-4, which might function independently of clathrin; a family of monomeric adaptors, the GGAs; and an ever-growing number of cargo-specific adaptors. The adaptors are targeted to the appropriate membrane at least in part by interacting with phosphoinositides, and, once on the membrane, they form interconnected networks to get different types of cargo into the same vesicle. Adaptors participate in trafficking pathways shared by all cells, and they are also used to generate specialized organelles and to influence cell fate during development.     

The building blocks for basolateral vesicles in polarized epithelial cells

After the discovery of basolateral sorting signals for polarized delivery in epithelial cells in the early 1990s, it was only about a decade later that the epithelial-cell-specific sorting adaptor AP-1B was discovered. AP-1B decodes a subclass of basolateral sorting signals and localizes to the recycling endosomes as opposed to the trans-Golgi network, suggesting that this is its major site of action. Furthermore, AP-1B does not simply select its cargo but also facilitates the recruitment of the exocyst complex needed for subsequent fusion with the plasma membrane. This review discusses our current knowledge of AP-1B function in cargo sorting to the basolateral membrane and its impact on our understanding of the similarities and differences between AP-1B-minus fibroblasts and AP-1B-positive epithelial cells.     

Cargo recognition during clathrin-mediated endocytosis: a team effort

Transmembrane proteins destined to endosomes are selectively accumulated in clathrin-coated pits at the plasma membrane and rapidly internalized in clathrin-coated vesicles. The recognition of specific sequence motifs in transmembrane cargo by coated-pit proteins confers specificity on the endocytic process. Interaction of membrane cargo with the clathrin adaptor protein complex AP-2 is the major mechanism of cargo sorting into coated pits in mammalian cells. Recent studies have revealed a variety of alternative mechanisms of cargo recruitment involving additional adaptor proteins. These alternative mechanisms appear to be particularly important during clathrin-mediated endocytosis of signaling receptors.     

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.     

Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical plasma membrane of fluid-transporting epithelia, where the plasma membrane abundance of CFTR is in part controlled by clathrin-mediated endocytosis. The protein networks that control CFTR endocytosis in epithelial cells have only been partially explored. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor critical for optimal clathrin coat formation. AP-2 is essential for recruitment of cargo proteins bearing the YXXΦ motif. Although AP-2 interacts directly with CFTR in vitro and facilitates CFTR endocytosis in some cell types, it remains unknown whether it is critical for CFTR uptake into clathrin-coated vesicles (CCVs). Disabled-2 (Dab2) is a clathrin-associated sorting protein (CLASP) that contributes to clathrin recruitment, vesicle formation, and cargo selection. In intestinal epithelial cells Dab2 was not found to play a direct role in CFTR endocytosis. By contrast, AP-2 and Dab2 were shown to facilitate CFTR endocytosis in human airway epithelial cells, although the specific mechanism remains unknown. Our data demonstrate that Dab2 mediates AP-2 independent recruitment of CFTR to CCVs in polarized human airway epithelial cells. As a result, it facilitates CFTR endocytosis and reduces CFTR abundance and stability in the plasma membrane. These effects are mediated by the DAB homology domain. Moreover, we show that in human airway epithelial cells AP-2 is not essential for CFTR recruitment to CCVs.     

Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins

The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment that also exhibits budding profiles positive for AP-1. AP-3 colocalizes with clathrin, but to a lesser extent than does AP-1. The AP-3- and AP-1-bearing tubular compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein receptor, and low amounts of the cation-independent mannose 6-phosphate receptor and the lysosome-associated membrane proteins (LAMPs) 1 and 2. Quantitative analysis revealed that of these distinct cargo proteins, only LAMP-1 and LAMP-2 are concentrated in the AP-3-positive membrane domains. Moreover, recycling of endocytosed LAMP-1 and CD63 back to the cell surface is greatly increased in AP-3-deficient cells. Based on these data, we propose that AP-3 defines a novel pathway by which lysosomal membrane proteins are transported from tubular sorting endosomes to lysosomes.     

A Phosphotyrosine Switch for Cargo Sequestration at Clathrin-coated Buds

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.     

Adaptor-related proteins.

Two new adaptor-related protein complexes, AP-3 and AP-4, have recently been identified, and both have been implicated in protein sorting at the trans-Golgi network (TGN) and/or endosomes. In addition, two families of monomeric proteins with adaptor-related domains, the GGAs and the stoned B family, have also been identified and shown to act at the TGN and plasma membrane, respectively. Together with the two conventional adaptors, AP-1 and AP-2, these proteins may act to direct different types of cargo proteins to different post-Golgi membrane compartments.     

Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling

Clathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. Here, we show that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact mu-homology domain, and functional AP-2 heterotetramers. Our data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling.     

Adaptor protein complexes and intracellular transport

The AP (adaptor protein) complexes are heterotetrameric protein complexes that mediate intracellular membrane trafficking along endocytic and secretory transport pathways. There are five different AP complexes: AP-1, AP-2 and AP-3 are clathrin-associated complexes; whereas AP-4 and AP-5 are not. These five AP complexes localize to different intracellular compartments and mediate membrane trafficking in distinct pathways. They recognize and concentrate cargo proteins into vesicular carriers that mediate transport from a donor membrane to a target organellar membrane. AP complexes play important roles in maintaining the normal physiological function of eukaryotic cells. Dysfunction of AP complexes has been implicated in a variety of inherited disorders, including: MEDNIK (mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis and keratodermia) syndrome, Fried syndrome, HPS (Hermansky–Pudlak syndrome) and HSP (hereditary spastic paraplegia).     

New directions for the clathrin adaptor AP-1 in cell biology and human disease

The clathrin adaptor protein complex-1 (AP-1) is a central player in cell physiology and human health. It is best known for its role in linking clathrin to its cargo at the trans-Golgi network and endosomes. It participates in traffic important for the correct function of a large number of organelles, including the trans-Golgi network, endosomes, lysosomes, lysosome-related organelles, and plasma membrane. Although it was one of the first clathrin adaptors identified, new discoveries about cargo and pathways that depend on AP-1 continue to emerge. This review summarizes new research into AP-1 that further illuminates its roles in the traffic of plasma membrane proteins, in maintaining TGN content, and in human disease.     

A di-leucine-based motif in the cytoplasmic tail of LIMP-II and tyrosinase mediates selective binding of AP-3.

Among the various coats involved in vesicular transport, the clathrin associated coats that contain the adaptor complexes AP-1 and AP-2 are the most extensively characterized. The function of the recently described adaptor complex AP-3, which is similar to AP-1 and AP-2 in protein composition but does not associate with clathrin, is not known. By monitoring surface plasmon resonance we observed that AP-3 is able to interact with the tail of the lysosomal integral membrane protein LIMP-II and that this binding depends on a DEXXXLI sequence in the LIMP-II tail. Furthermore, AP-3 bound to the cytoplasmic tail of the melanosome-associated protein tyrosinase which contains a related EEXXXLL sequence. The tails of LIMP-II and tyrosinase either did not interact, or only interacted poorly, with AP-1 or AP-2. In contrast, the cytoplasmic tails of other membrane proteins containing di-leucine and/or tyrosine-based sorting signals did not bind AP-3, but AP-1 and/or AP-2. This points to a function of AP-3 in intracellular sorting to lysosomes and melanosomes of a subset of cargo proteins via di-leucine-based sorting motifs.     

Disabled-2 exhibits the properties of a cargo-selective endocytic clathrin adaptor

Clathrin-coated pits at the cell surface select material for transportation into the cell interior. A major mode of cargo selection at the bud site is via the micro 2 subunit of the AP-2 adaptor complex, which recognizes tyrosine-based internalization signals. Other internalization motifs and signals, including phosphorylation and ubiquitylation, also tag certain proteins for incorporation into a coated vesicle, but the mechanism of selection is unclear. Disabled-2 (Dab2) recognizes the FXNPXY internalization motif in LDL-receptor family members via an N-terminal phosphotyrosine-binding (PTB) domain. Here, we show that in addition to binding AP-2, Dab2 also binds directly to phosphoinositides and to clathrin, assembling triskelia into regular polyhedral coats. The FXNPXY motif and phosphoinositides contact different regions of the PTB domain, but can stably anchor Dab2 to the membrane surface, while the distal AP-2 and clathrin-binding determinants regulate clathrin lattice assembly. We propose that Dab2 is a typical member of a growing family of cargo-specific adaptor proteins, including beta-arrestin, AP180, epsin, HIP1 and numb, which regulate clathrin-coat assembly at the plasma membrane by synchronizing cargo selection and lattice polymerization events.     

Sorting of major cargo glycoproteins into clathrin-coated vesicles

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.     

Clathrin-mediated endocytosis in AP-2-depleted cells

We have used RNA interference to knock down the AP-2 mu2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2-depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2-depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.     

ARF1.GTP,tyrosine-based signals,and phosphatidylinositol 4,5-bisphosphate constitute a minimal machinery to recruit the AP-1 clathrin adaptor to membranes

At the trans-Golgi network, clathrin coats containing AP-1 adaptor complexes are formed in an ARF1-dependent manner, generating vesicles transporting cargo proteins to endosomes. The mechanism of site-specific targeting of AP-1 and the role of cargo are poorly understood. We have developed an in vitro assay to study the recruitment of purified AP-1 adaptors to chemically defined liposomes presenting peptides corresponding to tyrosine-based sorting motifs. AP-1 recruitment was found to be dependent on myristoylated ARF1, GTP or nonhydrolyzable GTP-analogs, tyrosine signals, and small amounts of phosphoinositides, most prominently phosphatidylinositol 4,5-bisphosphate, in the absence of any additional cytosolic or membrane bound proteins. AP-1 from cytosol could be recruited to a tyrosine signal independently of the lipid composition, but the rate of recruitment was increased by phosphatidylinositol 4,5-bisphosphate. The results thus indicate that cargo proteins are involved in coat recruitment and that the local lipid composition contributes to specifying the site of vesicle formation.     

Oligomerization and dissociation of AP-1 adaptors are regulated by cargo signals and by ArfGAP1-induced GTP hydrolysis

The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1.GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 "GTPase timer." In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1.GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting.     

Clathrin adaptor AP-2 is essential for early embryonal development

The heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4 play key roles in transport vesicle formation and cargo sorting in post-Golgi trafficking pathways. Studies on cultured mammalian cells have shown that AP-2 mediates rapid endocytosis of a subset of plasma membrane receptors. To determine whether this function is essential in the context of a whole mammalian organism, we carried out targeted disruption of the gene encoding the mu2 subunit of AP-2 in the mouse. We found that mu2 heterozygous mutant mice were viable and had an apparently normal phenotype. In contrast, no mu2 homozygous mutant embryos were identified among blastocysts from intercrossed heterozygotes, indicating that mu2-deficient embryos die before day 3.5 postcoitus (E3.5). These results indicate that AP-2 is indispensable for early embryonic development, which might be due to its requirement for cell viability.     

Clathrin adaptors really adapt

The clathrin pathway is the principal route for receptor-mediated endocytosis and growth factor downregulation. Heterotetrameric clathrin adaptors directly link the clathrin coat with cargo transmembrane proteins that are sorted into coated pits and vesicles. A paper in this issue of Cell describes the atomic structure of the adaptor-protein 2 (AP-2) core, the portion that makes contacts with the membrane and cytosolic tails of cargo proteins.