Structural determinants controlling 14-3-3 recruitment to the endocytic adaptor Numb and dissociation of the Numb·α-adaptin complex.

Traffic of cargo across membranes helps establish, maintain, and reorganize distinct cellular compartments and is fundamental to many metabolic processes. The cargo-selective endocytic adaptor Numb participates in clathrin-dependent endocytosis by attaching cargoes to the clathrin adaptor α-adaptin. The phosphorylation of Numb at Ser265 and Ser284 recruits the regulatory protein 14-3-3, accompanied by the dissociation of Numb from α-adaptin and Numb's translocation from the cortical membrane to the cytosol. However, the molecular mechanisms underlying the Numb-α-adaptin interaction and its regulation by Numb phosphorylation and 14-3-3 recruitment remain poorly understood. Here, biochemical and structural analyses of the Numb·14-3-3 complex revealed that Numb phosphorylation at both Ser265 and Ser284 is required for Numb's efficient interaction with 14-3-3. We also discovered that an RQFRF motif surrounding Ser265 in Numb functions together with the canonical C-terminal DPF motif, required for Numb's interaction with α-adaptin, to form a stable complex with α-adaptin. Of note, we provide evidence that the phosphorylation-induced binding of 14-3-3 to Numb directly competes with the binding of α-adaptin to Numb. Our findings suggest a potential mechanism governing the dynamic assembly of Numb with α-adaptin or 14-3-3. This dual-site recognition of Numb by α-adaptin may have implications for other α-adaptin targets. We propose that the newly identified α-adaptin-binding site surrounding Ser265 in Numb functions as a triggering mechanism for the dynamic dissociation of the Numb·α-adaptin complex.

Structural basis for the recognition of two consecutive mutually interacting DPF motifs by the SGIP1 µ homology domain.

FCHo1, FCHo2, and SGIP1 are key regulators of clathrin-mediated endocytosis. Their µ homology domains (µHDs) interact with the C-terminal region of an endocytic scaffold protein, Eps15, containing fifteen Asp-Pro-Phe (DPF) motifs. Here, we show that the high-affinity µHD-binding site in Eps15 is a region encompassing six consecutive DPF motifs, while the minimal µHD-binding unit is two consecutive DPF motifs. We present the crystal structures of the SGIP1 µHD in complex with peptides containing two DPF motifs. The peptides bind to a novel ligand-binding site of the µHD, which is distinct from those of other distantly related µHD-containing proteins. The two DPF motifs, which adopt three-dimensional structures stabilized by sequence-specific intramotif and intermotif interactions, are extensively recognized by the µHD and are both required for binding. Thus, consecutive and singly scattered DPF motifs play distinct roles in µHD binding.

Phylogeny of endocytic components yields insight into the process of nonendosymbiotic organelle evolution.

The process by which some eukaryotic organelles, for example the endomembrane system, evolved without endosymbiotic input remains poorly understood. This problem largely arises because many major cellular systems predate the last common eukaryotic ancestor (LCEA) and thus do not provide examples of organellogenesis in progress. A model is emerging whereby gene duplication and divergence of multiple "specificity-" or "identity-" encoding proteins for the various endomembranous organelles produced the diversity of nonendosymbiotically derived cellular compartments present in modern eukaryotes. To address this possibility, we analyzed three molecular components of the endocytic membrane-trafficking machinery. Phylogenetic analyses of the endocytic syntaxins, Rab 5, and the beta-adaptins each reveal a pattern of ancestral, undifferentiated endocytic homologues in the LCEA. Subsequently, these undifferentiated progenitors independently duplicated in widely divergent lineages, convergently producing components with similar endocytic roles, e.g., beta1 and beta2-adaptin. In contrast, beta3, beta4, and all other adaptin complex subunits, as well as paralogues of the syntaxins and Rabs specific for the other membrane-trafficking organelles, all evolved before the LCEA. Thus, the process giving rise to the differentiated organelles of the endocytic system appears to have been interrupted by the major speciation event that produced the extant eukaryotic lineages. These results suggest that although many endocytic components evolved before the LCEA, other major features evolved independently and convergently after diversification into the primary eukaryotic supergroups. This finding provides an example of a basic cellular system that was simpler in the LCEA than in many extant eukaryotes and yields insight into nonendosymbiotic organelle evolution.

Solitary and repetitive binding motifs for the AP2 complex alpha-appendage in amphiphysin and other accessory proteins.

Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and beta-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the alpha-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the alpha-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the alpha-appendage.

Functional analysis of AP-2 alpha and mu2 subunits.

The AP-2 adaptor complex plays a key role in cargo recognition and clathrin-coated vesicle formation at the plasma membrane. To investigate the functions of individual binding sites and domains of the AP-2 complex in vivo, we have stably transfected HeLa cells with wild-type and mutant small interfering RNA-resistant alpha and mu2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of alpha, the phosphorylation site of mu2, or the YXXPhi binding site of mu2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of alpha, or mutating the PtdIns(4,5)P2 binding site of mu2, has no apparent effect. However, adding a C-terminal GFP tag to alpha renders it completely nonfunctional. These findings demonstrate that there is some functional redundancy in the binding sites of the various AP-2 subunits, because no single mutation totally abolishes function. They also help to explain why GFP-tagged AP-2 never appears to leave the plasma membrane in some live cell imaging studies. Finally, they establish a new model system that can be used both for additional structure-function analyses, and as a way of testing tagged constructs for function in vivo.

Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin.

Clathrin-mediated endocytosis is a well-studied uptake mechanism for nutrients and signalling receptors in mammalian cells that depends on the coordinated interaction of coat proteins and endocytic network proteins to perform the internalization. In this process AP180 promotes the assembly of clathrin triskelia into coated membrane patches at the plasma membrane, while alpha-adaptin interacts with various network proteins that are in turn required for the budding of the coated pits. The process of clathrin-mediated endocytosis in plants has not been dissected at the molecular level, nor have the members of an analogous uptake machinery been functionally described. In this respect, we have investigated the AP180 and alpha-adaptin orthologs from Arabidopsis thaliana: At-AP180 and At-alphaC-Ad. Both plant proteins display the same structural features as their mammalian counterparts and fulfill the same basic functions. To identify their interacting partners, the ear region of At-alphaC-Ad and the C-terminal region of At-AP180 were used as fusion proteins in pull-down experiments and plasmon-resonance measurements. At-alphaC-Ad binds several mammalian endocytic proteins, and its interaction with At-AP180 requires the DPF motif. At-AP180 functions as a clathrin assembly protein that promotes the formation of cages with an almost uniform size distribution. Deletion of the single DLL motif abolished the assembly activity of At-AP180 almost completely, but did not affect its binding to triskelia, suggesting the existence of additional binding determinants.

The vesicular acetylcholine transporter interacts with clathrin-associated adaptor complexes AP-1 and AP-2.

In neuronal cells the neurotransmitter acetylcholine is transferred from the cytoplasm into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). The cytoplasmic tail of VAChT has been shown to contain signals that direct its sorting and trafficking. The role of clathrin-associated protein complexes in VAChT sorting to synaptic vesicles has been examined. A fusion protein between the VAChT cytoplasmic tail and glutathione S-transferase was used to identify VAChT-clathrin-associated protein adaptor protein 1, adaptor protein 2 and adaptor protein 180 complexes from a rat brain extract. In vivo coimmunoprecipitation confirmed adaptin alpha and adaptin gamma complexes, but adaptor protein 180 complexes were not detected by this technique. Deletion and site directed mutagenesis show that the VAChT cytoplasmic tail contains multiple trafficking signals. These include a non-classical tyrosine motif that serves as the signal for adaptin alpha and a dileucine motif that serves as the signal for adaptin gamma. A classical tyrosine motif is also involved in VAChT trafficking, but does not interact with any known adaptor proteins. There appear to be two endocytosis motifs, one involving the adaptor protein 1 binding site and the other involving the adaptor protein 2 binding site. These results suggest a complex trafficking pathway for VAChT.

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.

Xenopus autosomal recessive hypercholesterolemia protein couples lipoprotein receptors with the AP-2 complex in oocytes and embryos and is required for vitellogenesis.

ARH is required for normal endocytosis of the low density lipoprotein (LDL) receptor in liver and mutations within this gene cause autosomal recessive hypercholesterolemia in humans. xARH is a localized maternal RNA in Xenopus with an unknown function in oogenesis and embryogenesis. Like ARH, xARH contains a highly conserved phosphotyrosine binding domain and a clathrin box. To address the function of xARH, we examined its expression pattern in development and used pull-down experiments to assess interactions between xARH, lipoprotein receptors and proteins in embryo extracts. xARH was detected concentrated at the cell periphery as well as in the perinuclear region of oocytes and embryos. In pull-down experiments, the xARH phosphotyrosine binding domain interacted with the LDL and vitellogenin receptors found in Xenopus oocytes and embryos. Mutations within the receptor internalization signal specifically abolished this interaction. The xARH C-terminal region pulled-down several proteins from embryo extracts including alpha- and beta-adaptins, subunits of the AP-2 endocytic complex. Mutations within either of the two Dvarphi(F/W) motifs found in xARH abolished binding to alpha- and beta-adaptins. Expression of a dominant negative mutant of xARH missing the clathrin box and one functional Dvarphi(F/W) motif severely inhibited endocytosis of vitellogenin in cultured oocytes. The data indicate that xARH acts as an adaptor protein linking LDL and vitellogenin receptors directly with the AP-2 complex. In oocytes, we propose that xARH mediates the uptake of lipoproteins from the blood for storage in endosomes and later use in the embryo. Our findings point to an evolutionarily conserved function for ARH in lipoprotein uptake.

Rab11-FIP2, an adaptor protein connecting cellular components involved in internalization and recycling of epidermal growth factor receptors.

Rab11-FIP2 is a member of a newly identified family of Rab11-binding proteins that have been implicated in the function of recycling endosomes. Here we show that Rab11-FIP2 may also be involved with the process of receptor-mediated endocytosis. First we demonstrate that Rab11-FIP2 contains an NPF motif that allows it to bind Reps1, a member of a family of EH domain proteins involved in endocytosis. We also show that Rab11-FIP2 associates with the alpha-adaptin subunit of AP-2 complexes, which are known to recruit receptors into clathrin-coated vesicles. Finally, we find that overexpression of Rab11-FIP2 suppresses the internalization of epidermal growth factor receptors, but not transferrin receptors, through binding sites that promote complex formation with Rab11, Reps1, and alpha-adaptin. These findings suggest that Rab11-FIP2 may participate in the coupling of receptor-mediated endocytosis to the subsequent sorting of receptor-containing vesicles in endosomes.