Structural Basis for Tetherin Antagonism as a Barrier to Zoonotic Lentiviral Transmission.

Tetherin is a host defense factor that physically prevents virion release from the plasma membrane. The Nef accessory protein of simian immunodeficiency virus (SIV) engages the clathrin adaptor AP-2 to downregulate tetherin via its DIWK motif. As human tetherin lacks DIWK, antagonism of tetherin by Nef is a barrier to simian-human transmission of non-human primate lentiviruses. To determine the molecular basis for tetherin counteraction, we reconstituted the AP-2 complex with a simian tetherin and SIV Nef and determined its structure by cryoelectron microscopy (cryo-EM). Nef refolds the first α-helix of the ß2 subunit of AP-2 to a ß hairpin, creating a binding site for the DIWK sequence. The tetherin binding site in Nef is distinct from those of most other Nef substrates, including MHC class I, CD3, and CD4 but overlaps with the site for the restriction factor SERINC5. This structure explains the dependence of SIVs on tetherin DIWK and consequent barrier to human transmission.

AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis.

Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here ß1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease.

Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells.

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit ß3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.

Basolateral sorting of the Mg²âº transporter CNNM4 requires interaction with AP-1A and AP-1B.

Ancient conserved domain protein/cyclin M (CNNM) 4 is an evolutionarily conserved Mg(2+) transporter that localizes at the basolateral membrane of the intestinal epithelia. Here, we show the complementary importance of clathrin adaptor protein (AP) complexes AP-1A and AP-1B in basolateral sorting of CNNM4. We first confirmed the basolateral localization of both endogenous and ectopically expressed CNNM4 in Madin-Darby Canine Kidney cells, which form highly polarized epithelia in culture. Single knockdown of µ1B, a cargo-recognition subunit of AP-1B, did not affect basolateral localization, but simultaneous knockdown of the µ1A subunit of AP-1A abrogated localization. Mutational analyses showed the importance of three conserved dileucine motifs in CNNM4 for both basolateral sorting and interaction with µ1A and µ1B. These results imply that CNNM4 is sorted to the basolateral membrane by the complementary function of AP-1A and AP-1B.

Clathrin adaptors. AP2 controls clathrin polymerization with a membrane-activated switch.

Clathrin-mediated endocytosis (CME) is vital for the internalization of most cell-surface proteins. In CME, plasma membrane-binding clathrin adaptors recruit and polymerize clathrin to form clathrin-coated pits into which cargo is sorted. Assembly polypeptide 2 (AP2) is the most abundant adaptor and is pivotal to CME. Here, we determined a structure of AP2 that includes the clathrin-binding ß2 hinge and developed an AP2-dependent budding assay. Our findings suggest that an autoinhibitory mechanism prevents clathrin recruitment by cytosolic AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate and cargo relieves this autoinhibition, triggering clathrin recruitment and hence clathrin-coated bud formation. This molecular switching mechanism can couple AP2's membrane recruitment to its key functions of cargo and clathrin binding.

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.

Identification of a gene encoding adaptin-like protein in the Paracoccidioides brasiliensis genome by random amplified polymorphic DNA analysis.

Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pb18) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pb18a by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein beta-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.

Src-dependent phosphorylation of beta2-adaptin dissociates the beta-arrestin-AP-2 complex.

Beta-arrestins are known to act as endocytic adaptors by recruiting the clathrin adaptor protein 2 (AP-2) complex to G-protein-coupled receptors (GPCRs), linking them to clathrin-coated pits (CCPs) for internalization. They also act as signaling molecules connecting GPCRs to different downstream effectors. We have previously shown that stimulation of the angiotensin II (Ang II) type 1 receptor (AGTR1, hereafter referred to as AT1R), a member of the GPCR family, promotes the formation of a complex between beta-arrestin, the kinase Src and AP-2. Here, we report that formation of such a complex is involved in the AT1R-mediated tyrosine phosphorylation of beta2-adaptin, the subunit of AP-2 involved in binding beta-arrestin. We identify a crucial tyrosine residue in the ear domain of beta2-adaptin and show in vitro that the phosphorylation of this site regulates the interaction between beta-arrestin and beta2-adaptin. Using fluorescently tagged proteins combined with resonance energy transfer and image cross-correlation spectroscopy approaches, we show in live cells that beta2-adaptin phosphorylation is an important regulatory process for the dissociation of beta-arrestin-AP-2 complexes in CCPs. Finally, we show that beta2-adaptin phosphorylation is involved in the early steps of receptor internalization. Our findings not only unveil beta2-adaptin as a new Src target during AT1R internalization, but also support the role of receptor-mediated signaling in the control of clathrin-dependent endocytosis of receptors.

Characterization of AP3B2_v2, a novel splice variant of human AP3B2.

A novel splice variant of human AP3B2, named AP3B2_v2, was isolated through the large-scale sequencing analysis of a human fetal brain cDNA library. The AP3B2_v2 cDNA is 1171 bp in length. Sequence analysis revealed AP3B2_v2 missed 22 exons that existed in AP3B2_v1, leading to a different putative protein. The deduced proteins were 145 amino acids (designated as AP3B2_v2) and 1082 amino acids (AP3B2_v1) in length, sharing the C-terminal 145 amino acids. RT-PCR analysis showed that human AP3B2_v2 were expressed in several human adult tissues analyzed. The expression levels of AP3B2_v2 were relatively high in brain and testis. In contrast, low levels of expression were detected in kidney, pancreas, spleen, thymus, prostate, ovary and small intestine.

AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells.

The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (mu1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). Mu1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that mu1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.5 d, sorted TfR correctly in the biosynthetic route but incorrectly in the recycling route. In contrast, these same cells missorted vesicular stomatitis virus G apically in the biosynthetic route. Strikingly, recently confluent MDCK cells (1-3 d) displayed AP1B-dependence in the biosynthetic route of TfR, which decreased with additional days in culture. Sucrose density gradient analysis detected AP1B predominantly in TfR-rich endosomal fractions in MDCK cells confluent for 1 and 4 d. Our results are consistent with the following model: AP1B sorts basolateral proteins in both biosynthetic and recycling routes of MDCK cells, as a result of its predominant functional localization in recycling endosomes, which constitute a post-Golgi station in the biosynthetic route of some plasma membrane proteins. TfR utilizes a direct route from Golgi to basolateral membrane that is established as the epithelial monolayer matures.

Expression of a novel beta adaptin subunit mRNA splice variant in human testes.

AIM: To identify a novel isoform of adaptin 2 beta subunit (named Ap2beta-NY) and to investigate its relationship with testicular development and spermatogenesis. METHODS: Using a human testis cDNA microarray, a clone (Ap2beta-NY), which was strongly expressed in adult testes but weakly expressed in embryo testes, was sequenced and analyzed. Using polymerase chain reaction (PCR), the tissue distribution and expression time pattern of Ap2beta-NY were determined. RESULTS: Ap2beta-NY was identified and has been deposited in the GenBank (AY341427). The expression level of Ap2beta-NY in the adult testis was about 3-fold higher than that in the embryo testis. PCR analysis using multi-tissue cDNA indicated that Ap2beta-NY was highly expressed in the testis, spleen, thymus, prostate, ovary, blood leukocyte and brain, but not in the heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. In addition, Ap2beta-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome, which implied that, in the testis, Ap2beta-NY was restrictively expressed in germ cells. CONCLUSION: Ap2beta-NY is an isoform of Ap2beta and may be involved in regulating the process of spermatogenesis and testis development.

Leishmania major: clathrin and adaptin complexes of an intra-cellular parasite.

To investigate the role of clathrin-mediated trafficking during the Leishmania lifecycle, open reading frames encoding clathrin heavy chain and the beta-adaptins, major components of the adaptor complexes, have been analysed both in silico and experimentally. The Leishmania genome encodes three beta-adaptins, which arose at a time predating speciation of these divergent trypanosomatids. Unlike Trypanosoma brucei, both clathrin heavy chain and beta-adaptin1 are constitutively expressed throughout the Leishmania life cycle. Clathrin relocalises in amastigotes relative to promastigotes, consistent with developmental alterations to the morphology of the endo-membrane system.

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.

The beta2-adaptin clathrin adaptor interacts with the mitotic checkpoint kinase BubR1.

The adaptor AP2 is a heterotetrameric complex that associates with clathrin and regulatory proteins to mediate rapid endocytosis from the plasma membrane. Here, we report the identification of the mitotic checkpoint kinase BubR1 as a novel binding partner of beta2-adaptin, one of the AP2 large subunits. Using two-hybrid experiments and in vitro binding assays, we show that beta2-adaptin binds to BubR1 through its amino-terminal beta2-'trunk' domain, while the beta2-binding region of BubR1 maps to the carboxy-terminal kinase domain. Subcellular immunolocalization studies suggest that the interaction between BubR1 and beta2-adaptin could take place in the cytosol at any time during the cell cycle. In addition, we found that BubR1 and the BubR1-related kinase, Bub1, also bind to beta-adaptins of other AP complexes. Together, these results support a model in which the mitotic checkpoint kinases BubR1 and BuB1, by binding to beta-adaptins, may play novel roles in the regulation of vesicular intracellular traffic.

Transforming growth factor-beta receptors interact with AP2 by direct binding to beta2 subunit.

Transforming growth factor-beta (TGF-beta) superfamily members regulate a wide range of biological processes by binding to two transmembrane serine/threonine kinase receptors, type I and type II. We have previously shown that the internalization of these receptors is inhibited by K(+) depletion, cytosol acidification, or hypertonic medium, suggesting the involvement of clathrin-coated pits. However, the involvement of the clathrin-associated adaptor complex AP2 and the identity of the AP2 subunit that binds the receptors were not known. Herein, we have studied these issues by combining studies on intact cells with in vitro assays. Using fluorescence photobleaching recovery to measure the lateral mobility of the receptors on live cells (untreated or treated to alter their coated pit structure), we demonstrated that their mobility is restricted by interactions with coated pits. These interactions were transient and mediated through the receptors' cytoplasmic tails. To measure direct binding of the receptors to specific AP2 subunits, we used yeast two-hybrid screens and in vitro biochemical assays. In contrast to most other plasma membrane receptors that bind to AP2 via the mu2 subunit, AP2/TGF-beta receptor binding was mediated by a direct interaction between the beta2-adaptin N-terminal trunk domain and the cytoplasmic tails of the receptors; no binding was observed to the mu2, alpha, or sigma2 subunits of AP2 or to mu1 of AP1. The data uniquely demonstrate both in vivo and in vitro the ability of beta2-adaptin to directly couple TGF-beta receptors to AP2 and to clathrin-coated pits, providing the first in vivo evidence for interactions of a transmembrane receptor with beta2-adaptin.

Subunit H of the V-ATPase involved in endocytosis shows homology to beta-adaptins.

The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that facilitates the acidification of intracellular compartments in eukaryotic cells and plays an important role in receptor-mediated endocytosis, intracellular trafficking processes, and protein degradation. In this study we show that the C-terminal fragment of 350 residues of the regulatory subunit H (V1H) of the V-ATPase shares structural and functional homologies with the beta-chains of adaptor protein complexes. Moreover, the fragment is similar to a region in the beta-subunit of COPI coatomer complexes, which suggests the existence of a shared domain in these three different families of proteins. For beta-adaptins, this fragment binds to cytoplasmic di-leucine-based sorting motifs such as in HIV-1 Nef that mediate endocytic trafficking. Expression of this fragment in cells blocks the internalization of transmembrane proteins, which depend on di-leucine-based motifs, whereas mutation of the consensus sequence GEY only partly diminishes the recognition of the sorting motif. Based on recent structural analysis, our results suggest that the di-leucine-binding domain consists of a HEAT or ARM repeat protein fold.

The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis.

betaarrestins mediate the desensitization of the beta2-adrenergic receptor (beta2AR) and many other G protein-coupled receptors (GPCRs). Additionally, betaarrestins initiate the endocytosis of these receptors via clathrin coated-pits and interact directly with clathrin. Consequently, it has been proposed that betaarrestins serve as clathrin adaptors for the GPCR family by linking these receptors to clathrin lattices. AP-2, the heterotetrameric clathrin adaptor protein, has been demonstrated to mediate the internalization of many types of plasma membrane proteins other than GPCRs. AP-2 interacts with the clathrin heavy chain and cytoplasmic domains of receptors such as those for epidermal growth factor and transferrin. In the present study we demonstrate the formation of an agonist-induced multimeric complex containing a GPCR, betaarrestin 2, and the beta2-adaptin subunit of AP-2. beta2-Adaptin binds betaarrestin 2 in a yeast two-hybrid assay and coimmunoprecipitates with betaarrestins and beta2AR in an agonist-dependent manner in HEK-293 cells. Moreover, beta2-adaptin translocates from the cytosol to the plasma membrane in response to the beta2AR agonist isoproterenol and colocalizes with beta2AR in clathrin-coated pits. Finally, expression of betaarrestin 2 minigene constructs containing the beta2-adaptin interacting region inhibits beta2AR endocytosis. These findings point to a role for AP-2 in GPCR endocytosis, and they suggest that AP-2 functions as a clathrin adaptor for the endocytosis of diverse classes of membrane receptors.