Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)–mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22a_Q64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22a_Q64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.     

Cross-talk between clathrin-dependent post-Golgi trafficking and clathrin-mediated endocytosis in Arabidopsis root cells

Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.     

The AP2 clathrin adaptor protein complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans

Regulation of glutamate receptor (GluR) abundance at synapses by clathrin-mediated endocytosis can control synaptic strength and plasticity. We take advantage of viable, null mutations in subunits of the clathrin adaptor protein 2 (AP2) complex in Caenorhabditis elegans to characterize the in vivo role of AP2 in GluR trafficking. In contrast to our predictions for an endocytic adaptor, we found that levels of the GluR GLR-1 are decreased at synapses in the ventral nerve cord (VNC) of animals with mutations in the AP2 subunits APM-2/μ2, APA-2/α, or APS-2/σ2. Rescue experiments indicate that APM-2/μ2 functions in glr-1–expressing interneurons and the mature nervous system to promote GLR-1 levels in the VNC. Genetic analyses suggest that APM-2/μ2 acts upstream of GLR-1 endocytosis in the VNC. Consistent with this, GLR-1 accumulates in cell bodies of apm-2 mutants. However, GLR-1 does not appear to accumulate at the plasma membrane of the cell body as expected, but instead accumulates in intracellular compartments including Syntaxin-13– and RAB-14–labeled endosomes. This study reveals a novel role for the AP2 clathrin adaptor in promoting the abundance of GluRs at synapses in vivo, and implicates AP2 in the regulation of GluR trafficking at an early step in the secretory pathway.     

Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

The adaptor protein-2 (AP-2) complex is a heterotetramer involved in clathrin-mediated endocytosis of cargo proteins from the plasma membrane in animal cells. The homologous genes of AP-2 subunits are present in the genomes of plants; however, their identities and roles in endocytic pathways are not clearly defined in plants. Here, we reveal the molecular composition of the AP-2 complex of Arabidopsis thaliana and its dynamics on the plasma membrane. We identified all of the α-, β-, σ-, and μ-subunits of the AP-2 complex and detected a weak interaction of the AP-2 complex with clathrin heavy chain. The μ-subunit protein fused to green fluorescent protein (AP2M-GFP) was localized to the plasma membrane and to the cytoplasm. Live-cell imaging using a variable-angle epifluorescence microscope revealed that AP2M-GFP transiently forms punctate structures on the plasma membrane. Homozygous ap2m mutant plants exhibited abnormal floral structures, including reduced stamen elongation and delayed anther dehiscence, which led to a failure of pollination and a subsequent reduction of fertility. Our study provides a molecular basis for understanding AP-2–dependent endocytic pathways in plants and their roles in floral organ development and plant reproduction.     

Leishmania adaptor protein-1 subunits are required for normal lysosome traffic, flagellum biogenesis, lipid homeostasis, and adaptation to temperatures encountered in the mammalian host

The adaptor protein-1 (AP-1) complex is involved in membrane transport between the Golgi apparatus and endosomes. In the protozoan parasite Leishmania mexicana mexicana, the AP-1 μ1 and σ1 subunits are not required for growth at 27°C but are essential for infectivity in the mammalian host. In this study, we have investigated the function of these AP-1 subunits in order to understand the molecular basis for this loss of virulence. The μ1 and σ1 subunits were localized to late Golgi and endosome membranes of the major parasite stages. Parasite mutants lacking either AP-1 subunit lacked obvious defects in Golgi structure, endocytosis, or exocytic transport. However, these mutants displayed reduced rates of endosome-to-lysosome transport and accumulated fragmented, sterol-rich lysosomes. Defects in flagellum biogenesis were also evident in nondividing promastigote stages, and this phenotype was exacerbated by inhibitors of sterol and sphingolipid biosynthesis. Furthermore, both AP-1 mutants were hypersensitive to elevated temperature and perturbations in membrane lipid composition. The pleiotropic requirements for AP-1 in membrane trafficking and temperature stress responses explain the loss of virulence of these mutants in the mammalian host.     

A Highlights from MBoC Selection: α-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

Extracellular signals regulate trafficking events to reorganize proteins at the plasma membrane (PM); however, few effectors of this regulation have been identified. β-Arrestins relay signaling cues to the trafficking machinery by controlling agonist-stimulated endocytosis of G-protein–coupled receptors. In contrast, we show that yeast α-arrestins, Aly1 and Aly2, control intracellular sorting of Gap1, the general amino acid permease, in response to nutrients. These studies are the first to demonstrate association of α-arrestins with clathrin and clathrin adaptor proteins (AP) and show that Aly1 and Aly2 interact directly with the γ-subunit of AP-1, Apl4. Aly2-dependent trafficking of Gap1 requires AP-1, which mediates endosome-to-Golgi transport, and the nutrient-regulated kinase, Npr1, which phosphorylates Aly2. During nitrogen starvation, Npr1 phosphorylation of Aly2 may stimulate Gap1 incorporation into AP-1/clathrin-coated vesicles to promote Gap1 trafficking from endosomes to the trans-Golgi network. Ultimately, increased Aly1-/Aly2-mediated recycling of Gap1 from endosomes results in higher Gap1 levels within cells and at the PM by diverting Gap away from trafficking pathways that lead to vacuolar degradation. This work defines a new role for arrestins in membrane trafficking and offers insight into how α-arrestins coordinate signaling events with protein trafficking.     

Assembly and Intracellular Targeting of the βγ Subunits of Heterotrimeric G Proteins

The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent α, β, and γ subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-β antibodies (ARC5 and ARC9), raised against immunoaffinity purified βγ complexes, recognize β subunits when not associated with γ and can thus be used to monitor assembly of βγ complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of βγ complexes with the plasma membrane fraction starts between 15–30 min of chase. Three pools of β subunits can be distinguished based on their association with γ subunits, their localization, and their detergent solubility. Association of β and α subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of βγ subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane.     

The Role of ADP-ribosylation Factor and Phospholipase D in Adaptor Recruitment

AP-1 and AP-2 adaptors are recruited onto the TGN and plasma membrane, respectively. GTPγS stimulates the recruitment of AP-1 onto the TGN but causes AP-2 to bind to an endosomal compartment (Seaman, M.N.J., C.L. Ball, and M.S. Robinson. 1993. J. Cell Biol. 123:1093–1105). We have used subcellular fractionation followed by Western blotting, as well as immunofluorescence and immunogold electron microscopy, to investigate both the recruitment of AP-2 adaptors onto the plasma membrane and their targeting to endosomes, and we have also examined the recruitment of AP-1 under the same conditions. Two lines of evidence indicate that the GTPγS-induced targeting of AP-2 to endosomes is mediated by ADP-ribosylation factor-1 (ARF1). First, GTPγS loses its effect when added to ARF-depleted cytosol, but this effect is restored by the addition of recombinant myristoylated ARF1. Second, adding constitutively active Q71L ARF1 to the cytosol has the same effect as adding GTPγS. The endosomal membranes that recruit AP-2 adaptors have little ARF1 or any of the other ARFs associated with them, suggesting that ARF may be acting catalytically. The ARFs have been shown to activate phospholipase D (PLD), and we find that addition of exogenous PLD has the same effect as GTPγS or Q71L ARF1. Neomycin, which inhibits endogenous PLD by binding to its cofactor phosphatidylinositol 4,5-bisphosphate, prevents the recruitment of AP-2 not only onto endosomes but also onto the plasma membrane, suggesting that both events are mediated by PLD. Surprisingly, however, neither PLD nor neomycin has any effect on the recruitment of AP-1 adaptors onto the TGN, even though AP-1 recruitment is ARF mediated. These results indicate that different mechanisms are used for the recruitment of AP-1 and AP-2.     

Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.

Eukaryotic cells have membrane-enclosed organelles, which carry out specialized functions, including compartmentalized biochemical reactions, metabolic channeling, and regulated signaling, inside a single cell. The transport of proteins, lipids, and other molecules between these organelles is mediated largely by small vesicular carriers that bud off at a donor compartment and fuse with the target membrane to deliver their cargo. The generation of these vesicles has been subject to extensive studies and has led to the identification of numerous coat proteins that are required for their formation at different sites (1, 2). Coat proteins can be monomers, but in most cases, they consist of several proteins, which form a heteromeric complex.Heterotetrameric adapter protein (AP) complexes are required at several endomembranes for cargo binding. Five well-conserved AP-complexes with differing functions have been identified in mammalian cells, named AP-1–AP-5, of which three (AP-1–AP-3) are conserved from yeast to human (3, 4). The three conserved adapter complexes function at different membranes along the endomembrane system. AP-1 is required for cargo transport between the Golgi and the endosome, AP-2 is required for cargo recognition and transport between the plasma membrane and the early endosome. Finally, AP-3 functions between the trans Golgi and the vacuole in yeast, whereas mammalian AP-3 localizes to a tubular endosomal compartment, in addition to or instead of the TGN (2, 5, 6).Each of the complexes consists of four different subunits: two large adaptins (named α−ζ and β1-5 respectively), a medium-sized subunit (μ1-5), and a small subunit (σ1-5). While μ- and σ-subunits together with the N-termini of the large adaptins build the membrane-binding core of the complex, the C-termini of both adaptins contain the ear domains, which are connected via flexible linkers (2). The recruitment of these complexes to membranes is not entirely conserved. They all require cargo binding, yet AP-1 binds Arf1-GTP with the γ and β1 subunit and phosphatidylinositol-4-phosphate (PI4P) via a proposed conserved site on its γ-subunit (7, 8). AP-2, on the other hand, interacts with PI(4,5)P₂ at the plasma membrane via its α, β2, and μ2 subunits (9, 10, 11).Several studies have uncovered how AP-3 functions in cargo sorting in yeast. AP-3 recognizes cargo at the Golgi via two sorting motifs in the cytosolic segments of membrane proteins: a Yxxφ sorting motif, as found in yeast in the SNARE Nyv1 or the Yck3 casein kinase, which binds to a site in μ3, as shown for mammalian AP-3, which is similar to μ2 in AP-2 (12, 13, 14), and dileucine motifs as found in the yeast SNARE Vam3 or the alkaline phosphatase Pho8, potentially also at a site comparable to AP-1 and AP-2 (15, 16). Unlike AP-1 and AP-2-coated vesicles, which depend on clathrin for their formation (2, 17), AP-3 vesicle formation in yeast does not require clathrin or the HOPS subunit Vps41 (18), yet Vps41 is required at the vacuole to bind AP-3 vesicles prior to fusion (19, 20, 21, 22). Studies in metazoan cells revealed that Vps41 and AP-3 function in regulated secretion (23, 24, 25), and AP-3 is required for biogenesis of lysosome-related organelles (26). This suggests that the AP-3 complex has features that are quite different from AP-1 and AP-2 complexes, which cooperate with clathrin in vesicle formation (2).Among the three conserved AP complexes, the function of the AP-3 complex is the least understood. Arf1 is necessary for efficient AP-3 vesicle generation in mammalian cells and shows a direct interaction with the β3 and δ subunits of AP-3 (27, 28). In addition, in vitro experiments on mammalian AP-3 using liposomes or enriched Golgi membranes suggest Arf1 as an important factor in AP-3 recruitment, whereas acidic lipids do not have a major effect, in contrast to what was found for AP-1 and AP-2 (7, 11, 29, 30). Another study showed that membrane recruitment of AP-3 depends on the recognition of sorting signals in cargo tails and PI3P (31), similar to AP-1 recruitment via cargo tails, Arf1 and PI4P (32).However, since AP-1 and AP-3 are both recruited to the trans-Golgi network (TGN) in yeast (33), the mechanism of their recruitment likely differs. Even though Arf1 is required, yeast AP-3 seems to be present at the TGN before the arrival of the Arf1 guanine nucleotide exchange factor (GEF) Sec7 (33). This implies the necessity for additional factors at the TGN and a distinct mechanism to allow for spatial and temporal separation of AP-1 and AP-3 recruitment to membranes. Structural data on mammalian AP-1 and AP-2 “core” complexes without the hinge and ear domains of their large subunits revealed that both exist in at least two very defined conformational states: a “closed” cytosolic state, where the cargo-binding sites are buried within the complex, and an “open” state, where the same sites are available to bind cargo (7, 8, 10, 34, 35). Binding of Arf1 to AP-1 or PI(4,5)P₂ in case of AP-2 induces a conformational change in the complexes that enables them to bind cargo molecules carrying a conserved acidic di-Leucine or a Tyrosine-based motif, as for all three AP complexes in yeast (8, 34). Additional conformational states and intermediates have been reported for both, mammalian AP-1 and AP-2 complex. AP-1, for example, can be hijacked by the human immunodeficiency virus-1 (HIV-1) proteins viral protein u (Vpu) and negative factor (Nef), resulting in a hyper-open conformation of AP-1 (36, 37).An emerging model over the past years has suggested that APs have several binding sites that allow for the stabilization of membrane binding and the open conformation of the complexes, but there are initial interactions required that dictate their recruitment to the target membrane. Although these interaction sites for mammalian AP-1 and AP-2 have been identified in great detail based on interaction analyses and structural studies (8, 10, 11, 35, 36, 38, 39), structural data for AP-3 is largely missing. The C-terminal part of the μ-subunit of mammalian AP-3 has been crystallized together with a Yxxφ motif-containing a cargo peptide, which revealed a similar fold and cargo-binding site as shown for AP-1 and AP-2 (14). However, positively charged binding surfaces required for PIP-interaction were not well conserved. Although the “trunk” segment of AP-1 and AP-2 is known quite well by now, information on hinge and ear domains in context of these complexes is largely missing. Crystal structures of the isolated ear domains of α-, γ- and β2-adaptin have been published (40, 41, 42), and a study on mammalian AP-3 suggested a direct interaction between δ-ear and δ3 that interfered with Arf1-binding (43). Furthermore, during tethering of AP-3 vesicles with the yeast vacuole, the δ−subunit Apl5 of the yeast AP-3 complex binds to the Vps41 subunit of the HOPS complex as a prerequisite of fusion (18, 19, 21, 22).In this study, we applied single particle electron cryo-microscopy (cryo-EM) to analyze the purified full-length AP-3 complex from yeast and unraveled the factors required for AP-3 recruitment to membranes by biochemical reconstitution. Our data reveal that a surprisingly flexible AP-3 complex requires a combination of cargo, PI4P, and Arf1 for membrane binding, which explains its function in selective cargo sorting at the Golgi.     

The polarized epithelia-specific mu 1B-adaptin complements mu 1A-deficiency in fibroblasts

The heterotetrameric AP-1A adaptor complex of clathrin-coated vesicles is ubiquitously expressed. The µ1-adaptin subunit of the complex exists as the ubiquitous µ1A and the polarized epithelia-specific µ1B, which are 80% identical. In polarized epithelia, µ1B is incorporated into the AP-1B complex, which is required for basolateral plasma membrane sorting of the low-density lipoprotein receptor. Binding of AP-1B to subdomains of the trans-Golgi network (TGN) appears to be part of the mechanism by which protein sorting is mediated. We expressed µ1B in µ1A-deficient fibroblasts to test for µ1B function in non-polarized cells. AP-1B complexes were formed and bound to the TGN and to endosomes. Moreover, AP-1B restored the AP-1A-dependent sorting of mannose 6-phosphate receptors between endosomes and the TGN. This demonstrates that µ1A and µ1B do have overlapping sorting functions and indicates that AP-1A and AP-1B mediate protein sorting along parallel pathways between the TGN and endosomes in polarized epithelia.     

Yeast Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins are but adaptor protein-1 is not required for cell-free transport of membrane proteins from the trans-Golgi network to the prevacuolar compartment

Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN–PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the β subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Δ membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Δ mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome.     

ABCG26-Mediated Polyketide Trafficking and Hydroxycinnamoyl Spermidines Contribute to Pollen Wall Exine Formation in Arabidopsis

Pollen grains are encased by a multilayered, multifunctional wall. The sporopollenin and pollen coat constituents of the outer pollen wall (exine) are contributed by surrounding sporophytic tapetal cells. Because the biosynthesis and development of the exine occurs in the innermost cell layers of the anther, direct observations of this process are difficult. The objective of this study was to investigate the transport and assembly of exine components from tapetal cells to microspores in the intact anthers of Arabidopsis thaliana. Intrinsically fluorescent components of developing tapetum and microspores were imaged in intact, live anthers using two-photon microscopy. Mutants of ABCG26, which encodes an ATP binding cassette transporter required for exine formation, accumulated large fluorescent vacuoles in tapetal cells, with corresponding loss of fluorescence on microspores. These vacuolar inclusions were not observed in tapetal cells of double mutants of abcg26 and genes encoding the proposed sporopollenin polyketide biosynthetic metabolon (ACYL COENZYME A SYNTHETASE5, POLYKETIDE SYNTHASE A [PKSA], PKSB, and TETRAKETIDE α-PYRONE REDUCTASE1), providing a genetic link between transport by ABCG26 and polyketide biosynthesis. Genetic analysis also showed that hydroxycinnamoyl spermidines, known components of the pollen coat, were exported from tapeta prior to programmed cell death in the absence of polyketides, raising the possibility that they are incorporated into the exine prior to pollen coat deposition. We propose a model where ABCG26-exported polyketides traffic from tapetal cells to form the sporopollenin backbone, in coordination with the trafficking of additional constituents, prior to tapetum programmed cell death.     

Phosphatidylinositol 3,4,5-trisphosphate Localization in Recycling Endosomes Is Necessary for AP-1B–dependent Sorting in Polarized Epithelial Cells

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell–specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits μ1A or μ1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of μ1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in μ1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P₃ formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B–dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P₃ formation in recycling endosomes is essential for AP-1B function.     

Repeated secondary loss of adaptin complex genes in the Apicomplexa

The Apicomplexa include parasites of devastating medical and economic consequence. While obviously essential for their parasitic mechanism, the molecular machinery underpinning membrane-trafficking in many apicomplexans is poorly understood. One potentially key set of players, the adaptins, selects cargo for incorporation into trafficking vesicles. Four distinct adaptin (AP) complexes exist in eukaryotes; AP1 and AP3 are involved in transport between the trans-Golgi Network (TGN) and endosomes, AP4 in TGN to cell surface transport, and AP2 in endocytosis from the cell surface. Of particular interest is the involvement of AP1 in Toxoplasma rhoptry biogenesis. The recent completion of several apicomplexan genomes should jump-start molecular parasitological studies and provide systems-level insight into the apicomplexan adaptin machinery. However, many of the encoded adaptin proteins are annotated conservatively and not to the necessary complex or subunit level. Prompted by previous evidence suggesting the lack of AP3 in Plasmodium falciparum, we undertook homology-searching and phylogenetic analysis to produce a rigorously annotated set of adaptin subunits encoded in diverse apicomplexan genomes. We found multiple losses of adaptins across the phylum; in particular Theileria, Babesia, and Cryptosporidium, but surprisingly not Plasmodium, appear to have lost the entirety of the AP3 complex. The losses correlate with a degenerate Golgi body structure and are reminiscent of recently reported secondary losses of additional endocytic components (i.e. the ESCRTs) in several Apicomplexa. These data may indicate a relaxation of the selective pressure on the apicomplexan endocytic system and, regardless, should greatly facilitate future molecular cell biological investigation of the role of adaptins in these important parasites.     

The Golgi-associated PDZ Domain Protein PIST/GOPC Stabilizes the β1-Adrenergic Receptor in Intracellular Compartments after Internalization

Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking.     

Adaptins: the final recount

Adaptins are subunits of adaptor protein (AP) complexes involved in the formation of intracellular transport vesicles and in the selection of cargo for incorporation into the vesicles. In this article, we report the results of a survey for adaptins from sequenced genomes including those of man, mouse, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, the plant Arabidopsis thaliana, and the yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. We find that humans, mice, and Arabidopsis thaliana have four AP complexes (AP-1, AP-2, AP-3, and AP-4), whereas D. melanogaster, C. elegans, S. cerevisiae, and S. pombe have only three (AP-1, AP-2, and AP-3). Additional diversification of AP complexes arises from the existence of adaptin isoforms encoded by distinct genes or resulting from alternative splicing of mRNAs. We complete the assignment of adaptins to AP complexes and provide information on the chromosomal localization, exon-intron structure, and pseudogenes for the different adaptins. In addition, we discuss the structural and evolutionary relationships of the adaptins and the genetic analyses of their function. Finally, we extend our survey to adaptin-related proteins such as the GGAs and stonins, which contain domains homologous to the adaptins.     

The Yeast Adaptor Protein Complex,AP-3, Is Essential for the Efficient Delivery of Alkaline Phosphatase by the Alternate Pathway to the Vacuole

A novel clathrin adaptor-like complex, adaptor protein (AP)-3, has recently been described in yeast and in animals. To gain insight into the role of yeast AP-3, a genetic strategy was devised to isolate gene products that are required in the absence of the AP-3 μ chain encoded by APM3. One gene identified by this synthetic lethal screen was VPS45. The Vps pathway defines the route that several proteins, including carboxypeptidase Y, take from the late Golgi to the vacuole. However, vacuolar alkaline phosphatase (ALP) is transported via an alternate, intracellular route. This suggested that the apm3-Δ vps45 synthetic phenotype could be caused by a block in both the alternate and the Vps pathways. Here we demonstrate that loss of function of the AP-3 complex results in slowed processing and missorting of ALP. ALP is no longer localized to the vacuole membrane by immunofluorescence, but is found in small punctate structures throughout the cell. This pattern is distinct from the Golgi marker Kex2p, which is unaffected in AP-3 mutants. We also show that in the apm3-Δ mutant some ALP is delivered to the vacuole by diversion into the Vps pathway. Class E vps mutants accumulate an exaggerated prevacuolar compartment containing membrane proteins on their way to the vacuole or destined for recycling to the Golgi. Surprisingly, in AP-3 class E vps double mutants these proteins reappear on the vacuole. We suggest that some AP-3–dependent cargo proteins that regulate late steps in Golgi to vacuole transport are diverted into the Vps pathway allowing completion of transfer to the vacuole in the class E vps mutant.The formation of vesicles for transport between membrane-bound organelles requires assembly of coat proteins that are recruited from the cytosol. These proteins direct the sequestration and concentration of cargo as well as invagination of the membrane. One of the best studied classes of coats involved in vesicle budding is comprised of clathrin and its adaptor proteins (APs)¹, AP-1 and AP-2 (Schmid, 1997). In clathrin-mediated vesicle transport the AP complexes play the dual role of cargo selection and recruitment of clathrin to the membrane. These adaptors are heterotetramers containing two large chains (adaptins, α or γ and β), one medium chain (μ), and one small chain (σ). AP-1 (γ, β1, μ1, and σ1) functions in sorting at the TGN, whereas AP-2 (α, β2, μ2, and σ2) is involved in receptor capture at the PM during endocytosis.Although there is a great deal of evidence supporting the involvement of adaptors in clathrin-mediated vesicle budding, recent studies in animal cells have led to the discovery of a novel adaptor-like complex, AP-3, that seems to function independently of clathrin (Newman et al., 1995; Simpson et al., 1996). AP-3 has identical subunit architecture to AP-1 and AP-2, with two adaptin-like subunits (δ and β3), a medium chain (μ3), and a small chain (σ3) (Simpson et al., 1996, 1997; Dell''Angelica et al., 1997a , b ). AP-3 antibodies label a perinuclear region, perhaps the TGN, and punctate structures extending to the cell periphery, which may be endosomal compartments (Simpson et al., 1996, 1997; Dell''Angelica et al., 1997a ). However, the mammalian AP-3 complex does not colocalize with clathrin or AP-1 and AP-2 adaptors in cells and it does not copurify with brain clathrin-coated vesicles (Newman et al., 1995; Simpson et al., 1996, 1997; Dell''Angelica et al., 1997b ). Clues to the function of AP-3 have come from the discovery that the garnet gene of Drosophila encodes a protein closely related to δ adaptin (Ooi et al., 1997; Simpson et al., 1997). Mutations in garnet cause decreased pigmentation of the eyes and other tissues and a reduced number of pigment granules, which may be lysosome-like organelles (Ooi et al., 1997; Simpson et al., 1997). Thus, AP-3 is proposed to function in clathrin-independent transport between the TGN, endosomes and/or lysosomes, although its exact sorting function is still not known.Over the last several years, yeast homologues of the mammalian adaptor subunits have been identified, allowing for the examination of specific functions of these proteins in a genetically tractable organism. Genes encoding subunits sufficient for at least three complete AP complexes have been identified by sequence homology (Phan et al., 1994; Rad et al., 1995; Stepp et al., 1995) or by function (Panek et al., 1997). APL1-APL6 encode large chain/ adaptin-related subunits, APM1-APM4 encode μ-like chains, and APS1-APS3 are genes for σ-related proteins. Apl2p (β), Apl4p (γ), Apm1p (μ1), and Aps1p (σ1) are thought to be subunits of an AP-1–like complex that functions with clathrin at the late Golgi/TGN (Phan et al., 1994; Rad et al., 1995; Stepp et al., 1995; Payne, G., personal communication). Mutations in the yeast AP-1 genes enhance the growth and the α-factor processing defects of a temperature sensitive (ts) allele of the clathrin heavy chain gene (Phan et al., 1994; Rad et al., 1995; Stepp et al., 1995; Payne, G., personal communication). The latter phenotype is a hallmark of clathrin-deficient yeast, in which late Golgi/ TGN proteins, such as the α-factor processing enzymes Kex2p and dipeptidyl amino peptidase-A (DPAP)-A, are not retained in the late Golgi but escape to the cell surface (Seeger and Payne, 1992b ). To date, no yeast adaptor subunit has been shown to be important for endocytosis, although Apl3p, Apm4p, and Aps2p are most homologous to mammalian AP-2 α, μ2 and σ2, respectively.Recently, a yeast adaptor related to AP-3 of animal cells was described (Panek et al., 1997). It is comprised of Apl5p, Apl6p, Apm3p, and Aps3p, which show preferential homology to mammalian δ, β3, μ3, and σ3, respectively. Mutations in each of these subunits were isolated by their ability to suppress the lethality resulting from loss of function of PM casein kinase 1 encoded by a gene pair, YCK1 and YCK2. Yck activity was found to be required for constitutive endocytosis of the a-factor receptor (Ste3p), and AP-3 subunit mutations partially rescued this internalization defect (Panek et al., 1997). However, the AP complex itself is not necessary for endocytosis, nor is it required for sorting of carboxypeptidase Y (CPY) or retention of late Golgi proteins. Furthermore, unlike disruption of the yeast AP-1 complex, loss of AP-3 function causes no synthetic phenotype in combination with chc1 mutations, suggesting it may function independently of clathrin. Although these data indicated that Apl5p, Apl6p, Apm3p, and Aps3p comprise an AP-3-like adaptor, its precise sorting role was still not known.In this report we describe a genetic approach to determine the function of the yeast AP-3 complex. A colony sectoring screen was performed to identify genes that are essential in the absence of Apm3p, the yeast AP-3 μ chain. Such synthetic lethal screens can be used to identify functional homologues, genes whose proteins function in intersecting or parallel pathways, and genes whose proteins physically interact (Bender and Pringle, 1991). We have cloned the gene for the apm three synthetic lethal mutant, mts1-1, and found it encodes Vps45p, a protein involved in vacuolar protein sorting (Vps; Cowles et al., 1994; Piper et al., 1994). The Vps pathway is defined by >40 complementation groups whose proteins are required for the transport of a number of soluble and membrane-bound proteins, including CPY, protease A (PrA), and carboxypeptidase S (CPS) from the late Golgi/TGN to the vacuole (Stack et al., 1995; Cowles et al., 1997). This pathway is also essential for proper assembly of the vacuolar ATPase (Raymond et al., 1992). However, the type II vacuolar membrane protein alkaline phosphatase (ALP) follows an alternate intracellular pathway to the vacuole (Raymond et al., 1992; Nothwehr et al., 1995; Cowles et al., 1997; Piper et al., 1997). Few vps mutants prevent localization of ALP to the vacuolar membrane and its arrival at the vacuole is not dependent upon transport through the cell surface. The requirement for Apm3p in the absence of Vps45p suggested the possibility that at least one of these routes to the vacuole must be functional for survival and led us to examine ALP sorting in the AP-3 mutants. We show here that yeast AP-3 is essential for the transport of ALP via the alternative pathway to the vacuole.     

ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlF_n, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.     

Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex

Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14–19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.     

Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex

The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat.