Pik1-ing clathrin adaptors

Clathrin adaptor proteins are essential for clathrin-coated vesicle biogenesis, yet the mechanisms governing their recruitment and interactions remain incompletely defined. The clathrin adaptors Gga and AP-1 are now shown to be recruited sequentially to the trans-Golgi network in two waves of clathrin coat assembly, coupled by Pik1-mediated phosphatidylinositol-4-phosphate synthesis. These findings reveal mechanistic insights into the functional and regulatory relationships between these clathrin adaptors.     

Ent3p and Ent5p exhibit cargo-specific functions in trafficking proteins between the trans-Golgi network and the endosomes in yeast

The phosphoinositide-binding proteins Ent3p and Ent5p are required for protein transport from the trans-Golgi network (TGN) to the vacuole in Saccharomyces cerevisiae. Both proteins interact with the monomeric clathrin adaptor Gga2p, but Ent5p also interacts with the clathrin adaptor protein 1 (AP-1) complex, which facilitates retention of proteins such as Chs3p at the TGN. When both ENT3 and ENT5 are mutated, Chs3p is diverted from an intracellular reservoir to the cell surface. However, Ent3p and Ent5p are not required for the function of AP-1, but rather they seem to act in parallel with AP-1 to retain proteins such as Chs3p at the TGN. They have all the properties of clathrin adaptors, because they can both bind to clathrin and to cargo proteins. Like AP-1, Ent5p binds to Chs3p, whereas Ent3p facilitates the interaction between Gga2p and the endosomal syntaxin Pep12p. Thus, Ent3p has an additional function in Gga-dependent transport to the late endosome. Ent3p also facilitates the association between Gga2p and clathrin; however, Ent5p can partially substitute for this function. We conclude that the clathrin adaptors AP-1, Ent3p, Ent5p, and the Ggas cooperate in different ways to sort proteins between the TGN and the endosomes.     

Coupled inositide phosphorylation and phospholipase D activation initiates clathrin-coat assembly on lysosomes.

Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis. PtdIns(4,5)P2 is generated on lysosomes by the sequential action of a lysosome-associated type II phosphatidylinositol 4-kinase and a soluble type I phosphatidylinositol 4-phosphate 5-kinase. Phosphatidic acid, which potently stimulates type I phosphatidylinositol 4-phosphate 5-kinase activity, is generated on the bilayer by a phospholipase D1-like enzyme located on the lysosomal surface. Quenching phosphatidic acid function with primary alcohols prevents the synthesis of PtdIns(4, 5)P2 and blocks coat assembly. Generating phosphatidic acid directly on lysosomes with exogenous bacterial phospholipase D in the absence of ATP still drives adaptor recruitment and limited coat assembly, indicating that PtdIns(4,5)P2 functions, at least in part, to activate the PtdIns(4,5)P2-dependent phospholipase D1. These results provide the first direct evidence for the involvement of anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators.     

The clathrin adaptor complex 1 directly binds to a sorting signal in Ste13p to reduce the rate of its trafficking to the late endosome of yeast

Yeast trans-Golgi network (TGN) membrane proteins maintain steady-state localization by constantly cycling to and from endosomes. In this study, we examined the trafficking itinerary and molecular requirements for delivery of a model TGN protein A(F-->A)-alkaline phosphatase (ALP) to the prevacuolar/endosomal compartment (PVC). A(F-->A)-ALP was found to reach the PVC via early endosomes (EEs) with a half-time of approximately 60 min. Delivery of A(F-->A)-ALP to the PVC was not dependent on either the GGA or adaptor protein 1 (AP-1) type of clathrin adaptors, which are thought to function in TGN to PVC and TGN to EE transport, respectively. Surprisingly, in cells lacking the function of both GGA and AP-1 adaptors, A(F-->A)-ALP transport to the PVC was dramatically accelerated. A 12-residue cytosolic domain motif of A(F-->A)-ALP was found to mediate direct binding to AP-1 and was sufficient to slow TGN-->EE-->PVC trafficking. These results suggest a model in which this novel sorting signal targets A(F-->A)-ALP into clathrin/AP-1 vesicles at the EE for retrieval back to the TGN.     

PI4P promotes the recruitment of the GGA adaptor proteins to the trans-Golgi network and regulates their recognition of the ubiquitin sorting signal

Phosphatidylinositol 4 phosphate (PI4P) is highly enriched in the trans-Golgi network (TGN). Here we establish that PI4P is a key regulator of the recruitment of the GGA clathrin adaptor proteins to the TGN and that PI4P has a novel role in promoting their recognition of the ubiquitin (Ub) sorting signal. Knockdown of PI4KIIalpha by RNA interference (RNAi), which depletes the TGN's PI4P, impaired the recruitment of the GGAs to the TGN. GGAs bind PI4P primarily through their GAT domain, in a region called C-GAT, which also binds Ub but not Arf1. We identified two basic residues in the GAT domain that are essential for PI4P binding in vitro and for the recruitment of GGAs to the TGN in vivo. Unlike wild-type GGA, GGA with mutated GATs failed to rescue the abnormal TGN phenotype of the GGA RNAi-depleted cells. These residues partially overlap with those that bind Ub, and PI4P increased the affinity of the GAT domain for Ub. Because the recruitment of clathrin adaptors and their cargoes to the TGN is mediated through a web of low-affinity interactions, our results show that the dual roles of PI4P can promote specific GGA targeting and cargo recognition at the TGN.     

The synaptojanin-like protein Inp53/Sjl3 functions with clathrin in a yeast TGN-to-endosome pathway distinct from the GGA protein-dependent pathway

Yeast TGN resident proteins that frequently cycle between the TGN and endosomes are much more slowly transported to the prevacuolar/late endosomal compartment (PVC) than other proteins. However, TGN protein transport to the PVC is accelerated in mutants lacking function of Inp53p. Inp53p contains a SacI polyphosphoinositide phosphatase domain, a 5-phosphatase domain, and a proline-rich domain. Here we show that all three domains are required to mediate "slow delivery" of TGN proteins into the PVC. Although deletion of the proline-rich domain did not affect general membrane association, it caused localization to become less specific. The proline-rich domain was shown to bind to two proteins, including clathrin heavy chain, Chc1p. Unlike chc1 mutants, inp53 mutants do not mislocalize TGN proteins to the cell surface, consistent with the idea that Chc1p and Inp53p act at a common vesicular trafficking step but that Chc1p is used at other steps also. Like mutations in the AP-1 adaptor complex, mutations in INP53 exhibit synthetic growth and transport defects when combined with mutations in the GGA proteins. Taken together with other recent studies, our results suggest that Inp53p and AP-1/clathrin act together in a TGN-to-early endosome pathway distinct from the direct TGN-to-PVC pathway mediated by GGA/clathrin.     

P4-ATPase requirement for AP-1/clathrin function in protein transport from the trans-Golgi network and early endosomes

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Delta cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.     

The yeast clathrin adaptor protein complex 1 is required for the efficient retention of a subset of late Golgi membrane proteins

In yeast, certain resident trans-Golgi network (TGN) proteins achieve steady-state localization by cycling through late endosomes. Here, we show that chitin synthase III (Chs3p), an enzyme involved in the assembly of the cell wall at the mother-bud junction, populates an intracellular reservoir that is maintained by a cycle of transport between the TGN and early endosomes. Traffic of Chs3p from the TGN/early endosome to the cell surface requires CHS5 and CHS6, mutant alleles of which trap Chs3p in the TGN/early endosome. Disruption of the clathrin adaptor protein complex 1 (AP-1) restores Chs3p transport to the plasma membrane. Similarly, in AP-1 deficient cells, the resident TGN/early endosome syntaxin, Tlg1p, is missorted. We propose that clathrin and AP-1 act to recycle Chs3p and Tlg1p from the early endosome to the TGN.     

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane.     

Novel binding sites on clathrin and adaptors regulate distinct aspects of coat assembly

Clathrin-coated vesicles (CCVs) sort proteins at the plasma membrane, endosomes and trans Golgi network for multiple membrane traffic pathways. Clathrin recruitment to membranes and its self-assembly into a polyhedral coat depends on adaptor molecules, which interact with membrane-associated vesicle cargo. To determine how adaptors induce clathrin recruitment and assembly, we mapped novel interaction sites between these coat components. A site in the ankle domain of the clathrin triskelion leg was identified that binds a common site on the appendages of tetrameric [AP1 and AP2] and monomeric (GGA1) adaptors. Mutagenesis and modeling studies suggested that the clathrin-GGA1 appendage interface is nonlinear, unlike other peptide-appendage interactions, but overlaps with a sandwich domain binding site for accessory protein peptides, allowing for competitive regulation of coated vesicle formation. A novel clathrin box in the GGA1 hinge region was also identified and shown to mediate membrane recruitment of clathrin, while disruption of the clathrin-GGA1 appendage interaction did not affect recruitment. Thus, the distinct sites for clathrin-adaptor interactions perform distinct functions, revealing new aspects to regulation of CCV formation.     

ENTH/ANTH domains expand to the Golgi

Clathrin-coated vesicles (CCVs) play important roles in nutrient uptake, downregulation of signaling receptors, pathogen invasion and biogenesis of endosomes and lysosomes. Although detailed models for endocytic CCV formation have emerged, the process of CCV formation at the Golgi and endosomes has been less clear. Key to endocytic CCV formation are proteins containing related phosphoinositide-binding ENTH and ANTH domains. Now, recent studies have identified novel ENTH/ANTH proteins that participate in CCV-mediated traffic between the trans-Golgi Network (TGN) and endosomes and have defined a molecular basis for interaction with AP-1 and GGA adaptors in clathrin coats of the TGN/endosomes. Thus, ENTH/ANTH domain proteins appear to be universal elements in nucleation of clathrin coats.     

Distinct roles for TGN/endosome epsin-like adaptors Ent3p and Ent5p

Clathrin adaptors are key factors in clathrin-coated vesicle formation, coupling clathrin to cargo and/or the lipid bilayer. A physically interacting network of three classes of adaptors participate in clathrin-mediated traffic between the trans-Golgi network (TGN) and endosomes: AP-1, Gga proteins, and epsin-like proteins. Here we investigate functional relationships within this network through transport assays and protein localization analysis in living yeast cells. We observed that epsin-like protein Ent3p preferentially localized with Gga2p, whereas Ent5p distributed equally between AP-1 and Gga2p. Ent3p was mislocalized in Gga-deficient but not in AP-1-deficient cells. In contrast, Ent5p retained localization in cells lacking either or both AP-1 and Gga proteins. The Ent proteins were dispensable for AP-1 or Gga localization. Synthetic genetic growth and alpha-factor maturation defects were observed when ent5Delta but not ent3Delta was introduced together with deletions of the GGA genes. In AP-1-deficient cells, ent3Delta and to a lesser extent ent5Delta caused minor alpha-factor maturation defects, but together resulted in a near-lethal phenotype. Deletions of ENT3 and ENT5 also displayed synthetic defects similar to, but less severe than, synthetic effects of AP-1 and Gga inactivation. These results differentiate Ent3p and Ent5p function in vivo, suggesting that Ent3p acts primarily with Gga proteins, whereas Ent5p acts with both AP-1 and Gga proteins but is more critical for AP-1-mediated transport. The data also support a model in which the Ent adaptors provide important accessory functions to AP-1 and Gga proteins in TGN/endosome traffic.     

Sorting it out: AP-2 and alternate clathrin adaptors in endocytic cargo selection

The AP-2 adaptor complex is widely viewed as a linchpin molecule in clathrin-mediated endocytosis, simultaneously binding both clathrin and receptors. This dual interaction couples cargo capture with clathrin coat assembly, but it has now been discovered that the association with cargo is tightly regulated. Remarkably, AP-2 is not obligatory for all clathrin-mediated uptake, and several alternate adaptors appear to perform similar sorting and assembly functions at the clathrin bud site.     

Structural basis for the accessory protein recruitment by the gamma-adaptin ear domain

The adaptor proteins AP-1 and GGA regulate membrane traffic between the trans-Golgi network (TGN) and endosomes/lysosomes through ARF-regulated membrane association, recognition of sorting signals, and recruitment of clathrin and accessory proteins. The gamma 1-adaptin subunits of AP-1 and GGA possess homologous ear domains involved in the recruitment of accessory proteins, gamma-synergin and Rabaptin-5. The crystal structure of the human gamma 1-adaptin ear domain consists solely of an immunoglobulin-like fold, unlike the alpha-adaptin ear domain. Structure-based mutational analyses reveal a binding site for the accessory proteins that is composed of conserved basic residues, indicating that the recruitment mechanism in gamma 1-adaptin and GGA is distinct from that in alpha-adaptin.     

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.     

Redistribution of clathrin-coated vesicle adaptor complexes during adipocytic differentiation of 3T3-L1 cells

Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio was reversed in coated vesicles purified from differentiated adipocytes. These results indicate that the relative abundance of TGN-derived clathrin lattices increases markedly during adipocytic differentiation. Subcellular fractionation indicated that cytosolic AP-1 and AP-2 adaptors comprised approximately 70% of the total cellular adaptor pool. Interestingly, neither the concentration nor the relative ratio of cytosolic AP-1 to AP-2 adaptors increased significantly during differentiation. These data suggest that the increase in TGN-derived lattices results from differentiation-induced mechanisms for enhanced assembly or stabilization of adaptors on Golgi membranes. Interestingly, double- immunofluorescence microscopy also revealed that whereas extensive colocalization between clathrin and beta adaptins occurred both in fibroblasts and adipocytes, structures stained only with anti-adaptin antibody could be detected. Taken together these results suggest that membranes coated with adaptors, but not clathrin, can exist in these cells.     

Yeast epsin-related proteins required for Golgi-endosome traffic define a gamma-adaptin ear-binding motif

Clathrin-coated vesicles (CCVs) are a central component of endocytosis and traffic between the trans-Golgi network (TGN) and endosomes. Although endocytic CCV formation is well characterized, much less is known about CCV formation at internal membranes. Here we describe two epsin amino-terminal homology (ENTH) domain-containing proteins, Ent3p and Ent5p, that are intimately involved in clathrin function at the Golgi. Both proteins associate with the clathrin adaptor Gga2p in vivo; Ent5p also interacts with the clathrin adaptor complex AP-1 and clathrin. A novel, conserved motif that mediates the interaction of Ent3p and Ent5p with gamma-ear domains of Gga2p and AP-1 is defined. Ent3p and Ent5p colocalize with clathrin, and cells lacking both Ent proteins exhibit defects in clathrin localization and traffic between the Golgi and endosomes. The findings suggest that Ent3p and Ent5p constitute a functionally related pair that co-operate with Gga proteins and AP-1 to recruit clathrin and promote formation of clathrin coats at the Golgi/endosomes. On the basis of our results and the established roles of epsin and epsin-related proteins in endocytosis, we propose that ENTH-domain-containing proteins are a universal component of CCV formation.     

Eps15 mediates vesicle trafficking from the trans-Golgi network via an interaction with the clathrin adaptor AP-1

Eps15 (EGFR pathway substrate clone 15) is well known for its role in clathrin-coated vesicle formation at the plasma membrane through interactions with other clathrin adaptor proteins such as AP-2. Interestingly, we observed that in addition to its plasma membrane localization, Eps15 is also present at the trans-Golgi network (TGN). Therefore, we predicted that Eps15 might associate with clathrin adaptor proteins at the TGN and thereby mediate the formation of Golgi-derived vesicles. Indeed, we have found that Eps15 and the TGN clathrin adaptor AP-1 coimmunoprecipitate from rat liver Golgi fractions. Furthermore, we have identified a 14-amino acid motif near the AP-2-binding domain of Eps15 that is required for binding to AP-1, but not AP-2. Disruption of the Eps15-AP-1 interaction via siRNA knockdown of AP-1 or expression of mutant Eps15 protein, which lacks a 14-amino acid motif representing the AP-1 binding site of Eps15, significantly reduced the exit of secretory proteins from the TGN. Together, these findings indicate that Eps15 plays an important role in clathrin-coated vesicle formation not only at the plasma membrane but also at the TGN during the secretory process.     

The clathrin adaptor AP-1A mediates basolateral polarity

Clathrin and the epithelial-specific clathrin adaptor AP-1B mediate basolateral trafficking in epithelia. However, several epithelia lack AP-1B, and mice knocked out for AP-1B are viable, suggesting the existence of additional mechanisms that control basolateral polarity. Here, we demonstrate a distinct role of the ubiquitous clathrin adaptor AP-1A in basolateral protein sorting. Knockdown of AP-1A causes missorting of basolateral proteins in MDCK cells, but only after knockdown of AP-1B, suggesting that AP-1B can compensate for lack of AP-1A. AP-1A localizes predominantly to the TGN, and its knockdown promotes spillover of basolateral proteins into common recycling endosomes, the site of function of AP-1B, suggesting complementary roles of both adaptors in basolateral sorting. Yeast two-hybrid assays detect interactions between the basolateral signal of transferrin receptor and the medium subunits of both AP-1A and AP-1B. The basolateral sorting function of AP-1A reported here establishes AP-1 as a major regulator of epithelial polarity.     

Adaptor autoregulation promotes coordinated binding within clathrin coats

Membrane traffic is an essential process that allows protein and lipid exchange between the endocytic, lysosomal, and secretory compartments. Clathrin-mediated traffic between the trans-Golgi network and endosomes mediates responses to the environment through the sorting of biosynthetic and endocytic protein cargo. Traffic through this pathway is initiated by the controlled assembly of a clathrin-adaptor protein coat on the cytosolic surface of the originating organelle. In this process, clathrin is recruited by different adaptor proteins that act as a bridge between clathrin and the transmembrane cargo proteins to be transported. Interactions between adaptors and clathrin and between different types of adaptors lead to the formation of a densely packed protein network within the coat. A key unresolved issue is how the highly complex adaptor-clathrin interaction and adaptor-adaptor interaction landscape lead to the correct spatiotemporal assembly of the clathrin coat. Here we report the discovery of a new autoregulatory motif within the clathrin adaptor Gga2 that drives synergistic binding of Gga2 to clathrin and the adaptor Ent5. This autoregulation influences the temporal and/or spatial location of the Gga2-Ent5 interaction. We propose that this synergistic binding provides built-in regulation to ensure the correct assembly of clathrin coats.