Auxilin depletion causes self-assembly of clathrin into membraneless cages in vivo

Auxilin is a cofactor for Hsc70-mediated uncoating of clathrin-coated vesicles (CCVs). However, small interfering RNA (siRNA) knockdown of the ubiquitous auxilin 2 in HeLa cells only moderately impairs clathrin-dependent trafficking. In this study, we show that HeLa cells also express auxilin 1, previously thought to be neuron specific, and that both auxilins need to be depleted for inhibition of clathrin-mediated endocytosis and intracellular sorting. Depleting both auxilins cause an ∼50% reduction in the number of clathrin-coated pits at the plasma membrane but enhances the association of clathrin and adaptors with intracellular membranes. CCV fractions isolated from auxilin-depleted cells have an ∼1.5-fold increase in clathrin content and more than fivefold increase in the amount of AP-2 adaptor complex and other endocytic machinery, with no concomitant increase in cargo. In addition, the structures isolated from auxilin-depleted cells are on average smaller than CCVs from control cells and are largely devoid of membrane, indicating that they are not CCVs but membraneless clathrin cages. Similar structures are observed by electron microscopy in intact auxilin-depleted HeLa cells. Together, these findings indicate that the two auxilins have overlapping functions and that they not only facilitate the uncoating of CCVs but also prevent the formation of nonproductive clathrin cages in the cytosol.     

EpsinR is an adaptor for the SNARE protein Vti1b

EpsinR is a clathrin-coated vesicle (CCV)-associated protein that binds to vti1b, suggesting that it may be a vti1b-selective adaptor. Depletion of epsinR to undetectable levels in HeLa cells using siRNA causes vti1b to redistribute from the perinuclear region to the cell periphery, but vti1a also redistributes in epsinR-depleted cells, and both vti isoforms redistribute in AP-1–depleted cells. As a more direct assay for epsinR function, we isolated CCVs from control and siRNA-treated cells and then looked for differences in cargo content. In clathrin-depleted cells, both coat and cargo proteins are greatly reduced in this preparation. Knocking down epsinR causes a ~50% reduction in the amount of AP-1 copurifying with CCVs and vice versa, indicating that the two proteins are dependent on each other for maximum incorporation into the coat. In addition, vti1b, but not vti1a, is reduced by >70% in CCVs from both epsinR- and AP-1–depleted cells. Because AP-1 knockdown reduces the amount of epsinR in CCVs, it is possible that its effect on vti1b may be indirect. These findings provide in vivo evidence that epsinR is an adaptor for vti1b, and they also show that CCV isolation can be used as an assay for adaptor function.     

Sorting of major cargo glycoproteins into clathrin-coated vesicles

The AP-1 and AP-2 complexes are the most abundant adaptors in clathrin-coated vesicles (CCVs), but clathrin-mediated trafficking can still occur in the absence of any detectable AP-1 or AP-2. To find out whether adaptor abundance reflects cargo abundance, we used lectin pulldowns to identify the major membrane glycoproteins in CCVs from human placenta and rat liver. Both preparations contained three prominent high molecular-weight proteins: the cation-independent mannose 6-phosphate receptor (CIMPR), carboxypeptidase D (CPD) and low-density lipoprotein receptor-related protein 1 (LRP1). To investigate how these proteins are sorted, we constructed and stably transfected CD8 chimeras into HeLa cells. CD8-CIMPR localized mainly to early/tubular endosomes, CD8-CPD to the trans Golgi network and CD8-LRP1 to late/multivesicular endosomes. All three constructs redistributed to the plasma membrane when clathrin was depleted by siRNA. CD8-CIMPR was also strongly affected by AP-2 depletion. CD8-CPD was moderately affected by AP-2 depletion but strongly affected by depleting AP-1 and AP-2 together. CD8-LRP1 was only slightly affected by AP-2 depletion; however, mutating an NPXY motif in the LRP1 tail caused it to become AP-2 dependent. These results indicate that all three proteins have AP-dependent sorting signals, which may help to explain the relative abundance of AP complexes in CCVs. However, the relatively low abundance of cargo proteins in CCV preparations suggests either that some of the APs may be empty or that the preparations may be dominated by empty coats.     

The role of cargo proteins in GGA recruitment

Coat proteins are recruited onto membranes to form vesicles that transport cargo from one compartment to another, but the extent to which the cargo helps to recruit the coat proteins is still unclear. Here we have examined the role of cargo in the recruitment of Golgi-localized, gamma-ear-containing, ADP ribosylation factor (ARF)-binding proteins (GGAs) onto membranes in HeLa cells. Moderate overexpression of CD8 chimeras with cytoplasmic tails containing DXXLL-sorting signals, which bind to GGAs, increased the localization of all three GGAs to perinuclear membranes, as observed by immunofluorescence. GGA2 was also expressed at approximately twofold higher levels in these cells because it was degraded more slowly. However, this difference only partially accounted for the increase in membrane localization because there was a approximately fivefold increase in GGA2 associated with crude membranes and a approximately 12-fold increase in GGA2 associated with clathrin-coated vesicles (CCVs) in cells expressing CD8-DXXLL chimeras. The effect of cargo proteins on GGA recruitment was reconstituted in vitro using permeabilized control and CD8-DXXLL-expressing cells incubated with cytosol containing recombinant GGA2 constructs. Together, these results demonstrate that cargo proteins contribute to the recruitment of GGAs onto membranes and to the formation of GGA-positive CCVs.     

Golgi-localized, γ-Ear-containing, ADP-Ribosylation Factor-binding Proteins: Roles of the Different Domains and Comparison with AP-1 and Clathrin

We have previously identified a novel family of proteins called the GGAs (Golgi-localized, gamma-ear-containing, ADP-ribosylation factor-binding proteins). These proteins consist of an NH(2)-terminal VHS domain, followed by a GAT domain, a variable domain, and a gamma-adaptin ear homology domain. Studies from our own laboratory and others, making use of both yeast and mammals cells, indicate that the GGAs facilitate trafficking from the trans-Golgi network to endosomes. Here we have further investigated the function of the GGAs. We find that GGA-deficient yeast are not only defective in vacuolar protein sorting but they are also impaired in their ability to process alpha-factor. Using deletion mutants and chimeras, we show that the VHS domain is required for GGA function and that the VHS domain from Vps27p will not substitute for the GGA VHS domain. In contrast, the gamma-adaptin ear homology domain contributes to GGA function but is not absolutely required, and full function can be restored by replacing the GGA ear domain with the gamma-adaptin ear domain. Deleting the gamma-adaptin gene together with the two GGA genes exacerbates the phenotype in yeast, suggesting that they function on parallel pathways. In mammalian cells, the association of GGAs with the membrane is extremely unstable, which may account for their absence from purified clathrin-coated vesicles. Double- and triple-labeling immunofluorescence experiments indicate that the GGAs and AP-1 are associated with distinct populations of clathrin-coated vesicles budding from the trans-Golgi network. Together with results from other studies, our findings suggest that the GGAs act as monomeric adaptors, with the four domains involved in cargo selection, membrane localization, clathrin binding, and accessory protein recruitment.     

Structural basis for binding of accessory proteins by the appendage domain of GGAs

The Golgi-associated, gamma-adaptin-related, ADP-ribosylation-factor binding proteins (GGAs) and adaptor protein (AP)-1 are adaptors involved in clathrin-mediated transport between the trans-Golgi network and endosomal system. The appendage domains of GGAs and the AP-1 gamma-adaptin subunit are structurally homologous and have been proposed to bind to accessory proteins via interaction with short sequences containing phenylalanines and acidic residues. Here we present the structure of the human GGA1 appendage in complex with its cognate binding peptide from the p56 accessory protein (DDDDFGGFEAAETFD) as determined by X-ray crystallography. The interaction is governed predominantly by packing of the first two phenylalanine residues of the peptide with conserved basic and hydrophobic residues from GGA1. Additionally, several main chain hydrogen bonds cause the peptide to form an additional beta-strand on the edge of the preexisting beta-sheet of the protein. Isothermal titration calorimetry was used to assess the affinities of different peptides for the GGA and gamma-appendage domains.     

Mammalian GGAs act together to sort mannose 6-phosphate receptors

The GGAs (Golgi-localized, gamma ear-containing, ADP ribosylation factor-binding proteins) are multidomain proteins implicated in protein trafficking between the Golgi and endosomes. We examined whether the three mammalian GGAs act independently or together to mediate their functions. Using cryo-immunogold electron microscopy, the three GGAs were shown to colocalize within coated buds and vesicles at the trans-Golgi network (TGN) of HeLa cells. In vitro binding experiments revealed multidomain interactions between the GGAs, and chemical cross-linking experiments demonstrated that GGAs 1 and 2 form a complex on Golgi membranes. RNA interference of each GGA resulted in decreased levels of the other GGAs and their redistribution from the TGN to cytosol. This was associated with impaired incorporation of the cation-independent mannose 6-phosphate receptor into clathrin-coated vesicles at the TGN, partial redistribution of the receptor to endosomes, and missorting of cathepsin D. The morphology of the TGN was also altered. These findings indicate that the three mammalian GGAs cooperate to sort cargo and are required for maintenance of TGN structure.     

GGA1 interacts with the adaptor protein AP-1 through a WNSF sequence in its hinge region

The Golgi-associated gamma-adaptin-related ADP-ribosylation factor-binding proteins (GGAs) are critical components of the transport machinery that mediates the trafficking of the mannose 6-phosphate receptors and associated cargo from the trans-Golgi network to the endosomes. The GGAs colocalize in vivo with the clathrin adaptor protein AP-1 and bind to AP-1 in vitro, suggesting that the two proteins may cooperate in packaging the mannose 6-phosphate receptors into clathrin-coated vesicles at the trans-Golgi network. Here, we demonstrate that the sequence, (382)WNSF(385), in the hinge region of GGA1 mediates its interaction with the AP-1 gamma-ear. The Trp and Phe constitute critical amino acids in this interaction. The binding of Rabaptin5 to the AP-1 gamma-ear, which occurs through a FXXPhi motif, is inhibited by a peptide encoding the GGA1 (382)WNSF(385) sequence. Moreover, mutations in the AP-1 gamma-ear that abolish its interaction with Rabaptin5 also preclude its association with GGA1. These results suggest that the GGA1 WXXF-type and Rabaptin5 FXXPhi-type motifs bind to the same or highly overlapping sites in the AP-1 gamma-ear. This binding is modulated by residues adjacent to the core motifs.     

The tyrosine-based lysosomal targeting signal in lamp-1 mediates sorting into Golgi-derived clathrin-coated vesicles.

Diversion of membrane proteins from the trans-Golgi network (TGN) or the plasma membrane into the endosomal system occurs via clathrin-coated vesicles (CCVs). These sorting events may require the interaction of cytosolic domain signals with clathrin adaptor proteins (APs) at the TGN (AP-1) or the plasma membrane (AP-2). While tyrosine- and di-leucine-based signals in several proteins mediate endocytosis via cell surface CCVs, segregation into Golgi-derived CCVs has so far only been documented for the mannose 6-phosphate receptors, where it is thought to require a casein kinase II phosphorylation site adjacent to a di-leucine motif. Although recently tyrosine-based signals have also been shown to interact with the mu chain of AP-1 in vitro, it is not clear if these signals also bind intact AP-1 adaptors, nor if they can mediate sorting of proteins into AP-1 CCVs. Here we show that the cytosolic domain of the lysosomal membrane glycoprotein lamp-1 binds AP-1 and AP-2. Furthermore, lamp-1 is present in AP-1-positive vesicles and tubules in the trans-region on the Golgi complex. AP-1 binding as well as localization to AP-1 CCVs require the presence of the functional tyrosine-based lysosomal targeting signal of lamp-1. These results indicate that lamp-1 can exit the TGN in CCVs and that tyrosine signals can mediate these sorting events.     

Identification of acidic dileucine signals in LRP9 that interact with both GGAs and AP-1/AP-2

The Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding family of monomeric clathrin adaptors (GGAs) is known to bind cargo molecules through short C-terminal peptide motifs conforming to the sequence DXXLL (X = any amino acid), while the heterotetrameric adaptors AP-1 and AP-2 utilize a similar but discrete sorting motif of the sequence [D,E]XXXL[L,I]. While it has been established that a single cargo molecule may contain either or both types of these acidic cluster-dileucine (AC-LL) sorting signals, there are no examples of cargo with overlapping GGA and AP-1/AP-2-binding motifs. In this study, we report that the cytosolic tail of low-density lipoprotein receptor-related protein (LRP)9 contains a bifunctional GGA and AP-1/AP-2-binding motif at its carboxy-terminus (EDEPLL). We further demonstrate that the internal EDEVLL sequence of LRP9 also binds to GGAs in addition to AP-2. Either AC-LL motif of LRP9 is functional in endocytosis. These findings represent the first study characterizing the trafficking of LRP9 and also have implications for the identification of additional GGA cargo molecules.     

A family of proteins with gamma-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome

We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH(2)-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal "ear" domain of gamma-adaptin. However, unlike gamma-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the gamma-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.     

The structure and function of GGAs, the traffic controllers at the TGN sorting crossroads

GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding proteins) are a family of monomeric clathrin adaptor proteins that are conserved from yeasts to humans. Data published during the past four years have provided detailed pictures of the localization, domain organization and structure-function relationships of GGAs. GGAs possess four conserved functional domains, each of which interacts with cargo proteins including mannose 6-phosphate receptors, the small GTPase ARF, clathrin, or accessory proteins including Rabaptin-5 and gamma-synergin. Together with or independent of the adaptor protein complex AP-1, GGAs regulate selective transport of cargo proteins, such as mannose 6-phosphate receptors, from the trans-Golgi network to endosomes mediated by clathrin-coated vesicles.     

Two WXXF-based motifs in NECAPs define the specificity of accessory protein binding to AP-1 and AP-2

The adaptor proteins AP-2 and AP-1/GGAs are essential components of clathrin coats at the plasma membrane and trans-Golgi network, respectively. The adaptors recruit accessory proteins to clathrin-coated pits, which is dependent on the adaptor ear domains engaging short peptide motifs in the accessory proteins. Here, we perform an extensive mutational analysis of a novel WXXF-based motif that functions to mediate the binding of an array of accessory proteins to the alpha-adaptin ear domain of AP-2. Using nuclear magnetic resonance and mutational studies, we identified WXXF-based motifs as major ligands for a site on the alpha-ear previously shown to bind the DPW-bearing proteins epsin 1/2. We also defined the determinants that allow for specific binding of the alpha-ear motif to AP-2 as compared to those that allow a highly related WXXF-based motif to bind to the ear domains of AP-1/GGAs. Intriguingly, placement of acidic residues around the WXXF cores is critical for binding specificity. These studies provide a structural basis for the specific recruitment of accessory proteins to appropriate sites of clathrin-coated vesicle formation.     

Characterization of a protein phosphatase 2A holoenzyme that dephosphorylates the clathrin adaptors AP-1 and AP-2

The AP-2 complex is a key factor in the formation of endocytic clathrin-coated vesicles (CCVs). AP-2 sorts and packages cargo membrane proteins into CCVs, binds the coat protein clathrin, and recruits numerous other factors to the site of vesicle formation. Structural information on the AP-2 complex and biochemical work have allowed understanding its function on the molecular level, and recent studies showed that cycles of phosphorylation are key steps in the regulation of AP-2 function. The complex is phosphorylated on both large subunits (alpha- and beta2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit mu2. Phosphorylation of mu2 is necessary for efficient cargo recruitment, whereas the functional context of the large subunit phosphorylation is unknown. Here, we show that the subunit phosphorylation of AP-2 exhibits striking differences, with calculated half-lives of <1 min for mu2, approximately 25 min for beta2, and approximately 70 min for alpha. We were also able to purify a phosphatase that dephosphorylates the mu2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic Cbeta subunit, a scaffolding Abeta subunit, and a regulatory Balpha subunit. RNA interference knock down of the latter subunit in HeLa cells resulted in increased levels of phosphorylated adaptors and altered endocytosis, showing that a specific PP2A holoenzyme is an important regulatory enzyme in CCV-mediated transport.     

CVAK104 is a novel regulator of clathrin-mediated SNARE sorting

Clathrin-coated vesicles (CCVs) mediate transport between the plasma membrane, endosomes and the trans Golgi network. Using comparative proteomics, we have identified coated-vesicle-associated kinase of 104 kDa (CVAK104) as a candidate accessory protein for CCV-mediated trafficking. Here, we demonstrate that the protein colocalizes with clathrin and adaptor protein-1 (AP-1), and that it is associated with a transferrin-positive endosomal compartment. Consistent with these observations, clathrin as well as the cargo adaptors AP-1 and epsinR can be coimmunoprecipitated with CVAK104. Small interfering RNA (siRNA) knockdown of CVAK104 in HeLa cells results in selective loss of the SNARE proteins syntaxin 8 and vti1b from CCVs. Morpholino-mediated knockdown of CVAK104 in Xenopus tropicalis causes severe developmental defects, including a bent body axis and ventral oedema. Thus, CVAK104 is an evolutionarily conserved protein involved in SNARE sorting that is essential for normal embryonic development.     

Phosphoinositide-mediated clathrin adaptor progression at the trans-Golgi network

Clathrin-coated vesicles mediate endocytosis and transport between the trans-Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo and membranes. Two main types of clathrin adaptor act in TGN-endosome traffic: GGA proteins and the AP-1 complex. Here we characterize the relationship between GGA proteins, AP-1 and other TGN clathrin adaptors using live-cell and super-resolution microscopy in yeast. We present evidence that GGA proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PtdIns(4)P) levels at the TGN slow or uncouple AP-1 coat assembly from GGA coat assembly. Conversely, enhanced PtdIns(4)P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PtdIns(4)-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PtdIns(4)P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN.     

A Role for Cargo in Arf-dependent Adaptor Recruitment

Membrane traffic requires the specific concentration of protein cargos and exclusion of other proteins into nascent carriers. Critical components of this selectivity are the protein adaptors that bind to short, linear motifs in the cytoplasmic tails of transmembrane protein cargos and sequester them into nascent carriers. The recruitment of the adaptors is mediated by activated Arf GTPases, and the Arf-adaptor complexes mark sites of carrier formation. However, the nature of the signal(s) that initiates carrier biogenesis remains unknown. We examined the specificity and initial sites of recruitment of Arf-dependent adaptors (AP-1 and GGAs) in response to the Golgi or endosomal localization of specific cargo proteins (furin, mannose-6-phosphate receptor (M6PR), and M6PR lacking a C-terminal domain M6PRΔC). We find that cargo promotes the recruitment of specific adaptors, suggesting that it is part of an upstream signaling event. Cargos do not promote adaptor recruitment to all compartments in which they reside, and thus additional factors regulate the cargo''s ability to promote Arf activation and adaptor recruitment. We document that within a given compartment different cargos recruit different adaptors, suggesting that there is little or no free, activated Arf at the membrane and that Arf activation is spatially and temporally coupled to the cargo and the adaptor. Using temperature block, brefeldin A, and recovery from each, we found that the cytoplasmic tail of M6PR causes the recruitment of AP-1 and GGAs to recycling endosomes and not at the Golgi, as predicted by steady state staining profiles. These results are discussed with respect to the generation of novel models for cargo-dependent regulation of membrane traffic.     

Phosphorylation-induced conformational changes regulate GGAs 1 and 3 function at the trans-Golgi network

The GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) are a family of multidomain proteins implicated in protein trafficking between the Golgi and the endosomes. All three GGAs (1, 2, and 3) bind to the mannose 6-phosphate receptor tail via their VHS domains, as well as to the adaptor protein complex-1 via their hinge domains. The latter interaction has been proposed to be important for cooperative packaging of cargo into forming clathrin-coated carriers at the trans-Golgi network. Here we present evidence that GGA1 function is highly regulated by cycles of phosphorylation and dephosphorylation. Cell fractionation showed that the phosphorylated pool of GGA1 resided predominantly in the cytosol and that recruitment onto membranes was associated with dephosphorylation. Okadaic acid inhibition studies and in vitro dephosphorylation assays indicated that dephosphorylation is mediated by a protein phosphatase 2A-like phosphatase. Dephosphorylation of GGA1 induced a change in the conformation to an "open" form as measured by gel filtration and sucrose gradient analyses. This was associated with enhanced binding to ligands because of release of autoinhibition and increased binding to the adaptor protein complex-1 gamma-appendage. A model is proposed for the regulation of GGA1 function at the trans-Golgi network.     

Binding partners for the COOH-terminal appendage domains of the GGAs and gamma-adaptin

The adaptor appendage domains are believed to act as binding platforms for coated vesicle accessory proteins. Using glutathione S-transferase pulldowns from pig brain cytosol, we find three proteins that can bind to the appendage domains of both the AP-1 gamma subunit and the GGAs: gamma-synergin and two novel proteins, p56 and p200. p56 elicited better antibodies than p200 and was generally more tractable. Although p56 and gamma-synergin bind to both GGA and gamma appendages in vitro, immunofluorescence labeling of nocodazole-treated cells shows that p56 colocalizes with GGAs on TGN46-positive membranes, whereas gamma-synergin colocalizes with AP-1 primarily on a different membrane compartment. Furthermore, in AP-1-deficient cells, p56 remains membrane-associated whereas gamma-synergin becomes cytosolic. Thus, p56 and gamma-synergin show very strong preferences for GGAs and AP-1, respectively, in vivo. However, the GGA and gamma appendages share the same fold as determined by x-ray crystallography, and mutagenesis reveals that the same amino acids contribute to their binding sites. By overexpressing wild-type GGA and gamma appendage domains in cells, we can drive p56 and gamma-synergin, respectively, into the cytosol, suggesting a possible mechanism for selectively disrupting the two pathways.     

The acidic cluster of the CK2 site of the cation-dependent mannose 6-phosphate receptor (CD-MPR) but not its phosphorylation is required for GGA1 and AP-1 binding

Lysosomal biogenesis depends on proper transport of lysosomal enzymes by the cation-dependent mannose 6-phosphate receptor (CD-MPR) from the trans-Golgi network (TGN) to endosomes. Trafficking of the CDMPR is mediated by sorting signals in its cytoplasmic tail. GGA1 (Golgi-localizing, gamma-ear-containing, ARF-binding protein-1) binds to CD-MPR in the TGN and targets the receptor to clathrin-coated pits for transport from the TGN to endosomes. The motif of the CD-MPR that interacts with GGA1 was shown to be 61DXXLL65. Reports on increased affinity of cargo, when phosphorylated by casein kinase 2 (CK2), to GGAs focused our interest on the effect of the CD-MPR CK2 site on binding to GGA1. Here we demonstrate that Glu58 and Glu59 of the CK2 site are essential for high affinity GGA1 binding in vitro, whereas the phosphorylation of Ser57 of the CD-MPR has no influence on receptor binding to GGA1. Furthermore, the in vivo interaction between GGA1 and CD-MPR was abolished only when all residues involved in GGA1 binding were mutated, namely, Glu58, Glu59, Asp61, Leu64, and Leu65. In contrast, the binding of adaptor protein-1 (AP-1) to CD-MPR required all the glutamates surrounding the phosphorylation site, namely, Glu55, Glu56, Glu58, and Glu59, but like GGA1 binding, was independent of the phosphorylation of Ser57. The binding affinity of GGA1 to the CD-MPR was found to be 2.4-fold higher than that of AP-1. This could regulate the binding of the two proteins to the partly overlapping sorting signals, allowing AP-1 binding to the CD-MPR only when GGA1 is released upon autoinhibition by phosphorylation.