Biochemical and structural characterization of the interaction of memapsin 2 (beta-secretase) cytosolic domain with the VHS domain of GGA proteins

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K(d), values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the K(d) decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.     

Memapsin 2 (beta-secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2

Memapsin 2, or beta-secretase, is a membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP) leading to the production of beta-amyloid peptide in the brain and the onset of Alzheimer's disease. Memapsin 2 and APP are both endocytosed into endosomes for cleavage. Here we show that the cytosolic domain of memapsin 2, but not that of memapsin 1, binds the VHS domains of GGA1 and GGA2. Gel-immobilized VHS domains of GGA1 and GGA2 also bound to full-length memapsin 2 from cell mammalian lysates. Mutagenesis studies established that Asp(496), Leu(499) and Leu(500) were essential for the binding. The spacing of these three residues in memapsin 2 is identical to those in the cytosolic domains of mannose-6-phosphate receptors, sortilin and low density lipoprotein receptor-related protein 3. These observations suggest that the endocytosis and intracellular transport of memapsin 2, mediated by its cytosolic domain, may involve the binding of GGA1 and GGA2.     

Internalization of exogenously added memapsin 2 (beta-secretase) ectodomain by cells is mediated by amyloid precursor protein

Memapsin 2 (beta-secretase) is the protease that initiates cleavage of amyloid precursor protein (APP) leading to the production of amyloid-beta (Abeta) peptide and the onset of Alzheimer's disease. Both APP and memapsin 2 are Type I transmembrane proteins and are endocytosed into endosomes where APP is cleaved by memapsin 2. Separate endocytic signals are located in the cytosolic domains of these proteins. We demonstrate here that the addition of the ectodomain of memapsin 2 (M2(ED)) to cells transfected with native APP or APP Swedish mutant (APPsw) resulted in the internalization of M2(ED) into endosomes with increased Abeta production. These effects were reduced by treatment with glycosylphosphatidylinositol-specific phospholipase C. The nontransfected parental cells had little internalization of M2(ED). The internalization of M2(ED) was dependent on the endocytosis signal in APP, because the expression of a mutant APP that lacks its endocytosis signal failed to support M2(ED) internalization. These results suggest that exogenously added M2(ED) interacts with the ectodomain of APP on the cell surface leading to the internalization of M2(ED), supported by fluorescence resonance energy transfer experiments. The interactions between the two proteins is not due to the binding of substrate APPsw to the active site of memapsin 2, because neither a potent active site binding inhibitor of memapsin 2 nor an antibody directed to the beta-secretase site of APPsw had an effect on the uptake of M2(ED). In addition, full-length memapsin 2 and APP, immunoprecipitated together from cell lysates, suggested that the interaction of these two proteins is part of the native cellular processes.     

Adaptor protein 2-mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

The β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER-Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.     

The GAT domains of clathrin-associated GGA proteins have two ubiquitin binding motifs

Ubiquitin (Ub) attachment to membrane proteins can serve as a sorting signal for lysosomal delivery. Recognition of Ub as a sorting signal can occur at the trans-Golgi network and is mediated in part by the clathrin-associated Golgi-localizing, gamma-adaptin ear domain homology, ARF-binding proteins (GGA). GGA proteins bind Ub via a three-helix bundle subdomain in their GAT (GGA and target of Myb1 protein) domain, which is also present in the Ub binding domain of target of Myb1 protein. Ubiquitin binding by yeast Ggas is required to direct sorting of ubiquitinated proteins such as general amino acid permease (Gap1) from the trans-Golgi network to endosomes. Using affinity chromatography and nuclear magnetic resonance spectroscopy, we have found that the human GGA3 GAT domain contains two Ub binding motifs that bind to the same surface of ubiquitin. These motifs are found within different helices within the three-helix GAT subdomain. When functionally analyzed in yeast, each motif was sufficient to mediate trans-Golgi network to endosomal sorting of Gap1, and mutation of both motifs resulted in defective Gap1 sorting without defects in other GGA-dependent processes.     

Interactions of GGA3 with the ubiquitin sorting machinery

The Golgi-localized, gamma-ear-containing, Arf-binding (GGA) proteins constitute a family of clathrin adaptors that are mainly associated with the trans-Golgi network (TGN) and mediate the sorting of mannose 6-phosphate receptors. This sorting is dependent on the interaction of the VHS domain of the GGAs with acidic-cluster-dileucine signals in the cytosolic tails of the receptors. Here we demonstrate the existence of another population of GGAs that are associated with early endosomes. RNA interference (RNAi) of GGA3 expression results in accumulation of the cation-independent mannose 6-phosphate receptor and internalized epidermal growth factor (EGF) within enlarged early endosomes. This perturbation impairs the degradation of internalized EGF, a process that is normally dependent on the sorting of ubiquitinated EGF receptors (EGFRs) to late endosomes. Protein interaction analyses show that the GGAs bind ubiquitin. The VHS and GAT domains of GGA3 are responsible for this binding, as well as for interactions with TSG101, a component of the ubiquitin-dependent sorting machinery. Thus, GGAs may have additional roles in sorting of ubiquitinated cargo.     

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.     

Molecular mechanism of membrane recruitment of GGA by ARF in lysosomal protein transport

GGAs are critical for trafficking soluble proteins from the trans-Golgi network (TGN) to endosomes/lysosomes through interactions with TGN-sorting receptors, ADP-ribosylation factor (ARF) and clathrin. ARF-GTP bound to TGN membranes recruits its effector GGA by binding to the GAT domain, thus facilitating recognition of GGA for cargo-loaded receptors. Here we report the X-ray crystal structures of the human GGA1-GAT domain and the complex between ARF1-GTP and the N-terminal region of the GAT domain. When unbound, the GAT domain forms an elongated bundle of three a-helices with a hydrophobic core. Structurally, this domain, combined with the preceding VHS domain, resembles CALM, an AP180 homolog involved in endocytosis. In the complex with ARF1-GTP, a helix-loop-helix of the N-terminal part of GGA1-GAT interacts with the switches 1 and 2 of ARF1 predominantly in a hydrophobic manner. These data reveal a molecular mechanism underlying membrane recruitment of adaptor proteins by ARF-GTP.     

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.     

GGA proteins bind ubiquitin to facilitate sorting at the trans-Golgi network

Ubiquitination functions as a sorting signal for lysosomal degradation of cell-surface proteins by facilitating their internalization from the plasma membrane and incorporation into lumenal vesicles of multivesicular bodies (MVBs). Ubiquitin may also mediate sorting of proteins from the trans-Golgi network (TGN) to the endosome, thereby preventing their appearance on the cell surface and hastening their degradation in the lysosome-vacuole. Substantiation of a direct ubiquitin-dependent TGN sorting pathway relies in part on identifying candidate machinery that may function as a ubiquitin-sorting 'receptor'at the TGN. Members of the GGA family of coat proteins localize to the TGN and promote the incorporation of proteins into clathrin-coated vesicles destined for transport to endosomes. We show that the GGA coat proteins bind directly to ubiquitin through their GAT domain and demonstrate that this interaction is required for the ubiquitin-dependent sorting of the Gap1 amino acid transporter from the TGN to endosomes. Thus, GGA proteins fulfill the role of ubiquitin sorting receptors at the TGN.     

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.     

GAT (GGA and Tom1) domain responsible for ubiquitin binding and ubiquitination

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins) are a family of monomeric adaptor proteins involved in membrane trafficking from the trans-Golgi network to endosomes. The GAT (GGA and Tom1) domains of GGAs have previously been shown to interact with GTP-bound ARF and to be crucial for membrane recruitment of GGAs. Here we show that the C-terminal subdomain of the GAT domain, which is distinct from the N-terminal GAT subdomain responsible for ARF binding, can bind ubiquitin. The binding is mediated by interactions between residues on one side of the alpha3 helix of the GAT domain and those on the so-called Ile-44 surface patch of ubiquitin. The binding of the GAT domain to ubiquitin can be enhanced by the presence of a GTP-bound form of ARF. Furthermore, GGA itself is ubiquitinated in a manner dependent on the GAT-ubiquitin interaction. These results delineate the molecular basis for the interaction between ubiquitin and GAT and suggest that GGA-mediated trafficking is regulated by the ubiquitin system as endosomal trafficking mediated by other ubiquitin-binding proteins.     

Yeast Gga Coat Proteins Function with Clathrin in Golgi to Endosome Transport

Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the "ear" domain of the clathrin adaptor AP-1 gamma subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Delta), the major Gga protein, accentuates growth and alpha-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Delta or a deletion of the AP-1 beta subunit gene (apl2Delta) alone are phenotypically normal, but cells carrying both gga2Delta and apl2Delta are defective in growth, alpha-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.     

Monoubiquitylation of GGA3 by hVPS18 regulates its ubiquitin-binding ability

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins), constitute a family of monomeric adaptor proteins and are associated with protein trafficking from the trans-Golgi network to endosomes. Here, we show that GGA3 is monoubiquitylated by a RING-H2 type-ubiquitin ligase hVPS18 (human homologue of vacuolar protein sorting 18). By in vitro ubiquitylation assays, we have identified lysine 258 in the GAT domain as a major ubiquitylation site that resides adjacent to the ubiquitin-binding site. The ubiquitylation is abolished by a mutation in either the GAT domain or ubiquitin that disrupts the GAT-ubiquitin interaction, indicating that the ubiquitin binding is a prerequisite for the ubiquitylation. Furthermore, the GAT domain ubiquitylated by hVPS18 no longer binds to ubiquitin, indicating that ubiquitylation negatively regulates the ubiquitin-binding ability of the GAT domain. These results suggest that the ubiquitin binding and ubiquitylation of GGA3-GAT domain are mutually inseparable through a ubiquitin ligase activity of hVPS18.     

Surface distribution of the mannose 6-phosphate receptors in epithelial Madin-Darby canine kidney cells

We have analyzed the surface polarity of both the cation-independent (CI-MPR) and the cation-dependent (CD-MPR) mannose 6-phosphate receptors in the epithelial Madin-Darby canine kidney (MDCK) cell line grown on polycarbonate filters. The surface localization was studied by plasma membrane domain-specific surface labeling methods and by confocal microscopy using MPR-specific antibodies. The CI-MPR was shown to be exclusively present on the basolateral cell surface. In contrast, the CD-MPR was expressed neither apically nor basolaterally. However, an intracellular pool of CD-MPR could be detected. In MDCKII-RCAr cells, cell surface CI-MPR was shown to recycle between the basolateral plasma membrane and the trans-Golgi network. After exogalactosylation, cell surface CI-MPR acquired sialic acid residues in a time-dependent manner. Furthermore, the basolateral CI-MPR was shown to be functional. Lysosomal enzymes, bearing the mannose 6-phosphate recognition marker, were taken up from the basolateral medium and endocytosed into the cells. Uptake of lysosomal enzymes from the apical side was insignificant and not MPR mediated. These results extend previous immunoelectron microscopic studies on the intracellular polarity of the CI-MPR (Parton, R. G., Prydz, K., Bomsel, M., Simons, K., and Griffiths, G. (1989) J. Cell Biol. 109, 3259-3272) which showed that the CI-MPR was present in basolateral early endosomes and in late endosomes but absent from apical early endosomes.     

Golgi-localizing, gamma-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains

GGA (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) proteins are potential effectors of ADP-ribosylation factors, are associated with the trans-Golgi network (TGN), and are involved in protein transport from this compartment. By yeast two-hybrid screening and subsequent two-hybrid and pull-down analyses, we have shown that GGA proteins, through their VHS (Vps27p/Hrs/STAM) domains, interact with acidic dileucine sequences found in the cytoplasmic domains of TGN-localized sorting receptors such as sortilin and mannose 6-phosphate receptor. A mutational analysis has revealed that a leucine pair and a cluster of acidic residues adjacent to the pair are mainly responsible for the interaction. A chimeric receptor with the sortilin cytoplasmic domain localizes to the TGN, whereas the chimeric receptor with a mutation at the leucine pair or the acidic cluster is mislocalized to punctate structures reminiscent of early endosomes. These results indicate that GGA proteins regulate the localization to or exit from the TGN of the sorting receptors.     

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.     

The yeast Vps10p cytoplasmic tail mediates lysosomal sorting in mammalian cells and interacts with human GGAs.

Yeast Vps10p is a receptor for transport of the soluble vacuolar hydrolase carboxypeptidase Y to the lysosome-like vacuole. Its functional equivalents in mammalian cells are the mannose 6-phosphate receptors that mediate sorting to lysosomes of mannose 6-phosphate-containing lysosomal proteins. A chimeric receptor was constructed by substituting the cytoplasmic domain of M(r) 300,000 mannose 6-phosphate receptor with the Vps10p cytoplasmic tail. Expression of the chimera in cells lacking endogenous mannose 6-phosphate receptors resulted in a subcellular receptor distribution and an efficiency in sorting of lysosomal enzymes similar to that of the wild type M(r) 300,000 mannose 6-phosphate receptor. Moreover, the cytoplasmic tail of the Vps10p was found to interact with GGA1 and GGA2, two mammalian members of a recently discovered family of clathrin-binding cytosolic proteins that participate in trans-Golgi network-endosome trafficking in both mammals and yeast. Our findings suggest a conserved machinery for Golgi-endosome/vacuole sorting and may serve as a model for future studies of yeast proteins.     

The GGA proteins: adaptors on the move

The GGA proteins are a family of ubiquitously expressed, Arf-dependent clathrin adaptors that mediate the sorting of mannose-6-phosphate receptors between the trans-Golgi network and endosomes. Recent studies have elucidated the biochemical and structural bases for the interaction of the GGA proteins with many binding partners, and have shed light on the molecular and cellular mechanisms by which the GGA proteins participate in protein sorting.     

Golgi-localized, gamma-ear-containing, Arf-binding protein adaptors mediate insulin-responsive trafficking of glucose transporter 4 in 3T3-L1 adipocytes

Small glucose transporter 4 (Glut4)-containing vesicles represent the major insulin-responsive compartment in fat and skeletal muscle cells. The molecular mechanism of their biogenesis is not yet elucidated. Here, we studied the role of the newly discovered family of monomeric adaptor proteins, GGA (Golgi-localized, gamma-ear-containing, Arf-binding proteins), in the formation of small Glut4 vesicles and acquisition of insulin responsiveness in 3T3-L1 adipocytes. In these cells, all three GGA isoforms are expressed throughout the differentiation process. In particular, GGA2 is primarily present in trans-Golgi network and endosomes where it demonstrates a significant colocalization with the recycling pool of Glut4. Using the techniques of immunoadsorption as well as glutathione-S-transferase pull-down assay we found that Glut4 vesicles (but not Glut4 per se) interact with GGA via the Vps-27, Hrs, and STAM (VHS) domain. Moreover, a dominant negative GGA mutant inhibits formation of Glut4 vesicles in vitro. To study a possible role of GGA in Glut4 traffic in the living cell, we stably expressed a dominant negative GGA mutant in 3T3-L1 adipocytes. Formation of small insulin-responsive Glut4-containing vesicles and insulin-stimulated glucose uptake in these cells were markedly impaired. Thus, GGA adaptors participate in the formation of the insulin-responsive vesicular compartment from the intracellular donor membranes both in vivo and in vitro.