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.     

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.     

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.     

Tollip and Tom1 form a complex and recruit ubiquitin-conjugated proteins onto early endosomes

Tom1 (target of Myb1) is a protein of unknown function. Tom1 and its relative Tom1L1 have an N-terminal VHS (Vps27p/Hrs/Stam) domain followed by a GAT (GGA and Tom1) domain, both of which are also found in the GGA (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor-binding protein) family of proteins. Although the VHS and GAT domains of GGA proteins bind to transmembrane cargo proteins and the small GTPase ADP-ribosylation factor, respectively, the VHS and GAT domains of Tom1 are unable to interact with these proteins. In this study, we show that the GAT domains of Tom1 and Tom1L1 interact with ubiquitin and Tollip (Toll-interacting protein). Ubiquitin bound the GAT domains of Tom1, Tom1L1, and GGA proteins, whereas Tollip interacted specifically with Tom1 and Tom1L1. Ubiquitin and Tollip bound to an overlapping region of the Tom1-GAT domain in a mutually exclusive manner. Tom1 was predominantly cytosolic when expressed in cells. On the other hand, Tollip was localized on early endosomes and recruited Tom1 and ubiquitinated proteins. These observations suggest that Tollip and Tom1 form a complex and regulate endosomal trafficking of ubiquitinated proteins.     

The trihelical bundle subdomain of the GGA proteins interacts with multiple partners through overlapping but distinct sites

The Golgi-localized, gamma-adaptin ear-containing, ARF-binding (GGA) proteins are monomeric clathrin adaptors that mediate the sorting of cargo at the trans-Golgi network and endosomes. The GGAs contain four different domains named Vps27, Hrs, Stam (VHS); GGAs and TOM1 (GAT); hinge; and gamma-adaptin ear (GAE). The VHS domain recognizes transmembrane cargo, whereas the hinge and GAE regions bind clathrin and accessory proteins, respectively. The GAT domain is a polyfunctional module that interacts with various partners including the small GTPase ARF, the endosomal fusion regulator Rabaptin-5, ubiquitin, and the product of the tumor susceptibility gene 101 (TSG101). Previous x-ray crystallographic analyses showed that the GAT region is composed of two subdomains, an N-terminal helix-loop-helix containing the ARF binding site, and a C-terminal triple alpha-helical (trihelical) bundle. In this study, we define the Rabaptin-5 binding site on the GGA1-GAT domain and its relationship to the binding sites for ubiquitin and TSG101. Our observations show that Rabaptin-5, ubiquitin, and TSG101 bind to overlapping but distinct binding sites on the trihelical bundle. The different GAT binding partners engage in both competitive and cooperative interactions that may be important for the function of the GGAs in protein sorting.     

K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway

A growing number of yeast and mammalian plasma membrane proteins are reported to be modified with K63-linked ubiquitin (Ub) chains. However, the relative importance of this modification versus monoubiquitylation in endocytosis, Golgi to endosome traffic, and sorting into the multivesicular body (MVB) pathway remains unclear. In this study, we show that K63-linked ubiquitylation of the Gap1 permease is essential for its entry into the MVB pathway. Carboxypeptidase S also requires modification with a K63-Ub chain for correct MVB sorting. In contrast, monoubiquitylation of a single target lysine of Gap1 is a sufficient signal for its internalization from the cell surface, and Golgi to endosome transport of the permease requires neither its ubiquitylation nor the Ub-binding GAT (Gga and Tom1) domain of Gga (Golgi localizing, gamma-ear containing, ARF binding) adapter proteins, the latter being crucial for subsequent MVB sorting of the permease. Our data reveal that K63-linked Ub chains act as a specific signal for MVB sorting, providing further insight into the Ub code of membrane protein trafficking.     

Structural Requirements for Function of Yeast GGAs in Vacuolar Protein Sorting, α-Factor Maturation, and Interactions with Clathrin

The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are a family of multidomain adaptor proteins involved in protein sorting at the trans-Golgi network of eukaryotic cells. Here we present results from a functional characterization of the two Saccharomyces cerevisiae GGAs, Gga1p and Gga2p. We show that deletion of both GGA genes causes defects in sorting of carboxypeptidase Y (CPY) and proteinase A to the vacuole, vacuolar morphology, and maturation of alpha-factor. A structure-function analysis reveals a requirement of the VHS, GAT, and hinge for function, while the GAE domain is less important. We identify putative clathrin-binding motifs in the hinge domain of both yeast GGAs. These motifs are shown to mediate clathrin binding in vitro. While mutation of these motifs alone does not block function of the GGAs in vivo, combining these mutations with truncations of the hinge and GAE domains diminishes function, suggesting functional cooperation between different clathrin-binding elements. Thus, these observations demonstrate that the yeast GGAs play important roles in the CPY pathway, vacuole biogenesis, and alpha-factor maturation and identify structural determinants that are critical for these functions.     

The interaction of the human GGA1 GAT domain with rabaptin-5 is mediated by residues on its three-helix bundle

GGA proteins regulate clathrin-coated vesicle trafficking by interacting with multiple proteins during vesicle assembly. As part of this process, the GAT domain of GGA is known to interact with both ARF and Rabaptin-5. Particularly, the GAT domains of GGA1 and -2, but not of GGA3, specifically bind with a coiled-coil region of Rabaptin-5. Rabaptin-5 interacts with Rab5 and is an essential component of the fusion machinery for targeting endocytic vesicles to early endosomes. The recently determined crystal structure of the GGA1 GAT domain has provided insights into its interactions with partner proteins. Here, we describe mutagenesis studies on the GAT-Rabaptin-5 interaction. The results demonstrate that a hydrophobic surface patch on the C-terminal three-helix bundle motif of the GAT domain is directly involved in Rabaptin-5 binding. A GGA3-like mutation, N284S, in this Rabaptin-5 binding patch of GGA1 led to a reduced level of Rabaptin-5 binding. Furthermore, a reversed mutation, S293N, in GGA3 partially establishes Rabaptin-5 binding ability in its GAT domain. These results provide a structural explanation for the binding affinity difference among GGA proteins. The current results also suggest that the binding of GAT to Rabaptin-5 is independent of its interaction with ARF.     

Crystal structure of the human GGA1 GAT domain

GGAs are a family of vesicle-coating regulatory proteins that function in intracellular protein transport. A GGA molecule contains four domains, each mediating interaction with other proteins in carrying out intracellular transport. The GAT domain of GGAs has been identified as the structural entity that binds membrane-bound ARF, a molecular switch regulating vesicle-coat assembly. It also directly interacts with rabaptin5, an essential component of endosome fusion. A 2.8 A resolution crystal structure of the human GGA1 GAT domain is reported here. The GAT domain contains four helices and has an elongated shape with the longest dimension exceeding 80 A. Its longest helix is involved in two structural motifs: an N-terminal helix-loop-helix motif and a C-terminal three-helix bundle. The N-terminal motif harbors the most conservative amino acid sequence in the GGA GAT domains. Within this conserved region, a cluster of residues previously implicated in ARF binding forms a hydrophobic surface patch, which is likely to be the ARF-binding site. In addition, a structure-based mutagenesis-biochemical analysis demonstrates that the C-terminal three-helix bundle of this GAT domain is responsible for the rabaptin5 binding. These structural characteristics are consistent with a model supporting multiple functional roles for the GAT domain.     

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.     

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.     

Ubiquitin interactions of NZF zinc fingers

Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of the NZF domain from Npl4 in complex with Ub. The structure reveals that three key NZF residues (13TF14/M25) surrounding the zinc coordination site bind the hydrophobic 'Ile44' surface of Ub. Mutations in the 13TF14/M25 motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub. However, substitution of the 13TF14/M25 motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold. Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole. Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes.     

Structural basis for recognition of ubiquitinated cargo by Tom1-GAT domain

Tom1 (Target of Myb1) is suggested to be involved in the transport of ubiquitinated proteins, through the interaction of its GAT (GGA and Tom1) domain with ubiquitin. Here, we demonstrate that the three-helix bundle of Tom1-GAT has two ubiquitin-binding sites recognizing the hydrophobic Ile44 surface of ubiquitin. The complex crystal structure demonstrates that the first site is a hydrophobic patch on helices alpha1 and alpha2. NMR and biochemical data revealed that the N-terminal half of helix alpha3 of Tom1-GAT constitutes the second, stronger binding site. The double-sided ubiquitin binding enhances the efficiency of recognition of ubiquitinated proteins by Tom1.     

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.     

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.     

Crystal structure of human GGA1 GAT domain complexed with the GAT-binding domain of Rabaptin5

GGA proteins coordinate the intracellular trafficking of clathrin-coated vesicles through their interaction with several other proteins. The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5. The GGA-Rabaptin5 interaction is believed to function in the fusion of trans-Golgi-derived vesicles to endosomes. We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT-binding domain. In this structure, the Rabaptin5 domain is a 90-residue-long helix. At the N-terminal end, it forms a parallel coiled-coil homodimer, which binds one GAT domain of GGA1. In the C-terminal region, it further assembles into a four-helix bundle tetramer. The Rabaptin5-binding motif of the GGA1 GAT domain consists of a three-helix bundle. Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle-helix bundle interaction. The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis. The four-helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles.     

DOA1/UFD3 plays a role in sorting ubiquitinated membrane proteins into multivesicular bodies

Ubiquitin (Ub) is a sorting signal that targets integral membrane proteins to the interior of the vacuole/lysosome by directing them into lumenal vesicles of multivesicular bodies (MVBs). The Vps27-Hse1 complex, which is homologous to the Hrs-STAM complex in mammalian cells, serves as a Ub-sorting receptor at the surface of early endosomes. We have found that Hse1 interacts with Doa1/Ufd3. Doa1 is known to interact with Cdc48/p97 and Ub and is required for maintaining Ub levels. We find that the Hse1 Src homology 3 domain binds directly to the central PFU domain of Doa1. Mutations in Doa1 that block Hse1 binding but not Ub binding do not alter Ub levels but do result in the missorting of the MVB cargo GFP-Cps1. Loss of Doa1 also causes a synthetic growth defect when combined with loss of Vps27. Unlike the loss of Doa1 alone, the doa1Delta vps27Delta double mutant phenotype is not suppressed by Ub overexpression, demonstrating that the effect is not due to indirect consequence of lowered Ub levels. Loss of Doa1 results in a defect in the accumulation of GFP-Ub within yeast vacuoles, implying that there is a reduction in the flux of ubiquitinated membrane proteins through the MVB pathway. This defect was also reflected by an inability to properly sort Vph1-GFP-Ub, a modified subunit of the multiprotein vacuolar ATPase complex, which carries an in-frame fusion of Ub as an MVB sorting signal. These results reveal novel roles for Doa1 in helping to process ubiquitinated membrane proteins for sorting into MVBs.     

Interactions of TOM1L1 with the multivesicular body sorting machinery

Tom1L1 (Tom1-like1) and related proteins Tom1 (Target of Myb1) and Tom1L2 (Tom1-like2) constitute a new protein family characterized by the presence of a VHS (Vps27p/Hrs/Stam) domain in the N-terminal portion followed by a GAT (GGA and Tom) domain. Recently it was demonstrated that the GAT domain of both Tom1 and Tom1L1 binds ubiquitin, suggesting that these proteins might participate in the sorting of ubiquitinated proteins into multivesicular bodies (MVBs). Here we report a novel interaction between Tom1L1 and members of the MVB sorting machinery. Specifically, we found that the VHS domain of Tom1L1 interacts with Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate), whereas a PTAP motif, located between the VHS and GAT domain of Tom1L1, is responsible for binding to TSG101 (tumor susceptibility gene 101). Myc epitope-tagged Tom1L1 showed a cytosolic distribution but was recruited to endosomes following Hrs expression. In addition, Tom1L1 possesses several tyrosine motifs at the C-terminal region that mediate interactions with members of the Src family kinases and other signaling proteins such as Grb2 and p85. We showed that a fraction of Fyn kinase localizes at endosomes and that this distribution becomes more evident after epidermal growth factor internalization. Moreover, expression of a constitutive active form of Fyn also promoted the recruitment of Tom1L1 to enlarged endosomes. Taken together, we propose that Tom1L1 could act as an intermediary between signaling and degradative pathways.     

Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome

Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting "receptors," the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.