GGA Proteins Research Articles

Tom1, a VHS Domain-containing Protein, Interacts with Tollip, Ubiquitin, and Clathrin

The gene for Tom1 was initially identified as a specific target of the oncogene v-myb. The Tom1 protein belongs to the VHS domain-containing protein family, and it has a GAT domain in a central part as well as an N-terminal VHS domain. VHS domain-containing proteins, including Hrs/Vps27, STAM, and GGA proteins, have been implicated in intracellular trafficking and sorting, but the role of Tom1 has not yet been elucidated. In this study, we found that Tom1 binds directly with ubiquitin chains and Tollip, which was initially isolated as a mediator of interleukin-1 signaling and has a capacity to bind ubiquitin chains. Gel filtration and subsequent Western blot analysis showed that endogenous Tom1 associates with Tollip to form a complex. In addition, Tom1 was found to be capable of binding to clathrin heavy chain through a typical clathrin-binding motif. Fluorescence microscopic analysis revealed that green fluorescent protein-Tom1 was localized predominantly in the cytoplasm, whereas its mutant with deletion of the clathrin-binding motif had a diffuse localization throughout the cell. Thus, we propose that a Tom1-Tollip complex functions as a factor that links polyubiquitinated proteins to clathrin.

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.

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.

The Structure of the GGA1-GAT Domain Reveals the Molecular Basis for ARF Binding and Membrane Association of GGAs

The GGAs are a family of clathrin adaptor proteins involved in vesicular transport between the trans-Golgi network and endosomal system. Here we confirm reports that GGAs are targeted to the Golgi via interaction between the GGA-GAT domain and ARF-GTP, and we present the structure of the GAT domain of human GGA1, completing the structural description of the folded domains of GGA proteins. The GGA-GAT domain possesses an all α-helical fold with a “paper clip” topology comprising two independent subdomains. Structure-based mutagenesis demonstrates that ARF1-GTP binding by GGAs is exclusively governed by the N-terminal “hook” subdomain, and, using an in vitro recruitment assay, we show that ARF-GTP binding by this small structure is required and sufficient for Golgi targeting of GGAs.

タンパク質結晶学のブレイクスルー ２． 構造生物学のブレイクスルー 輸送系の構造生物学

Many eukaryiotic proteins are glycosylated after translation to become mature proteins. Efficient sorting of such glycosylated proteins is essential for the cell's function and is achieved by vesicle transport. Mannose 6-phosphate (M6P) modification of lysosomal proteins is recognized by a M6P receptor (MPR) which incorporates the lysosomal proteins to vesicles. Recently GGA proteins have been identified and later verified as a new family of adapter proteins distinct from well known AP-1 to 4 complexes. They are responsible for vesicle transport of glycosylated human proteins from the Golgi apparatus to early endosomesl lysosomes. We have determined the crystal structures of the VHS domain of human GGA 1 and the ear domain of human γ1 adaptin. Combined with biochemical and cell-biological data, these structures reveal the recognition mechanisms of the acidic dileucine motif signal of mannose-6-phophate receptor by the VHS domain of human GGA protein and the interaction between the ear domain of γ1 adaptin of the AP-1 complex and γ-synergin and Rabaptin-5. They constitute part of a rapidly growing ensemble of structures of transport proteins, where protein-protein interactions, as elucidated by X-ray structural analyses, play critical roles.

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.

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.

GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADP-ribosylation factors.

ADP-ribosylation factors (ARFs) are a family of small GTPases that are involved in various aspects of membrane trafficking events. These include ARF1-ARF6, which are divided into three classes on the basis of similarity in the primary structure: Class I, ARF1-ARF3; Class II, ARF4 and ARF5; and Class III, ARF6. Previous studies identified a novel family of potential ARF effectors, termed GGA1-GGA3, which interact specifically with GTP-bound ARF1 and ARF3 and are localized to the trans-Golgi network (TGN) or its related compartment(s) (GGA is an abbreviation for Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding protein). In the present study we have shown that ARF proteins belonging to the three classes, ARF1, ARF5 and ARF6, can interact with all GGA proteins in a yeast two-hybrid assay, in vitro and in vivo. Segmentation of GGA proteins and isolation of GGA mutants defective in ARF binding have revealed that a limited region within the GGA homology domain, which is conserved in the GGA family, is essential for ARF binding. Expression in cells of GTPase-restricted mutants of ARF1 and ARF5 blocks dissociation of GGA proteins from membranes induced by brefeldin A. However, neither of the ARF mutants recruits GGA mutants defective in ARF binding. On the basis of these observations, we conclude that at least ARF1 (Class I) and ARF5 (Class II) in their GTP-bound state cause recruitment of GGA proteins on to TGN membranes. In contrast, on the basis of similar experiments, ARF6 (Class III) may be involved in recruitment of GGA proteins to other compartments, possibly early endosomes.

ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization.

Golgi-localized gamma-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ~25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs and VPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

Dominant-negative mutant of BIG2, an ARF–guanine nucleotide exchange factor, specifically affects membrane trafficking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins

BIG2 is one of the guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and AP-1 coat protein complexes and GGA proteins. Brefeldin A (BFA), an ARF–GEF inhibitor, causes redistribution of the coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). We have recently shown that BIG2 overexpression blocks BFA-induced redistribution of the AP-1 complex but not TGN membrane tubulation. In the present study, we constructed a dominant-negative BIG2 mutant and found that when expressed in cells it induced redistribution of AP-1 and GGA1 and membrane tubulation of the TGN. By contrast, the mutant did not induce COPI redistribution or Golgi membrane tubulation. These observations indicate that BIG2 is involved in trafficking from the TGN by regulating membrane association of AP-1 and GGA through activating ARF.

Phosphoregulation of sorting signal-VHS domain interactions by a direct electrostatic mechanism.

Phosphorylation of the cytosolic tails of transmembrane receptors can regulate their intracellular trafficking. The structural basis for such regulation, however, has not been explained in most cases. The cytosolic tail of the cation-independent mannose 6-phosphate receptor contains a serine residue within an acidic-cluster dileucine signal that is important for the function of the receptor in the biosynthetic sorting of lysosomal hydrolases. We show here that phosphorylation of this Ser enhances interactions of the signal with its recognition module, the VHS domain of the GGA proteins. Crystallographic analyses demonstrate that the phosphoserine residue interacts electrostatically with two basic residues on the VHS domain of GGA3, thus providing an additional point of attachment of the acidic-cluster dileucine signal to its recognition module.

Single-handed recognition of a sorting traffic motif by the GGA proteins

Selective transport of cargo between membrane-bound organelles is vital for the well-being of cells. The crystal structure of a short peptide signal from the cytoplasmic tail of the mannose-6-phosphate receptor bound to the VHS domain of GGA proteins gives hints to how sorting works.

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GGA proteins: new players in the sorting game.

The GGA proteins are a novel family of proteins that were discovered nearly simultaneously by several labs studying very different aspects of membrane trafficking. Since then, several studies have described the GGA proteins and their functions in yeast and mammalian cells. Four protein domains are present in all GGA proteins, as defined by sequence homology and function. These different domains interact directly with ARF proteins, cargo and clathrin. Alteration of the levels of GGA proteins by gene knockout or overexpression affects specific trafficking events between the trans-Golgi network and endosomes. These data suggest that GGAs function as ARF-dependent, monomeric clathrin adaptors to facilitate cargo sorting and vesicle formation at the trans-Golgi network.

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.

GGAs Tie Up the Loose Ends

Cargo receptors, which recruit cargo proteins into transport vesicles, must themselves be sorted into appropriate vesicles budding from the correct cellular compartment. The job of sorting CI-MPR and CD-MPR cargo receptors into newly forming vesicles at the trans-Golgi network (TGN) has been widely attributed to the adaptor protein AP-1. Now, as [Tooze][1] explains in her Perspective, the job of sorting MPRs into TGN vesicles appears to be carried out instead by members of the GGA protein family ([ Zhu et al .][2], [ Puertollano et al .][3]). [1]: http://www.sciencemag.org/cgi/content/full/292/5522/1663 [2]: http://www.sciencemag.org/cgi/content/short/292/5522/1716 [3]: http://www.sciencemag.org/cgi/content/short/292/5522/1712

A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins.

Pep12p is a yeast syntaxin located primarily in late endosomes. Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole. When this signal is mutated, Pep12p instead passes to early endosomes, a step that is determined by its transmembrane domain. Surprisingly, Pep12p is then specifically retained in early endosomes and does not go on to late endosomes. By testing appropriate chimeras in mutant strains, we found that FSDSPEF-dependent sorting was abolished in strains lacking Gga1p and Gga2p, Golgi-associated coat proteins with homology to gamma adaptin. In the gga1 gga2 double mutant endogenous Pep12p cofractionated with the early endosome marker Tlg1p, and recycling of Snc1p through early endosomes was defective. Pep12p sorting was also defective in cells lacking the clathrin heavy or light chain. We suggest that specific and direct delivery of proteins to early and late endosomes is required to maintain the functional heterogeneity of the endocytic pathway and that the GGA proteins, probably in association with clathrin, help create vesicles destined for late endosomes.

Adaptor γ Ear Homology Domain Conserved in γ-Adaptin and GGA Proteins That Interact with γ-Synergin

We identified a novel family of proteins that have a VHS domain and an AGEH (adaptor γ ear homology) domain that is homologous to the ear domain of the γ-adaptin subunit of the AP-1 clathrin adaptor. When overexpressed, the proteins, called GGA1, GGA2, and GGA3, localized to the trans-Golgi network (TGN) and often caused fragmentation and vacuolation of the compartment. Yeast two-hybrid analysis showed that the AGEH domains of the GGA proteins as well as those of γ-adaptins are able to interact with γ-synergin, which was previously shown to localized in the TGN region and interact with γ-adaptin. Furthermore, γ-synergin and either of the GGA proteins coexpressed were colocalized in the TGN region. These results suggest that the GGA proteins regulate the function of the TGN or membrane trafficking from this compartment and that the AGEH domains of GGAs and γ-adaptins, like the ear domain of α-adaptin, are involved in interaction with molecules that modulate their functions.

Sorting and Membrane Traffic

CELL BIOLOGY Soluble and membrane proteins destined for lysosomes or vacuoles are sorted from other proteins in the trans Golgi network (TGN). Three groups—Hirst et al. , Dell'Angelica et al. , and Boman et al. —have isolated proteins (named GGAs) that are involved in this process, most likely in the formation of coats on transport vesicles. The GGA proteins are structurally related to another class of coat proteins, the γ-adaptins, which are involved in trafficking proteins from the TGN to the plasma membrane. Immunoelectron microscopy and immunofluorescence microscopy localized the GGA proteins to the TGN. These proteins also contain a VHS domain, thought to be involved in membrane traffic and signal transduction, and a GAT domain that appears to be involved in targeting the GGA protein to the TGN membrane and in binding ARF1, a small GTP-binding protein already implicated in membrane traffic. In yeast strains lacking the GGA proteins vacuolar targeting of a hydrolase was defective. The identification of other coat proteins that interact with the GGAs should lead to a better understanding of the sorting machinery at the exit of the Golgi.— SMH J. Cell Biol. 149 , 67 (2000); J. Cell Biol. 149 , 81 (2000); Mol. Biol. Cell 11 , 1241 (2000).

Yeast GGA proteins interact with GTP-bound Arf and facilitate transport through the Golgi.

ARF proteins regulate the formation of transport vesicles at many steps of the secretory and endocytic pathways. A recently identified family of ARF effectors, named GGAs, appears to regulate membrane traffic exiting the trans-Golgi network in mammalian cells (Boman et al., 2000). We have identified two GGA homologues in the yeast S. cerevisiae. These previously uncharacterized open reading frames, YDR358w and YHR108w, have been named GGA1 and GGA2, respectively. Using the two-hybrid assay and GST-affinity chromatography, we show that Gga1p and Gga2p interact with Arf1p and Arf2p in a GTP-dependent manner, suggesting that both are functional homologues of the human GGA proteins. The Arf-binding domain resides in the amino-terminal half of Gga1p (amino acids 170-330), and the carboxy-terminal 100 amino acids resemble the gamma-adaptin 'ear domain'. Gene deletion experiments indicate that GGA1 and GGA2 are not essential genes, as single and double knockouts are viable at both 30 degrees C and 37 degrees C. However, cells lacking GGA1 and GGA2 exhibit defects in invertase processing and CPY sorting, but not endocytosis. We conclude that yeast Gga proteins are effectors of Arf in yeast that facilitate traffic through the late Golgi.