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Originally published In Press as doi:10.1074/jbc.M700936200 on March 6, 2007

J. Biol. Chem., Vol. 282, Issue 18, 13282-13289, May 4, 2007
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Clathrin Adaptor GGA1 Polymerizes Clathrin into Tubules*

Fang Zhang1, Yang-In Yim1, Sarah Scarselletta, Mark Norton, Evan Eisenberg, and Lois E. Greene2

From the Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-0301

Received for publication, January 31, 2007 , and in revised form, March 2, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
GGAs, a class of monomeric clathrin adaptors, are involved in the sorting of cargo at the trans-Golgi network of eukaryotic cells. They are modular structures consisting of the VHS, the GAT, hinge, and GAE domains, which have been shown to interact directly with cargo, ARF, clathrin, and accessory proteins, respectively. Previous studies have shown that GGAs interact with clathrin both in solution and in the cell, but it has yet been shown whether they assemble clathrin. We find that GGA1 promoted assembly of clathrin with complete assembly achieved when one GGA1 molecule is bound per heavy chain. In the presence of excess GGA1, we obtained the unusual stoichiometry of five GGA1s per heavy chain, and even at this stoichiometry the binding was not saturated. The assembled structures were mostly baskets, but ~10% of the structures were tubular with an average length of 180 ± 40 nm and width of ~50 nm. The truncated GGA1 fragment consisting of the hinge+GAE domains bound to clathrin with similar affinity as the full-length molecule and polymerized clathrin into baskets. Unlike the full-length molecule, this fragment saturated the lattices at one molecule per heavy chain and assembled clathrin only into baskets. The separated hinge and GAE domains bound much weaker to clathrin than the intact molecule, and these domains do not significantly polymerize clathrin into baskets. We conclude that clathrin adaptor GGA1 is a clathrin assembly protein, but it is unique in its ability to polymerize clathrin into tubules.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The family of GGA proteins are ubiquitously expressed monomeric clathrin adaptor proteins that function in the sorting and trafficking of cargo. Humans have three members of this family, whereas yeast has two. The GGAs have four domains, the N-terminal VHS domain that binds to proteins containing the DXXLL sorting sequence such as the mannose 6-phosphate receptor, the GAT domain that binds ARF1-GTP, the hinge domain that binds clathrin, and the C-terminal GAE3 domain that binds accessory protein such as rabaptin-5 and {gamma}-synergin (1–4). High resolution structures have been obtained for all of the domains except the hinge domain, which is rather unstructured (2).

In mammalian cells, the GGA proteins bind primarily to the TGN where they colocalize with AP1 on clathrin-coated buds (5, 6). In addition, GGAs are present on transport intermediates emanating from the TGN and endosomes (7, 8). GGAs function to sort the mannose 6-phosphate receptors as they traffic between the TGN and the late endosome in mammalian cells (8–11). Similarly, in yeast, GGAs are important in the trafficking of carboxypeptidase Y and carboxypeptidase S from the TGN to the vacuole (5, 12). Recently, the GAT domain of GGA was found to bind a monoubiquitin motif on proteins. GGA uses this motif to sort epidermal growth factor receptor from the early endosome to the multivesicular body in mammalian cells and the GAP1 amino acid permease from the late Golgi to the vacuole in yeast (13, 14).

The association of GGA with clathrin on the TGN and transport vesicles has been demonstrated by electron microscopy using immunogold labeling (5, 6). Binding of GGA to these structures is dependent on activated ARF1 (6). Biochemical studies have established the direct interaction between GGAs and clathrin. GST-tagged fragments of the hinge and GAE domains from GGA1, GGA2, and GGA3 bound to clathrin in GST-pulldown assays. In addition, recombinant full-length GGA1 mediated the GTP-dependent binding of clathrin to liposomes containing ARF1 (15). GGA1 and GGA2 were also found on microsomes but not on CCVs purified from rat liver (5). Aside from genetic evidence suggesting interaction between GGA proteins and clathrin in yeast, direct physical association of GGA proteins and clathrin has been demonstrated for yeast proteins (12). This direct binding of yeast clathrin to yeast GGAs was demonstrated using GST-pulldown experiments, immunoprecipitation experiments, and chromatography of a high speed pellet enriched in yeast CCVs.

Members of the family of monomeric clathrin adaptors include AP180 and beta-arrestin, in addition to GGA proteins. In vitro experiments have shown that AP180 and beta-arrestin have very different clathrin-binding properties. Specifically, AP180 binds with high affinity to clathrin and polymerizes clathrin with a stoichiometry of one AP180 per clathrin triskelion (16). On the other hand, although beta-arrestin binds to clathrin with high affinity, it does not polymerize clathrin into baskets (17). As for the monomeric GGAs, although biochemical studies have established they bind to clathrin, there are contradictory observations regarding their strength of interaction with clathrin. In vivo studies have established that overexpression of GGA increases clathrin recruitment to the TGN ~3-fold suggesting strong binding of GGA to clathrin (15). On the other hand, when cells are permeabilized, GGAs readily dissociate from the TGN suggesting weak binding of GGAs to clathrin (18). A biochemical study of the interaction of GGA with clathrin should help clarify these observations.

In the present study we examined the ability of GGA1 and the domains of GGA1 to assemble clathrin and to bind to preassembled baskets. These studies show that, although GGA1 is able to assemble clathrin, it binds more weakly to clathrin than the multimeric clathrin adaptors AP1 and AP2 and the monomeric clathrin adaptor AP180. GGA1 also has a very unusual stoichiometry of binding to clathrin baskets with as many as five GGA1 molecules bound per CHC. Finally, we found that, unlike other adaptors, GGA1 polymerizes clathrin both into long tubules as well as into baskets. Unlike the full-length molecule, a fragment of GGA1 consisting of just the hinge and GAE domains bind with a stoichiometry of one molecule per CHC and only polymerizes clathrin into clathrin baskets.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Constructs and Purification—We used a bacterial expression system to purify full-length human GGA1 and the following domains of GGA1, hinge+GAE domain (amino acids 315–639), hinge domain (amino acids 315–514), and GAE domain (amino acids 515–639) (15). VHS plus GAT (amino acids 1–314) was cloned into the pGEX-4T-1 vector, and the human GGA2 (clone number: MGC-1002, ATCC, Manassas, VA) was subcloned into pGEX-6P-1 vector. The fusion proteins were expressed in Escherichia coli BL21. Bacteria were grown for 2 h before expression was induced with 0.5 mM isopropyl 1-thio-beta-D-galactopyranoside for another 4–5 h at 25 °C. The full-length His6-GGA1 was purified on TALON metal affinity resin (Clontech, Mountain View, CA). GST fusion proteins were purified on Glutathione-Sepharose (GE Healthcare, Piscataway, NJ). Protease inhibitor tablets (BD Biosciences) were added in the initial extraction step when preparing the GGA1 proteins. The GGA1 (hinge+GAE) construct and GGA2 in the pGEX-6P-1 vector were used to obtain the fusion protein in which we cleaved the GST moiety. To cleave GST from these constructs, we washed the matrix with cleavage buffer, added PreScission Protease (GE Healthcare) to cleave the GST from the fusion protein-bound Glutathione-Sepharose matrix, and then incubated the mixture at 4 °C overnight. On the next day, the eluate was collected and dialyzed against 50 mM Tris, 10 mM NaCl, 1 mM dithiothreitol, pH 7.0. His6-AP180(C58) plasmid, comprising the 58-kDa C-terminal domain of AP180, was constructed and prepared as described previously (16).

Preparation of Hsc70, Clathrin, CCVs, Clathrin Triskelia, and Clathrin Baskets—Hsc70 was prepared from bovine brains, and clathrin-coated vesicles were prepared from either fresh bovine brains or frozen rat livers (Pel-Freeze Biologicals, Rogers, AK) as described previously (19). Clathrin was extracted from brain CCVs using 0.5 M Tris, pH 7.0, and the high speed supernatant containing clathrin and APs was concentrated and loaded onto a preparative Superose 6 filtration column (2.5 x 90 cm) equilibrated in 0.5 M Tris, pH 7.0, at 25 °C to purify the clathrin. Pure clathrin baskets, and clathrin baskets assembled using APs were made as described previously (16). The AP180(C58)-clathrin baskets had a composition of one AP180(C58) per clathrin triskelion (16). The concentrations of clathrin, Hsc70, and GGA proteins were determined from their molar extinction coefficient and absorbance at 280 nm or from the Coomassie Blue intensity of these proteins on SDS gels using Hsc70 as a standard.


Figure 1
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  		FIGURE 1.
SDS gel (4–12%) of the purified recombinant GGA1 protein and domains of GGA1 stained with Coomassie Blue. Lane 1, 6-histidine-tagged full-length GGA1; lane 2, GST-(hinge+GAE) domains of GGA1; lane 3, GST-GAE domain; lane 4, GST-hinge domain; lane 5, hinge+GAE domains after cleavage of the GST protein with PreScission Protease.

 
Clathrin Binding and Clathrin Assembly Assays—The binding of full-length GGA and its domains to assembled clathrin baskets or CCVs was performed by incubating the proteins at 25 °C for 30 min in 0.15 M NaCl, 1 mM MgCl2, 1 mM dithiothreitol, and either 20 mM imidazole (pH 7.0) or 20 mM MES (pH 6.0). The assembly of clathrin was performed by adding various concentrations of GGA to a fixed concentration of clathrin, followed by overnight dialysis at 4 °C against polymerization buffer, 0.1 M MES, pH 6.5, 1 mM dithiothreitol (16). The clathrin and GGA1 solutions were centrifuged in a TL-100 ultracentrifuge at 400,000 x g x for 6 min at 4 °C. The supernatants were then run on SDS-PAGE gel electrophoresis on 4–12% gels (Invitrogen). Varying dilutions of the supernatants were used in running the SDS gels to ensure that the proteins were always in the linear range of the Coomassie Blue scale. The clathrin and GGA in the supernatant were quantified from the Coomassie Blue intensity measured using the ChemiImager densitometer (Alpha Innotech Corp., San Leandro, CA).

Electron Microscopy—For negatively stained images, 5-µl samples were applied to Formvar/carbon-coated grids and stained with 2% uranyl acetate. Preparations of proteins were fixed with 2.5% glutaraldehyde/1% paraformaldehyde in 0.1 M cacodylate buffer for 1 h at room temperature and overnight at 4 °C, post-fixed in 1% OsO4, and stained with 2% uranyl acetate and 1% tannic acid before embedding in Epon. The thin sections were stained further with 2% uranyl acetate and Reynold's lead citrate solution. Micrographs were taken by using a JEOL EX II electron microscope at 80 kV.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The clathrin adaptor GGA1 and its domains were made as recombinant proteins in E. coli. The full-length GGA1 protein was expressed with a 6-histidine tag, whereas the hinge-GAE region, the hinge region alone, and the GAE region alone were expressed as GST fusion proteins. Fig. 1 shows an SDS-PAGE gel of the proteins after purification by column chromatography. Full-length GGA1 (lane 1) has two bands; the lower molecular weight band is a proteolytic fragment of the full-length protein. To minimize degradation, the full-length GGA1 was always used within 24 h of preparation. The GST fusion constructs of the hinge+GAE region, the hinge region alone, and the GAE region alone yielded single bands (lanes 2–4). The hinge+GAE domain construct without the GST tag (lane 5) was prepared by cleaving the GST fusion protein with PreScission Protease.

We first examined the interaction of the full-length GGA1 with clathrin by determining whether GGA1 assembles clathrin into clathrin baskets. After overnight dialysis of GGA1 and clathrin against polymerization buffer, the clathrin-GGA1 solution was centrifuged to sediment the polymerized clathrin. The amount of free clathrin remaining in the supernatant was determined by SDS-gel electrophoresis. In the absence of GGA1, there was no significant sedimentation of clathrin under this condition (Fig. 2A, lanes 1 and 2). When 6 µM GGA1 was added to 0.4 µM clathrin triskelia (1.2 µM CHC), most of the clathrin sedimented, leaving only a small amount of the total clathrin in the supernatant (lanes 3 and 4). These results show that GGA1 induces sedimentation of the clathrin, presumably due to polymerization of clathrin.

Fig. 2B shows the concentration dependence of clathrin polymerization by GGA1. When 1 µM GGA1 was added to 0.3 µM clathrin triskelia, 50% of the GGA1 bound to the clathrin and correspondingly ~50% of the clathrin polymerized. To completely polymerize 0.3 µM clathrin triskelia, it was necessary to add ~2 µM GGA1. Under these conditions, one GGA1 molecule was bound per CHC (Fig. 2B). These results also indicate that GGA1 binds relatively weakly to clathrin with a dissociation constant in the micromolar range.

From the intensities of the unbound GGA1 bands in the SDS gel, the amount of GGA1 bound to the polymerized clathrin was calculated at each concentration of added GGA1. When the clathrin was completely polymerized, the stoichiometry of binding was one GGA1 per CHC. Surprisingly, however, as more GGA1 was added, it continued to bind to the polymerized clathrin reaching an unusually high stoichiometry of binding. For example, when 0.4 µM clathrin triskelia were polymerized by 6 µM GGA1, there was negligible GGA1 left in the supernatant, and this was not due to sedimentation of the GGA1 alone (Fig. 2A, lanes 5 and 6). Rather, the intensities of the bands on the SDS gel showed that ~5 GGA1 molecules bound per CHC. Furthermore, there was no indication that the GGA1 binding was approaching saturation as the GGA1 concentration was increased.

We also examined the binding of GGA1 to preformed clathrin lattices. In particular, we examined whether this binding also shows much more than one GGA1 molecule bound per CHC. The concentration dependence of GGA1 binding was measured using the following substrates: pure clathrin baskets at pH6.0, AP180(C58)-clathrin baskets at pH 7.0, or rat liver CCVs at pH 7.0. Rat liver CCVs are enriched in lipid and AP1 and are therefore the most physiological substrate for GGA1, a clathrin adaptor found at the TGN. As shown in Fig. 2C, essentially the same binding isotherms were obtained with the different substrates. Specifically, as the GGA1 concentration was increased, more than five GGA1 molecules bound per CHC with no indication of the binding reaching saturation. Therefore, we observed the same unusual binding properties of GGA1 to preassembled clathrin lattices as to clathrin assembled into lattices by the GGA1 itself. Furthermore, the GGA1 binding to clathrin did not appear to be affected by varying conditions such as pH, the presence of other clathrin adaptors, or the presence of membrane.


Figure 2
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  		FIGURE 2.
Interaction of full-length GGA1 with clathrin. A, GGA1 promotes clathrin assembly. After overnight dialysis against polymerization buffer, clathrin and GGA1 solutions were centrifuged. The dialyzed solution (lanes 1, 3, and 5) and the supernatant following centrifugation (lanes 2, 4, and 6) were run on SDS gels (4–12%). Lanes 1 and 2, clathrin alone (0.4µM triskelia or 1.2 µM CHC); lanes 3 and 4, clathrin (0.4 µM triskelia) and GGA1 (6 µM); lanes 5 and 6, GGA1 alone (6 µM). All samples were diluted 3-fold prior to electrophoresis to ensure the Coomassie Blue staining was in the linear range for densitometry measurements. B, polymerization of clathrin as a function of GGA1 concentration. Varying concentrations of GGA1 (0–5 µM) were added to 0.3 µM clathrin triskelia, and the solutions were dialyzed overnight. After centrifugation, the concentration of clathrin and GGA1 was measured in the supernatant to calculate the fraction of clathrin polymerized and the molecules of GGA1 bound per CHC. Data are plotted as either fraction clathrin polymerized (squares) or GGA1 bound per CHC (diamonds) as a function of GGA1 concentration. C, binding of GGA1 to various clathrin lattices. Varying concentrations of GGA1 (0–6 µM) were added to 0.3 µM clathrin triskelia using the following substrates: pure clathrin baskets at pH 6.0 (circles), AP180 (C58)-clathrin baskets at pH 7.0 (squares), and rat liver CVs at pH 7.0 (triangles). Data are plotted as GGA1 bound per CHC as a function of GGA1 concentration. The concentrations of clathrin and GGA1 in the supernatant were determined as described under "Experimental Procedures."

 


Figure 3
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  		FIGURE 3.
Interaction of full-length GGA2 with clathrin. A, GGA2 promotes clathrin assembly. After overnight dialysis against polymerization buffer, clathrin and GGA2 solutions were centrifuged. The dialyzed solution (lanes 1, 3, and 5) and the supernatant following centrifugation (lanes 2, 4, and 6) were run on SDS gels (4–12%). Lanes 1 and 2, clathrin alone (0.3 µM triskelia or 0.9 µM CHC); lanes 3 and 4, clathrin (0.3 µM triskelia) and GGA2 (6 µM); lanes 5 and 6, GGA2 alone (6 µM). B, binding of GGA2 to pure clathrin baskets at pH 6.0. Lanes 1 and 2, clathrin baskets alone (0.3 µM triskelia or 0.9 µM CHC); lanes 3 and 4, clathrin baskets (0.3 µM triskelia) and GGA2(6 µM); lanes 5 and 6, GGA2 alone (6 µM). All samples were diluted 3-fold prior to electrophoresis to ensure the Coomassie Blue staining was in the linear range for densitometry measurements.

 
We next examined whether GGA2 shows the same unusual stoichiometry of binding to clathrin as GGA1. First, the ability of GGA2 to polymerize clathrin into clathrin baskets was measured using 6 µM GGA2 and 0.3 µM clathrin triskelia. As expected, GGA2 polymerized clathrin just like GGA1, but with a very different stoichiometry of binding as indicated by all the free GGA2 in the supernatant (Fig. 3A, lane 4). The stoichiometry of binding, calculated from the total and free concentrations of GGA2, was 1.0 molecule of GGA2 bound per molecule of CHC in the polymerized baskets. Similar results were obtained for the binding of GGA2 to pure clathrin baskets measured using 6 µM GGA2 and 0.3 µM pure clathrin baskets at pH 6.0 (Fig. 3B). The stoichiometry of binding was calculated to be 0.6 molecule GGA2 per CHC, again very different from that obtained with GGA1.

To verify the unusual stoichiometry of GGA1 binding, we measured the stoichiometry directly by resuspending the GGA1-clathrin pellets obtained from both the assembly and binding experiments. After SDS electrophoresis of the resuspended pellets, the clathrin and GGA1 concentrations were then calculated from the Coomassie Blue intensities. In the assembly experiment, when 5 µM GGA1 was used to assemble 0.3 µM clathrin triskelia, the clathrin and GGA1 bands have similar intensities in both the original sample and resuspended pellet (Fig. 4A). From the staining intensities, the pellet had a ratio of 3.8 GGA1 molecules per CHC. In the binding experiment, when 4 µM GGA1 was added to 0.3 µM pure clathrin baskets, the resuspended pellet had a ratio of 3.2 GGA1 molecules bound per CHC. These results are in agreement with the stoichiometry of binding calculated from measuring the free GGA1 in the supernatant.


Figure 4
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  		FIGURE 4.
Binding of GGA1 to clathrin lattices and soluble clathrin. A, determination of stoichiometry of GGA1 binding to clathrin from resuspension of the clathrin-GGA1 pellet. In an assembly experiment, clathrin triskelia (0.3 µM) and GGA1 (5 µM) were dialyzed overnight and centrifuged, and the pellet after sedimentation was resuspended. In a binding experiment, pure clathrin baskets (0.3 µM clathrin triskelia) was mixed with GGA1 (4 µM), and the pellet after sedimentation was resuspended. The concentration of clathrin and the GGA1 in the resuspended pellet was determined to calculate the ratio of GGA1 per CHC. Lane 1, samples before spin; lane 2, resuspended pellets. B, Superose 12 column profile obtained from incubating soluble clathrin with GGA1. Clathrin (1.0 µM triskelia) and GGA1 (10 µM) were incubated for 30 min at 25 °C in 0.15 M NaCl, 20 mM imidazole (pH 7.0), 1 mM dithiothreitol prior to chromatography. Fractions were run on SDS gels to determine the percentage of clathrin and GGA1 in each fraction.

 
To examine whether GGA1 either self-assembles or assembles on soluble clathrin triskelia, we performed column chromatography experiments. Fig. 4B shows the elution profile from the Superose 12 column obtained from incubating 10 µM GGA1 with 1 µM clathrin triskelia. The monomeric clathrin, which eluted in the void volume, showed no interaction with GGA1. The GGA1 eluted in the same fraction in the presence and absence of clathrin (data not shown). Moreover, GGA1 alone was not self-assembling, because it eluted in the same position as the 70-kDA protein, Hsc70. Therefore, the unusual binding stoichiometry of GGA1 to clathrin only occurs with polymerized clathrin.


Figure 5
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  		FIGURE 5.
Electron micrographs of clathrin baskets. A, clathrin structures formed from dialysis against polymerizing buffer of 0.3 µM clathrin triskelia and 10 µM GGA1 (5 µM). Inset, enlarged image of the polymerized clathrin basket. B, enlarged image of clathrin tubule polymerized by GGA1. C, AP180-clathrin baskets, enlarged image of clathrin basket. D, AP-180 baskets incubated with 10 µM GGA1. Inset, enlarged image of clathrin basket. E, pure clathrin baskets. F, pure clathrin baskets incubated with 10 µM GGA1.

 
Given the unusual binding properties of GGA1 to polymerized clathrin, we examined by electron microscopy whether clathrin polymerized by GGA1 formed clathrin baskets. The dialysate obtained after overnight dialysis of 0.3 µM clathrin with 10 µM GGA was imaged using negative staining. As shown in Fig. 5, GGA1 clearly polymerized the clathrin into baskets, but in addition to baskets, we obtained an unusual tubule-shaped clathrin lattice. These structures, which were 10% of the population in 230 images, had an average length of 180 ± 40 nm and a width of ~50 nm. As shown in Fig. 5B, some of the clathrin tubules had a very ordered appearance and frequently we could detect rows of hexagons in these tubules. The clathrin baskets polymerized by GGA1 had a denser appearance and a less well defined edge than baskets polymerized in the absence of GGA1. This is evident by comparing baskets polymerized by AP180 (C58) to the identical baskets incubated with 10 µM GGA1 (Fig. 5, C and D). Similar dense appearance of baskets was obtained from incubating pure clathrin baskets with 10 µM GGA1 (Fig. 5, E and F). In addition, we found that there was a reduction in the diameter of AP180 (C58) baskets in the presence of GGA1. In the absence of GGA1, the baskets had a diameter of 99 ± 11 nm (n = 100), whereas the diameter was reduced to 73 ± 8nm(n = 100) in the presence of GGA1. This suggests that the baskets are more rigid in the presence of GGA1 and do not flatten on the grid when they are processed for negative staining. Polymerization of clathrin by GGA2 produced baskets (data not shown). Tubules were only observed when clathrin was polymerized in the presence of excess GGA1. They were not found when clathrin was polymerized by excess GGA2 or when GGA1 was added to preformed baskets.

We next investigated whether the GST-(hinge+GAE) construct shows the same high stoichiometry of binding to clathrin as full-length GGA1. The graph in Fig. 6A shows that the GST-(hinge+GAE) construct polymerized clathrin with a stoichiometry of one hinge+GAE molecule bound per CHC, the same binding stoichiometry as occurs with full-length GGA1. Unlike the intact molecule, the binding was saturated as evident by the constant stoichiometry with increasing concentrations of the hinge+GAE construct. Electron microscopy verified that the GST-(hinge+GAE) construct assembled clathrin into baskets, but interestingly no tubule formation was observed (data not shown).

We also measured the binding of a GST-(hinge+GAE) construct to pure clathrin baskets. As shown in Fig. 6B, the GST-(hinge+GAE) construct bound to clathrin with a stoichiometry of one hinge+GAE construct per CHC, and again this binding saturated at one hinge+GAE construct per CHC. To verify the stoichiometry of binding of the GST-(hinge+GAE) construct to clathrin, we resuspended and analyzed the pellets from both the assembly experiment and the binding experiments (Fig. 6C). After overnight dialysis of 0.35 µM clathrin triskelia and 8 µM GST-(hinge+GAE) construct, the resuspended pellet showed 1.2 GST-(hinge+GAE) construct bound per CHC. Similarly, in the binding experiment to preformed baskets using 0.35 µM clathrin triskelia and 10 µM GST-(hinge+GAE) construct, the pellet had 1.1 GST-(hinge+GAE) construct bound per CHC. Therefore, the GST-(hinge+GAE) construct does not show the high stoichiometry of binding observed with full-length GGA1, nor does it induce formation of clathrin tubules.

Because GST fusion proteins dimerize we were concerned that the stoichiometry of binding might be affected by the presence of GST. Therefore, to ensure that the GST-(hinge+GAE) construct bound with a stoichiometry of one molecule per CHC, the GST tag was cleaved using PreScission Protease. Baskets were then assembled by overnight dialysis of 0.3 µM clathrin triskelia and 5 µM hinge+GAE construct against polymerization buffer, and the resuspended pellet was analyzed by SDS gel. The results showed a binding stoichiometry of 1.35 hinge+GAE construct bound per CHC. Similarly, when 5 µM hinge+GAE construct was added to 0.3 µM pure clathrin baskets at pH 6.0, the resuspended pellet showed a stoichiometry of 0.9 hinge+GAE construct domain bound per CHC. Therefore, in contrast to full-length GGA1, which bound with an unusually high stoichiometry to clathrin, we obtained a stoichiometry of binding of one hinge+GAE construct per CHC.

Next, because the hinge domain of GGA1 contains the clathrin-binding motif, LLDDE, we measured the ability of the GST-hinge domain of GGA1 without the adjacent GAE region to bind to and polymerize clathrin. As shown in Fig. 7A, the hinge domain alone was much less effective than the full-length molecule or hinge+GAE construct in polymerizing clathrin. Even at a concentration of 10 µM hinge, only one-third of the added clathrin sedimented. Electron microscopy of this preparation showed that there were some clathrin baskets that formed in the midst of the free hinge and clathrin triskelia (data not shown). We also measured the binding of the GST-hinge domain to preformed clathrin baskets at pH 6.0. In this experiment we raised the clathrin concentration to 1 µM so that we could detect the weak binding of the hinge domain to the baskets. Although 50% of the hinge-GAE domain bound to clathrin when 1 µM hinge-GAE domain was added to 0.3 µM clathrin baskets, 4.5 µM hinge domain alone had to be added to 1.5 µM clathrin baskets to obtain 50% hinge domain binding (Fig. 7B). Therefore, the hinge domain without the adjacent GAE binds clathrin weakly, which accounts for the poor polymerizing properties of the hinge domain. These results indicate that the GAE domain contributes to the binding of the hinge domain to clathrin.


Figure 6
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  		FIGURE 6.
Interaction of the hinge+GAE domain of GGA1 with clathrin. A, polymerization of clathrin by the GST-(hinge+GAE) domain of GGA1. Varying concentrations (0–8 µM) of GST-(hinge+GAE) domain were added to 0.3 µM clathrin triskelia in a 0.5-ml volume. The solutions were dialyzed overnight against polymerization buffer and then centrifuged. The clathrin and the GST-(hinge+GAE) domain in the supernatant were used to determine the clathrin polymerized and the stoichiometry of GST-(hinge+GAE) binding per CHC. The data are plotted either as fraction clathrin polymerized (triangles) or as GST-(hinge+GAE) bound per CHC (circles) versus GST-(hinge+GAE) concentration. B, binding of GST-(hinge+GAE) domain to pure clathrin baskets. Varying concentrations of GST-(hinge+GAE) construct (0–10 µM) were added to 0.3 µM clathrin baskets. The concentration of GST-(hinge+GAE) in the supernatant was determined after centrifugation. C, stoichiometry of GST-(hinge+GAE) to CHC in resuspended pellets. In the assembly experiment, clathrin (0.35 µM) and GST-hinge+GAE domain (8 µM) were dialyzed overnight and centrifuged, and the pellet after sedimentation was resuspended. In the binding experiment, GST-(hinge+GAE) domain (10 µM) was incubated with pure clathrin baskets (0.35 µM) at pH 6.0 for 30 min at 25 °C prior to centrifugation. D, the same as in C, except that for the polymerization and the binding experiments we used 0.3 µM clathrin baskets and 5 µM hinge+GAE domain, which was digested of its GST using PreScission Protease. In C and D, lane 1 is the sample before spin and lane 2 is the resuspended pellet.

 
We also examined the ability of the GST-GAE domain of GGA1 to assemble clathrin and to bind to perform clathrin baskets. As shown in Fig. 7C, there was neither detectable polymerization of clathrin by 10 µM GST-GAE. Likewise, there was no binding of GST-GAE to 1.0 µM pure clathrin baskets (Fig. 7D). Therefore, although the GAE domain alone did not bind to clathrin, the presence of this domain significantly strengthened the binding of the hinge domain, which contained the clathrin binding site. The last construct we examined was the GST-VHS-GAT domain of GGA1, which has no known clathrin-binding motif. As expected, there was no significant binding of this construct to clathrin baskets (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study has shown that, although the clathrin adaptor GGA1 acts as a clathrin assembly protein, it differs in its properties from other common adaptor proteins such as the multimeric adaptors, AP1 and AP2, and the monomeric adaptor, AP180. Based on the biochemical properties of GGA1 that we observed, several apparently contradictory findings can now be explained. First, although GGA1 is localized to the TGN of cells, it is not found in isolated rat liver CCVs (5, 18). Moreover, it readily dissociates from the Golgi as soon as the cell is permeabilized (18). In this study, we found that GGA1 did not polymerize clathrin stoichiometrically, but rather it bound relatively weakly with the addition of 1 µM GGA1 polymerizing only 50% of the 0.3 µM clathrin present. This indicated that the dissociation constant of GGA1 is ~0.5 µM, a relatively weak dissociation constant that could explain why GGA1 readily dissociates from clathrin when the cell is permeabilized. The affinity of GGA1 for clathrin was not affected by the presence of other clathrin adaptors or the presence of lipid in the CCVs.

Our biochemical studies may also explain other in vivo observations made on GGA1. Puertollano et al. (15) observed that excess GGA1 recruits 3-fold more clathrin to the Golgi. In addition, in vivo studies have shown that the transport carriers containing GGA1, clathrin, and AP1 that emanate from the TGN are much larger than coated vesicles and are often tubular (13). These tubules are even observed in the absence of overexpressed GGAs (20). The ability of GGA1 to tubulate clathrin in vitro may promote the initiation of the long tubules observed emanating from the TGN in vivo. To obtain long tubules of clathrin, the clathrin in the tubes would be organized predominantly in a hexagonal array, because pentagons induce the clathrin to form closed vesicles. Using correlative light electron microscopy technique, transport carriers emanating from the Golgi ranged from the typical CCVs to larger, convoluted tubular-vesicular structures displaying several coated buds. As expected, clathrin was found associated with buds and on vesicles, whereas in addition to these structures, GGA1 was also found to decorate tubules (21).


Figure 7
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  		FIGURE 7.
The interaction of clathrin with either the GST-hinge domain of GGA1 or the GST-GAE domain of GGA1. A, polymerization of clathrin by the GST-hinge domain of GGA1. Varying concentrations of GST-hinge (0–10 µM) were added to clathrin (0.3 µM triskelia) and polymerized by overnight dialysis. Following centrifugation, the clathrin and the hinge domain in the supernatant were determined by SDS electrophoresis. B, binding of GST-hinge domain to pure clathrin baskets. Pure clathrin baskets (1 µM) were mixed with either 1.5 µM or 4.5 µM GST-hinge for 30 min at 25 °C. The total clathrin baskets (lane 1), 1.5 µM GST-hinge alone (lane 2), and 4.5 µM GST-hinge alone (lane 4) were used. The supernatant obtained from the mixture of GGA1 and clathrin baskets is shown in lane 3 using 1.5 µM GST-hinge, and lane 5 using 4.5 µM GST-hinge. C, polymerization of clathrin by GST-GAE domain of GGA1. Clathrin (0.3 µM triskelia), either in the absence of GGA1 (lanes 1 and 2) or presence of 10 µM GST-GAE (lanes 3 and 4), was dialyzed overnight against polymerization buffer. The SDS gel represents the samples before spin (lanes 1 and 3) and supernatant after centrifugation (lanes 2 and 4). D, pure clathrin baskets (1 µM) were mixed with either 1.5 µM or 4.5 µM GST-GAE for 30 min at 25 °C. Lane 1, the total clathrin baskets; lane 2, 1.5 µM GST-GAE alone; and lane 4, 4.5 µM GST-GAE alone. The supernatant obtained from the mixture of GST-GAE and clathrin baskets is shown in lane 3 using 1.5 µM GST-GAE and lane 5 using 4.5 µM GST-GAE.

 
Interestingly, overexpression of other monomeric clathrin adaptors produces a very different phenotype. When AP180 or its homolog, CALM (clathrin assembly lymphoid myeloid leukemia protein), is overexpressed in the cell, there is a loss of clathrin from the TGN (22). Similarly, overexpression of auxilin or GAK, the clathrin binding Hsc70 cochaperones, results in a loss of clathrin from the TGN (23). All of these adaptors sequestered clathrin in granules in the cytosol. Interestingly, GGA2 does not show the same clathrin-binding properties as GGA1 in that, unlike GGA1, it binds with a stoichiometry of one GGA2 per clathrin heavy chain. The structure of GGA2 is known to differ from that of GGA1. First, unlike GGA1 and GGA3, the clathrin hinge of GGA2 does not bind to its own VHS domain to create a closed conformation (24). Furthermore, a recent report from the Brodsky laboratory (25) has identified two other motifs that interact with clathrin in addition to the LLDDE canonical clathrin-binding motif in the hinge region of GGA1 (6). One of these clathrin-binding sites is also located in the hinge region and binds to the N-terminal domain just like the canonical motif. Based on sequence homology, it does not appear to be in GGA2. The other binding site, which is located in the GAE domain of GGA1, binds to the clathrin ankle and not the clathrin N-terminal domain (25). It is unclear whether this site exists in the GAE domain of GGA2, because the interface between clathrin and the GAE domain is not a linear motif (25).

Unlike the full-length molecule, the hinge+GAE construct of GGA1 did not induce the formation of long tubules. Moreover, it bound with a stoichiometry of one hinge+GAE domain per CHC. This suggests that there is another region in GGA1 not present in the full-length GGA2 that must account for the unusual properties of the full-length molecule. GGA1 can fold back on itself and bind to its own VHS domain (24); it thus creates a closed conformation that can be reopened by dephosphorylation of a serine adjacent to the clathrin binding site in the hinge region. Because the hinge region is known to interact with the VHS domain in GGA1, the high stoichiometry of GGA1 binding to clathrin may be due to a domain-swapping mechanism in which the hinge domain of one molecule binds to the VHS domain of an adjacent molecule (26), thereby causing formation of an interlocking GGA1 oligomer. This oligomerization would only occur after a GGA1 monomer binds to a clathrin basket provided that the binding produces the open conformation necessary for the binding of additional GGA1 molecules. This could explain why GGA1 does not form oligomeric structures in the absence of clathrin baskets even when monomeric clathrin is present. Because the results of this study show that GGA1, unlike GGA2, binds with a high stoichiometry of clathrin and forms tubules, this suggests that the effects of GGA1 are specific and therefore are likely to be physiological. In the cell, it has been suggested that dephosphorylation produces the open form of GGA1 that binds to cargo (27). The results of this study show that this conformation also promotes tubule formation. Therefore, GGA1 may be having a dual function when dephosphorylated in the cell in initiating the tubules observed emanating from the TGN containing GGA1 and mannose 6-phosphate receptor (21) and binding to the mannose 6-phosphate receptor.

The hinge domain alone bound much more weakly to clathrin than the hinge+GAE construct showing that, even though the hinge domain has the clathrin binding site, the GAE domain contributes to the binding. This is in agreement with previous studies also observed that the hinge+GAE domain interacted with clathrin in the cytosol better than the hinge domain (15). These results agree with the recent observation from the Brodsky laboratory (25) that the GAE domain actually contributes to the pocket where clathrin binds.

Although we have obtained insight into the biochemical interactions of GGA1 and clathrin, the interaction of GGA1 with the many other proteins in the cell would no doubt affect its interaction with clathrin. GGAs are recruited to the plasma membrane by activated ARF1. In addition, GGAs not only bind to clathrin but also to the multimeric assembly protein, AP1 (27). The AP1 binding site of GGA1 has been localized to the WNSF sequence in the GGA1 hinge region (28). In addition, the binding of cargo and other proteins such as {gamma}-synergin and rabaptin may also alter the interaction of GGA with clathrin. Sorting out these interactions and how they are regulated will ultimately provide an understanding of how GGAs interact with clathrin and the role of GGAs in the budding of clathrin-coated transport carriers from the TGN.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Laboratory of Cell Biology, NHLBI, National Institutes of Health, 50 South Dr., Rm. 2537, MSC 8017, Bethesda, MD 20892–8017. Tel.: 301-496-1228; Fax: 301-402-1519; E-mail: greenel{at}helix.nih.gov.

3 The abbreviations used are: GAE, {gamma}-adaptin ear homology domain of GGA; TGN, trans-Golgi network; AP1, assembly protein 1; CCV, clathrin-coated vesicles; CHC, clathrin heavy chain; GST, glutathione S-transferase; MES, 4-morpholineethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Juan Bonifacino and Rosa Puertollano for the expression constructs of GGA1 and its domains, Dr. Rosa Puertollano for providing critical comments of this manuscript, Dr. Xiaohong Zhao for technical assistance, and Myoung-Soon Cho for the electron microscopy.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Robinson, M. S., and Bonifacino, J. S. (2001) Curr. Opin. Cell Biol. 13, 444–453[CrossRef][Medline] [Order article via Infotrieve]
  2. Bonifacino, J. S. (2004) Nat. Rev. Mol. Cell. Biol. 5, 23–32[CrossRef][Medline] [Order article via Infotrieve]
  3. Boman, A. L. (2001) J. Cell Sci. 114, 3413–3418[Abstract/Free Full Text]
  4. Nakayama, K., and Wakatsuki, S. (2003) Cell Struct. Funct. 28, 431–442[CrossRef][Medline] [Order article via Infotrieve]
  5. Hirst, J., Lui, W. W., Bright, N. A., Totty, N., Seaman, M. N., and Robinson, M. S. (2000) J. Cell Biol. 149, 67–80[Abstract/Free Full Text]
  6. Dell'Angelica, E. C., Puertollano, R., Mullins, C., Aguilar, R. C., Vargas, J. D., Hartnell, L. M., and Bonifacino, J. S. (2000) J. Cell Biol. 149, 81–94[Abstract/Free Full Text]
  7. Puertollano, R., van der Wel, N. N., Greene, L. E., Eisenberg, E., Peters, P. J., and Bonifacino, J. S. (2003) Mol. Biol. Cell 14, 1545–1557[Abstract/Free Full Text]
  8. Puertollano, R., Aguilar, R. C., Gorshkova, I., Crouch, R. J., and Bonifacino, J. S. (2001) Science 292, 1712–1716[Abstract/Free Full Text]
  9. Nielsen, M. S., Madsen, P., Christensen, E. I., Nykjaer, A., Gliemann, J., Kasper, D., Pohlmann, R., and Petersen, C. M. (2001) EMBO J. 20, 2180–2190[CrossRef][Medline] [Order article via Infotrieve]
  10. Takatsu, H., Katoh, Y., Shiba, Y., and Nakayama, K. (2001) J. Biol. Chem. 276, 28541–28545[Abstract/Free Full Text]
  11. Zhu, Y., Drake, M. T., and Kornfeld, S. (2001) Methods Enzymol. 329, 379–387[Medline] [Order article via Infotrieve]
  12. Costaguta, G., Stefan, C. J., Bensen, E. S., Emr, S. D., and Payne, G. S. (2001) Mol. Biol. Cell 12, 1885–1896[Abstract/Free Full Text]
  13. Puertollano, R., and Bonifacino, J. S. (2004) Nat. Cell Biol. 6, 244–251[Medline] [Order article via Infotrieve]
  14. Scott, P. M., Bilodeau, P. S., Zhdankina, O., Winistorfer, S. C., Hauglund, M. J., Allaman, M. M., Kearney, W. R., Robertson, A. D., Boman, A. L., and Piper, R. C. (2004) Nat. Cell Biol. 6, 252–259[Medline] [Order article via Infotrieve]
  15. Puertollano, R., Randazzo, P. A., Presley, J. F., Hartnell, L. M., and Bonifacino, J. S. (2001) Cell 105, 93–102[CrossRef][Medline] [Order article via Infotrieve]
  16. Ma, Y., Greener, T., Pacold, M. E., Kaushal, S., Greene, L. E., and Eisenberg, E. (2002) J. Biol. Chem. 277, 49267–49274[Abstract/Free Full Text]
  17. Goodman, O. B., Jr., Krupnick, J. G., Gurevich, V. V., Benovic, J. L., and Keen, J. H. (1997) J. Biol. Chem. 272, 15017–15022[Abstract/Free Full Text]
  18. Hirst, J., Lindsay, M. R., and Robinson, M. S. (2001) Mol. Biol. Cell 12, 3573–3588[Abstract/Free Full Text]
  19. Greene, L. E., and Eisenberg, E. (1990) J. Biol. Chem. 265, 6682–6687[Abstract/Free Full Text]
  20. Waguri, S., Dewitte, F., Le, B. R., Rouille, Y., Uchiyama, Y., Dubremetz, J. F., and Hoflack, B. (2003) Mol. Biol. Cell 14, 142–155[Abstract/Free Full Text]
  21. Polishchuk, R. S., San, P. E., Di, P. A., Tete, S., and Bonifacino, J. S. (2006) Traffic. 7, 1092–1103[CrossRef][Medline] [Order article via Infotrieve]
  22. Tebar, F., Bohlander, S. K., and Sorkin, A. (1999) Mol. Biol. Cell 10, 2687–2702[Abstract/Free Full Text]
  23. Zhao, X., Greener, T., Al-Hasani, H., Cushman, S. W., Eisenberg, E., and Greene, L. E. (2001) J. Cell Sci. 114, 353–365[Abstract]
  24. Doray, B., Bruns, K., Ghosh, P., and Kornfeld, S. A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 8072–8077[Abstract/Free Full Text]
  25. Knuehl, C. Y., Chen, C. Y., Manalo, V., Hwang, P. K., Ota, and Brodsky, F. M. (2006) Traffic 7, 1688–1700[CrossRef][Medline] [Order article via Infotrieve]
  26. Bennett, M. J., Schlunegger, M. P., and Eisenberg, D. (1995) Protein Sci. 4, 2455–2468[Medline] [Order article via Infotrieve]
  27. Doray, B., Ghosh, P., Griffith, J., Geuze, H. J., and Kornfeld, S. (2002) Science 297, 1700–1703[Abstract/Free Full Text]
  28. Bai, H., Doray, B., and Kornfeld, S. (2004) J. Biol. Chem. 279, 17411–17417[Abstract/Free Full Text]

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