The GAT Domains of Clathrin-associated GGA Proteins Have TwoUbiquitin Binding Motifs*

Received for publication, June 15, 2004, and in revised form, October 18, 2004Published, JBC Papers in Press, October 19, 2004, DOI 10.1074/jbc.M406654200

Patricia S. Bilodeau‡, Stanley C. Winistorfer‡, Margaret M. Allaman§¶, Kavitha Surendhran‡,William R. Kearney§¶, Andrew D. Robertson�, and Robert C. Piper‡**

From the ‡Departments of Physiology and Biophysics and �Biochemistry, University of Iowa and the §University ofIowa College of Medicine NMR Facility, Iowa City, Iowa 52242

Ubiquitin (Ub) attachment to membrane proteins canserve as a sorting signal for lysosomal delivery. Recog-nition of Ub as a sorting signal can occur at the trans-Golgi network and is mediated in part by the clathrin-associated Golgi-localizing, �-adaptin ear domainhomology, ARF-binding proteins (GGA). GGA proteinsbind Ub via a three-helix bundle subdomain in theirGAT (GGA and target of Myb1 protein) domain, which isalso present in the Ub binding domain of target of Myb1protein. Ubiquitin binding by yeast Ggas is required todirect sorting of ubiquitinated proteins such as generalamino acid permease (Gap1) from the trans-Golgi net-work to endosomes. Using affinity chromatography andnuclear magnetic resonance spectroscopy, we havefound that the human GGA3 GAT domain contains twoUb binding motifs that bind to the same surface of ubiq-uitin. These motifs are found within different heliceswithin the three-helix GAT subdomain. When function-ally analyzed in yeast, each motif was sufficient tomediate trans-Golgi network to endosomal sorting ofGap1, and mutation of both motifs resulted in defectiveGap1 sorting without defects in other GGA-dependentprocesses.

Ubiquitin (Ub)1 can serve as a specific lysosomal sortingmotif when covalently attached to membrane proteins (1).Ubiquitinated membrane proteins that have passed qualitycontrol at the endoplasmic reticulum are delivered to internalmembranes of multivesiculated bodies where they can undergocomplete degradation by lysosomal proteases. Ubiquitinationoccurs at many different compartments within the secretory/endocytic pathway, including the cell surface and trans-GolgiNetwork (TGN) and is likely to occur at endosomes as well (1).Ubiquitin may also act as a specific sorting signal at various

intracellular locales, each of which contributes to the finaldelivery of cargo to the lysosome. Ubiquitin can serve as aninternalization signal for proteins such as Ste2 and Fc�RIIreceptor (2, 3). Recognition of Ub as an internalization signal islikely mediated in part by epidermal growth factor receptorpathway substrate 15 (Eps15) and Ent1 and Ent2. In yeast,mutation of the Ub binding domains of these proteins slows therate of Ste2 internalization (4, 5). Ubiquitin also serves as aspecific signal at the endosome for incorporating proteins intothe lumenal membranes of multivesicular bodies. Ubiquitin isrecognized at the endosome by the Vps27-Hse1 (Hrs-STAM inmammalian cells) complex, the ESCRT-I complex, and possiblythe ESCRT II complex (7–9). Ubiquitin can also facilitate thesorting of proteins from the Golgi directly to endosomes, thusforcing ubiquitinated proteins to bypass the cell surface (1).The general amino acid permease, Gap1, and the uracil per-mease, Fur4, are well studied examples of proteins that un-dergo this type of sorting (10, 11). When their substrate islimited, these transporters are delivered to the cell surfacewhere they are relatively stable. However, when substrate isavailable, these cell surface transporters are down-regulatedand delivered to the lysosome/vacuole, and newly synthesizedtransporters are routed directly to endosomes where they arethen sorted into the degradative multivesicular body pathway.

Recently, we have shown that the Gga proteins (Golgi-local-izing, �-adaptin ear domain homology, ARF-binding protein)contribute to the sorting of ubiquitinated proteins from theTGN to the endosome (12). Gga proteins are modular clathrin-associated proteins containing an N-terminal VHS (Vps27,Hrs, STAM) domain, an Arf-binding GAT (GGA and Tom1)domain, a clathrin binding hinge region followed by a domainresembling the C-terminal “ear” of �-adaptin (13). The humangenome encodes three GGA genes, GGA1, GGA2, and GGA3,with the latter found in both a short and long form because ofalternative splicing (13). In mammalian cells, GGA1 and GGA2proteins directly bind to dileucine motifs within the cytosolictails of proteins such as the cation-independent mannose-6-phosphate receptor and facilitate their incorporation into AP-1-coated vesicles targeted for transport to endosomes. Theshort splice variant of GGA3, which is expressed at high levels(14), and the yeast Gga proteins lack a portion of the VHSdomain required for interaction with dileucine motif-contain-ing receptor tails (12), implying that these proteins may serveother functions. Saccharomyces cerevisiae has two GGA genes,GGA1 and GGA2, which are expressed at slightly differentlevels but are otherwise functionally interchangeable. Deletionof both GGA genes delays endocytic delivery of Gap1 to thevacuole and causes a Vacuolar Protein Sorting (Vps) phenotypecharacterized by secretion of carboxypeptidase Y (12, 15).

Gga proteins also bind Ub via a subregion of their GAT

* This work was supported in part by National Institutes of HealthGrants GM58202 (to R. C. P.) and GM46869 (to A. D. R.). The costs ofpublication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¶ Supported by the University of Iowa College of Medicine.** To whom correspondence should be addressed. Tel.: 319-335-7842;

Fax: 319-335-7330; E-mail: robert-piper@uiowa.edu.1 The abbreviations and trivial terms used are: Ub, ubiquitin; ADCB,

L-azetidine-2-carboxylic acid; ARF, ADP-ribosylation factor; CUE, cou-pling of ubiquitin to endoplasmic reticulum degradation; E3, ubiquitin-protein isopeptide ligase; Gap, general amino acid permease; GAT,GGA and Tom1; GFP, green fluorescent protein; GGA, Golgi-localizing,�-adaptin ear domain homology, ARF-binding protein; GST, glutathi-one S-transferase; Hrs, hepatocyte growth factor-regulated tyrosinekinase substrate; HSQC, heteronuclear single quantum correlation;TGN, trans-Golgi Network; Tom1, target of Myb 1; TSI, Total ShiftIndex; UIM, ubiquitin-interacting motif; VPS, vacuolar protein sorting;VHS, Vps27p/Hrs/Stam.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 52, Issue of December 24, pp. 54808–54816, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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domains, which is encompassed by a C-terminal three-helixbundle that is distinct from the N-terminal region required forArf�GTP binding (12, 16). This three-helix bundle region, butnot the Arf�GTP binding region, is also present in the Tom1protein where it contributes to Ub binding (17, 18). Yeastbearing GGA2 carrying a deletion of this three-helix bundle astheir sole copy of GGA are specifically defective in divertingGap1 toward endosomes under nitrogen replete conditions,whereas other GGA-dependent functions remain unaltered(12). Gga proteins are also required to divert ubiquitinatedmutant forms of the Pma1 ATPase from the TGN to endosomes(19). These data support a model whereby Gga proteins bindubiquitinated cargo at the TGN and usher it into transportvesicles targeted to endosomes, thus subverting the delivery ofubiquitinated cargo to the plasma membrane. Of the humanGGA proteins, hGGA3 binds most strongly to Ub (12, 20).Interestingly, a point mutation in the three-helix bundle of thehGGA3 GAT domain (L276A) that compromises Ub bindingalso perturbs the transport of ubiquitinated epidermal-derivedgrowth factor receptor from the cell surface to late endosomes,possibly indicating another role for the recognition of Ub byGGAs (20).

Previous mutagenesis studies have mapped the putative sitewithin the human GGA3 GAT domain that interacts with Ub.This region corresponds to a small area within the third helixcentering around Leu-276 and Leu-280 in the long form ofhGGA3 (17, 21). However, our previous studies with the yeasttwo-hybrid assay demonstrated that deletion of this region inyeast Gga1 did not completely block Ub binding (12). Further-more, failure to find point mutations that abolished Ub bindingby random mutagenesis coupled with a “reverse two-hybrid”screen indicated that other regions of the GAT domain mightcontribute to Ub binding. Using hGGA3 as a model, we haveshown that GAT domains can have two distinct Ub bindingmotifs. In the context of hGGA3 GAT, each is sufficient for Ubbinding and each binds to a similar surface on Ub. Usingchimeras of yeast Gga2 containing wild-type and mutant hu-man GGA3 GAT domains, we have shown that each motif cancontribute to Ub-dependent Gap1 trafficking.

MATERIALS AND METHODS

Plasmids and Strains—The YAB538 MAT � gga1�::TRP1gga2�::HIS3 leu2 ura3 strain derived from BY4735 and BY4704 aspreviously described (22). Yeast with stably integrated GGA2-HA orgga2-�GAT-HA as their sole copy of GGA (PLY3084 and PLY3085) weremade as previously described (12). The apl2�::Kanr gga1�::TRP1gga2�::HIS3 leu2 ura3 strain was made by replacing the APL2 openreading frame with a KanMX cassette in YAB538 cells carrying theURA3-GGA2 plasmid pAB491. To make gga� end3� cells, PLY3085cells were selected for Ade� Leu- to remove the gga2-�GAT-HA allele.These cells grew more slowly than their parental strain and weretemperature-sensitive for growth at 37 °C.

C-terminal HA epitope-tagged yeast Gga2p were expressed undertheir endogenous promoter using the previously described plasmids(pAB491) or versions converted to a LEU2-based plasmid (22). Thegga2-�Arf allele containing mutations in the Arf�GTP binding regionwas previously described (12, 22). Mutations and fragments of humanGGA3 were based on the long isoform (GenBankTM NP 619525). Plas-mids expressing GAT domains were made by cloning PCR amplifiedregions corresponding to hGGA3 residues 166–318 (133–285 of theshort hGGA3 isoform) into the pET151D-TOPO vector (Invitrogen) toproduce pPL2277 as described (12). Mutant hGGA3 GAT domains weremade by PCR recombination and also cloned into the pET151D-TOPOvector (Invitrogen). GST�ARFGTP and GST�ARFGDP plasmids were madeby subcloning the open reading frame of human Arf containing a Gln-70or Asn-31 mutation into pGEX-3X.

Plasmids encoding GGA2 chimeric proteins containing the GAT do-mains of hGGA3 were made using gap repair recombination of pPL2117cut with StuI and PstI cotransformed with hGGA3 GAT domains PC-R-amplified with the following oligos: GGTAAACCTGAAGATTTGAG-GGAAGCTAACAAATTAATGAAAATCATGGTGAAGGAAGACGA-

GGC, AGCAGAAACATGACTTGGATGTATCTGCGAAGCAGCGTTG-GAGTCCCCTTCAATAATTGTTTTGT. pPL2117 is similar to GGA-2-HA (pAB491) except that it contains the hGGA1 GAT domain. Thisreplaces residues 226–321 in yeast Gga2 with residues 200–298 ofhGGA3 corresponding to the three-helix bundle C-terminal region ofthe GAT domain. Plasmids were rescued from yeast and verifiedby sequencing.

NMR Analysis—Recombinant Ub was expressed and purified as de-tailed previously (23, 24). The 15N-labeling medium was from SpectraStable Isotopes (Columbia, MD). 15N-Ub was mixed with either purifiedhGGA3 GAT domain or yeast GGA2 GAT domain in 40 mM NaPO4, pH7.2, containing 10% D2O at 25 °C. Resonance assignments were used asdescribed (25). GAT domains were produced from pET151D-TOPO-basedvectors in BL21 bacterial cells upon induction with isopropyl 1-thio-�-D-galactopyranoside. Proteins were purified over nickel-agarose and dia-lyzed in 40 mM NaPO4, pH 7.2. The Ub surface was generated fromProtein Data Bank accession number 1ogw. Peak assignments were madeusing the Sparky software package (T. D. Goddard and D. G. Kneller,SPARKY 3, University of California, San Francisco). Chemical shift dif-ferences were calculated using the formula (0.2 �N2 � �H2)1/2. To repre-sent the magnitude of chemical shift changes, the values derived from (0.2�N2 � �H2)1/2 for each residue were normalized to 0.3439 ppm to represent100%. This value represented the maximal chemical shift change in Ubbound to wild-type hGGA3 GAT at 64 �M. In subsequent figures, valuesgreater than 75% of this value were plotted as maximal (red), valueswithin 60–75% were plotted as orange, 50–60% yellow, 40–50% green,30–40% dark blue, 20–30% light blue. A value of 0.068 or less wasconsidered insignificant. Peaks that disappeared altogether were as-signed red. As a rough estimate for the level of binding for each GGA-GATmutant with Ub, we calculated a Total Shift Index (TSI). Although thechange in chemical shift for each residue was proportional to the level ofbinding as determined by titration experiments with wild-type hGGA3GAT, the results for the wild-type could not be used to estimate thebinding affinity of Ub for mutant GAT domains because the relativechemical shift differences for individual residues in Ub varied among thedifferent GAT mutants. In an effort to average out these variations, theTSI was calculated by summing the magnitude of chemical shift differ-ences ((0.2 �N2 � �H2)1/2) induced in 15N-Ub upon GAT binding. Residueswhose peaks disappeared were assigned a value of 200% of the maximalshift difference of wild-type GAT domain (0.3439 ppm). Summed shiftchanges were then expressed as a function of the number of residuesanalyzed. Using the peak positions of Glu-18, Ser-20, Thr-22, and Leu-56in Ub, which do not undergo chemical shift changes when bound to GAT,we estimated a S.E. for measuring peak positions within all the data of0.008 ppm or 0.75% of the normalized shift.

Glutathione-Agarose Affinity Chromatography—A GST fusion of Ub,GST�Arf, or GST alone was expressed in bacteria and purified as de-scribed (9, 12). GST affinity chromatography experiments were per-formed with bacterial lysates or purified proteins as described (9, 12).

Analysis of Gap1 and Fur4—For ADCB sensitivity assays, yeastwere first grown in S.D. ammonia, serially (3-fold) diluted, and platedonto S.D. ammonia plates containing L-azetidine-2-carboxylic acid(ADCB; Sigma) as described previously (12). To control for cell density,cell dilutions were also plated onto S.D. ammonia plates in the absenceof ADCB. Cells were photographed after 2 days of growth. For fluores-cence assays, GGA2-HA end3�, gga2-�GAT end3�, or gga� end3� cellscarrying various GGA2-GAThGGA3 chimeric genes on low copy plasmidswere transformed with a CUP1-GAP1-GFP plasmid or CUP1-FUR4-GFP plasmid made by homologous recombination from the CUP1-GAP1-GFP plasmid. Cells were grown overnight in S.D. media lackingleucine and uracil and containing 100 �M bathocupoinedisulfonic acid.Cells were pelleted and resuspended in 200 �M CuSO4 in either YPD(for Gap1�GFP analysis) or S.D. media containing 20 �g/ml uracil (forFur4�GFP analysis). Cells were visualized after resuspension in 100 mM

Tris, pH 8.0, 0.2% NaN3, and NaF.Structure Models—A putative GGA3-GAT structure was made using

Swiss-Model (www.expasy.org) using the crystal structure of humanGGA1 GAT domain (26). Models were rendered with VMD software(www.ks.uiuc.edu/Research/vmd/). Manual interactive docking and en-ergy minimization was performed with the SYBYL®/Base (Tripos Inc.)software to provide a model of the hGGA3 GAT domain in a complexwith Ub.

RESULTS

Fig. 1 shows a sequence alignment of the hGGA3 (humanGGA3) GAT domain together with the corresponding regionfrom yGga2 (yeast Gga2) and Tom1, both of which bind Ub. The

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FIG. 1. Human GGA3 has two conserved ubiquitin binding motifs. A, a predicted model of the C-terminal three-helix bundle of the humanGGA3 GAT domain. Shown in blue are two regions conserved among other ubiquitin-binding proteins with GAT domains. Also shown are residuesproposed to comprise Ub binding interfaces. Residues in Site1 are present on the N-terminal �-helix and are colored orange. Residues in Site2 arered. B, an alignment of GAT domains from human GGA3, Tom1, and yeast GGA2. The numbering corresponds to the residue position of the longform of hGGA3. The two conserved regions in blue in panel A are underscored with a blue bar. Residues predicted to be on the same external faceof an �-helix are designated with a dot. C, sequences of two putative Ub binding motifs are aligned at the top. Predicted surface residues aredesignated with a dot. Middle, the region of the GAT domain with both Ub binding motifs. Bottom, views of each �-helical region showing thealignment of Glu-219, Leu-227, and Glu-230 as well as Asp-273, Leu-280, and Asp-284 on the external face of the helix. D, bacterial lysatescontaining wild-type or mutant GAT domains were incubated with 100 �l of GSH-agarose bound with GST alone (ø) or GST�Ub (Ub). Beads were

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aligned regions correspond to the C-terminal portion of theGAT domain that forms a three-helix bundle in the case ofGGA1. Because hGGA3 is found in both a long and short splicevariant, all numbering is referenced to the long form of GGA3in contrast to our previous work where referencing was to theshort form. Also shown is a predicted model of the three-dimensional structure of this region of hGGA3 GAT (Fig. 1A)generated using the known crystal structure of hGGA1, whichalso binds Ub. Previous studies identified a region within theC-terminal portion of the three-helix bundle subdomain of thehGGA3 GAT domain. Using the residue numbering corre-sponding to the long splice variant of hGGA3, mutation ofLeu-276 to Ser or Ala, Leu-280 to Arg, or Asp-285 to Gly causedloss of Ub binding (16, 20). Similarly, mutation of the Tom1GAT domain residues L285R and D289G, which correspondpositionally to Leu-280 and Asp-285 in hGGA3, also resulted ina loss of Ub binding by Tom1 (17). These residues fall withinthe C-terminal �-helix of the GAT subdomain. Our previousyeast two-hybrid analysis showed that a VHS-GAT domain ofyeast Gga1 lacking this region (residues 1–260) was still able tointeract with Ub, albeit less strongly than a VHS-GAT frag-ment containing the entire GAT domain (12). This 1–260 Gga1fragment would correspond to an hGGA3 fragment containingonly residues 1–241 and thus lacking the C-terminal �-helix.Therefore, we reexamined the GAT domain to more preciselydefine the regions responsible for Ub binding. Using the align-ment in Fig. 1B, we found two regions in separate helices thatwere relatively conserved among all the GAT domains.Throughout the course of this work we designated the N-ter-minal-most region Site1 and the other Site2. Site1 encom-passes residues 218–232 and Site2 encompasses residues 272–286 of the long form of hGGA3. The modeled tertiarystructure of the hGGA3 GAT domain helped predict whichresidues were likely to be exposed to the surface and thuspotentially involved in Ub binding. Intriguingly, we foundthat both regions could be aligned such that exposed residuesshared a high level of identity. Both helical regions showedan acidic residue (Glu) and a bulky hydrophobic residue (Leu)followed by another acidic residue (Glu or Asp) on the sameside of the helix (Fig. 1C). The latter two residues in Site2correspond to Leu-280 and Asp-284, which were found to beimportant for Ub binding in other studies. Also, the last fourresidues of both putative motifs end with Ser-Acidic-X-Leu.Because of the similarity of these regions and our previoustwo-hybrid results, we hypothesized that each formed a motifcapable of binding Ub.

As a first test of this hypothesis we mutated conserved res-idues in Site1 or Site2 of the hGGA3 GAT domain. Site1 wasaltered with four alanine substitutions from 226RLLSE to226AALAA and Site2 was altered with four alanine substitu-tions from 279ILQASD to 279AAQAAA. The mutant or wild-typehGGA3 GAT domains (residues 166–318, which include theN-terminal Arf binding region and the three-helix C-terminalregion) were expressed in Escherichia coli behind a hexohis-tidine and V5 epitope tag and measured for binding toGST�Ub fusion and GST alone (Fig. 1D). As found previously,the wild-type hGGA3 GAT domain bound well to GST�Ub.The GAT domain with mutations in Site1 showed much lessUb binding (�10 times less than wild-type). Mutations inSite2 also diminished binding to Ub, although to a lesserextent than mutations in Site1. A mutant GAT domain car-

rying four alanine substitutions in both Site1 and Site2showed no binding to Ub. These data showed that each Sitewas sufficient for Ub binding and that these regions togetheraccounted for the Ub binding activity of the hGGA3 GATdomain. We next tested whether the residues predicted to beon the external surface of the helices were responsible for Ubbinding. We mutated Leu-227 and Glu-230 in Site1 in thecontext of a mutant GAT containing the four alanine substi-tution mutations in Site2 that we believed completely inac-tivate Site2 binding. Fig. 1D shows that this mutant alsoshowed no binding of GST�Ub, indicating the importance ofthese predicted surface residues in Site1.

From these results we then refined our mutational analysisto evaluate the contribution of the surface-exposed Leu-227and Glu-230 of Site1 and the Leu-280 and Asp-284 of Site2. InGST�Ub binding experiments, loss of Ub binding was observedwhen Site1 or Site2 was mutated by alanine substitution ofeither Leu-227 or Leu-280 singly or in combination with theadjacent acidic residue (Glu-230 or Asp-284). In agreementwith data obtained with the four alanine substitution mutants,mutations in Site1 caused a greater loss in Ub binding thanthose in Site2, although the effects were comparable overall.Combining mutations in Site1 and Site2 led to a complete lossof detectable Ub binding. No difference was observed betweendouble Site1 and Site2 mutants containing alanine replace-ments of the leucine residues alone (L227A and L280A) or incombination with the acidic residues (E230A and D284A). Pre-vious analysis has shown an L276A or L276S mutation in Site2can also cause loss of Ub binding (16, 20). However, we did nottest these corresponding residues because a hydrophobic resi-due is not present at this position in Site1 nor is this residueconserved between yGGA2 and Tom1 in Site2. Together, thesedata show that the surface-exposed residues of both Site1 andSite2 together are required for Ub binding.

To confirm these results we used NMR spectroscopy tomonitor chemical shift differences in Ub upon binding todifferent mutant hGGA3 GAT domains. This was done notonly to avoid any potential artifacts from the GST�Ub bindingexperiments but also to compare the binding surface of Ubused by Site1 and Site2. In principle, these experiments coulddistinguish whether Site1 and Site2 bound similarly to Ub,supporting the idea that they function as autonomous redun-dant sites, or whether each provides unique contacts with Ubto form a bipartite binding interface in the complete hGGA3GAT domain. Purified wild-type and mutant hGGA3 GATdomains were mixed with 15N-labeled Ub (110 �M) and ana-lyzed by 15N HSQC. Fig. 2A shows a titration experiment inwhich we followed the chemical shift differences Ub had uponbinding increasing amounts of hGGA3 GAT domain. As ex-pected, increasing amounts of hGGA3 increased the magnitudeof chemical shift changes in Ub. Several residues showed a grad-ual increase in the titration scheme, and these changes werefitted to a curve from which we were able to estimate a Kd of10–50 �M. As reported previously, the largest chemical shiftchanges were observed in three regions of Ub: residues 7–13,45–48, and 68–72. These regions describe a surface of Ub thatincludes residues Leu-8, Thr-12, Lys-48, His-68, Val-70, and Arg-72. Several surface residues did not show large changes in chem-ical shift, including Ile-44 and Arg-42, which are involved inbinding UIM domains, and Gln-62 and Glu-64, which are in-volved in binding TSG101 and Vps23.

washed and immunoblotted together with the indicated fraction of the starting lysate with anti-V5 antibodies directed to an N-terminalhexohistidine/V5 epitope tag. GAT domains with mutations in Site1 and/or Site2 are underlined. E, wild-type and mutant GAT domains werepurified over nickel-agarose and incubated (10 �g) with 100 �l of GSH-agarose bound with GST alone (ø) or GST�Ub (Ub). Beads were washed andimmunoblotted together with the indicated fraction of the starting lysate with anti-V5 antibodies. GAT domains with mutations in Site1 and/orSite2 are underlined.

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We next looked at the binding of various mutant GAT do-mains in which a single alanine was substituted for the sur-face-exposed leucine either separately (Leu-227 or Leu-280) orin combination with the adjacent surface acidic residue (Glu-

230 or Asp-284) (Fig. 2). The mutant GAT domains were ana-lyzed at a concentration of 64 �M, at which the wild-type GATinduces large chemical shift changes in Ub. We then colori-metrically plotted the magnitude of chemical shift differences

FIG. 2. Both GAT ubiquitin binding motifs bind the same surface. A, the HSQC spectra of 15N-labeled Ub (110 �M) were measured in thepresence of increasing quantities of wild-type GAT domain (shown on right) as well as in the presence of mutant GAT domains. The chemical shiftdifferences induced by GAT binding were quantified using (0.2 �N2 � �H2)1/2, and values were normalized using the maximal shift change observedwith wild-type GAT domain at 64 �M. The magnitude of chemical shift change was then plotted colorimetrically for each residue using the colorscale indicated. Each mutant GAT domain was measured at 64 �M except for the one indicated mutant measured at 96 �M. GAT domain mutantswith a single alanine substitution of Leu-227 (Site1) and/or Leu-280 (Site2) are designated as L�A. Mutants with double mutations in Site 1(L227A,E230A) and/or Site2 (L280A,D284A) are designated L�A, E/D �A. B, using the color scale in panel A, residues undergoing chemical shiftchanges were plotted onto the surface of Ub. Also pictured (upper right) is the secondary structure of Ub overlaid with the surface. The structureof corresponding GAT domains that induced chemical shift changes on each Ub surface are modeled in the inset. Mutant GAT domains containingsingle or double alanine substitutions in either Site1 or Site2 follow the same nomenclature and are designated L�A or L�A, E/D�A, respectively.

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along the length of Ub and represented those changes on thethree-dimensional surface of Ub. Consistent with the GST�Ubbinding experiments, we found that GAT domains containingeither single or double alanine substitution mutations in eitherSite1 or Site2 alone were still able to bind Ub. The trend wasthat GAT domains with an intact Site1 bound to Ub better thanGAT domains with only Site2 intact. This is most evident incomparing the Site1 L227A mutant to the Site2 L280A mutant(Fig. 2). Mutation of both leucine residues in Site1 and Site2resulted in very low binding. Only minor chemical shiftchanges were observed with this mutant, consistent with theGST�Ub binding experiments in Fig. 1. When both leucines(Leu-227 and Leu-280) and the acidic residues (Glu-230 andAsp-284) were mutated in Site1 and Site2 together, no signif-icant chemical shift differences were observed. These data in-dicate that the acidic residues contribute to Ub binding becausetheir loss in the context of the L227A,L280A mutant resulted incomplete loss of chemical shift differences in Ub. Given thelevel of sensitivity measured in titration of the wild-type GATdomain, the affinity of the L227A,E230A,L280A,D284A mutantis less than 5% of wild-type.

In general, each of the Ub binding sites on the GAT domaininduced similar chemical shift changes in Ub, indicating thatthey largely interact with the same surface of Ub. There weresome differences between the two Sites, particularly in regardto the ratio of chemical shift changes induced in one residueversus another. These differences most likely reflect subtledifferences in the geometry of the binding interface rather thanradically different binding configurations. Because of the dif-ferences between Site1 and Site2 binding, however, we couldnot accurately use these data to estimate Kd. This was becausethe HSQC spectral changes induced by GAT domains contain-ing either Site1 or Site2 alone did not match the chemical shiftspectra of wild-type GAT. The likely explanation is that thechemical shift changes observed with wild-type GAT domainbinding represent a mixture of both Site1 and Site2 bound toUb. Furthermore, there may be a third configuration in whichtwo Ubs could be bound to a single GAT. Using the magnitudeof chemical shift differences observed on the surface of Ub as anindicator, we estimate that mutation of Site1 caused a strongerdefect in Ub binding than mutation of Site2 (Fig. 2B). Summingthe chemical shift differences across all residues of Ub to esti-mate a TSI indicated that the ordering of strongest binding toweakest binding was as follows: WT (TSI � 0.172 ppm) �Site2L280A (TSI � 0.113 ppm) �Site2 L280A,D284A (TSI � 0.076ppm) �Site1 L227A,E230A (TSI � 0.066 ppm) �Site1 L227A(TSI � 0.060 ppm) �Site1 � 2 L280A,L227A (TSI � 0.027)�Site1 � 2 L280A,D284A,L227A,E230A (TSI � 0.007): allvalues � 0.008 ppm.

We next evaluated the function of these Ub binding motifs invivo. Previous experiments examined the role of yeast Ggaproteins in the Ub-dependent trafficking of Gap1. When nitro-gen is limiting, Gap1 is transported to the cell surface where itcan facilitate the transport of a variety of nitrogen sources intothe cell. However, when nitrogen is readily available, newlysynthesized Gap1 is transported from the Golgi to endosomes,thus bypassing the cell surface (11, 27, 28). This transport steprequires ubiquitination of Gap1. Consequently, in relativelyrich nitrogen conditions, wild-type cells are resistant to thetoxic proline analog ADCB, a substrate of Gap1. In contrast,gga� cells are highly sensitive to ADCB not only because Gap1is transported to the cell surface but also because its rapidendocytosis and delivery to the vacuole is compromised (12). Asa result, there are high levels of Gap1 at the surface in gga�cells. In cells carrying a mutant Gga2 protein lacking the entirethree-helix bundle portion of the GAT domain (gga2-�GAT),

Gap1 is endocytosed to the vacuole normally but newly synthe-sized Gap1 is delivered to the cell surface, rendering the cellsmoderately sensitive to ADCB. These data indicated that theUb binding activity of Gga was specifically responsible forsorting ubiquitinated Gap1 from the TGN to endosomes. Thestructural data we obtained in the present study allowed us tomore precisely correlate loss of Ub binding with loss of Ub-de-pendent Gap1 sorting to endosomes. This would serve as amore rigorous evaluation of the Ub binding function of Ggaproteins as opposed to other functions that could be ascribed tothe conserved GAT domain. Therefore, we assessed the func-tion of chimeric yGga2 proteins, which contained either wild-type or mutant hGGA3 GAT domains (Fig. 3A). Using growthon ADCB as an index of Gap1 sorting, we found that mutationof both Site1 and Site2 was required to confer sensitivity toADCB to the level observed for the gga2-�GAT allele. A veryslight growth defect was seen when Site1 was mutated, but nodefect was observed when Site2 alone was altered. This trendwas observed regardless of whether each Ub binding site wasaltered by alanine substitution of one, two, or four residues. Toconfirm the effect of these chimeric yGga2-hGGA3 proteins onGap1 sorting, we assessed distribution of Gap1�GFP in end3�cells that expressed chimeric Gga2 proteins with either wild-type or mutant hGGA3 GAT domains as performed previously(12). Gap1�GFP was expressed under the copper inducible con-trol of the CUP1 promoter in cells grown in rich nitrogenconditions where Gap1 is normally sorted directly from theGolgi to the vacuole, thus bypassing the cell surface. As shownin Fig. 3C, wild-type Gga2-GAThGGA3 supported this directsorting step because newly synthesized Gap1�GFP did not ac-cumulate at the plasma membrane but rather the vacuole. Incontrast, Gap1�GFP was easily detected at the cell surfaceof end3� cells when the hGGA3 GAT domain of the Gga2-GAThGGA3 chimera carried the L227A,E230A,L280A,D284Amutations (L�A, E/D �A) or the more extensive 226RLLSE to226AALAA and 279ILQASD to 279AAQAAA mutations (4�A).Similar effects were found on the sorting of Fur4 uracil per-mease (Fig. 3C), which also undergoes direct Ub-dependentsorting from the Golgi to the vacuole when cells are grown inexcess uracil (10). In end3� cells with wild-type GGA2 (data notshown) or GGA2-hGGA3GAT alleles grown in the presence ofuracil, Fur4 was efficiently delivered to the vacuole. However,when the ubiquitin binding sites were altered in the Gga2-hGGA3GAT protein, Fur4�GFP was found at the cell surface.Similar results were found using end3� cells carrying the gga2-�GAT allele missing the entire three-helix bundle. In additionto cell surface Fur4�GFP, Fur4�GFP was also found in thevacuole, implying that there are other factors besides the Ggathat may contribute to the sorting of ubiquitinated Fur4 simi-lar to what we observed previously for Gap1 (12). Thesephenotypes were not because of altered levels of expression ofthe different chimeras as all were expressed at the same levelby immunoblot analysis (data not shown). Furthermore, muta-tions in the Ub binding regions of the hGGA3 GAT domain didnot affect its ability to bind Arf-GTP (Fig. 3D). We also foundthat the GGA2-hGGA3GAT allele as well as the mutantchimeras supported other GGA functions. Previously, we foundthat the gga2-�GAT allele as the sole GGA resulted in normalendocytosis and sorting of vacuolar proteases. Other experi-ments have shown that deletion of GGA1 and GGA2 from yeastis lethal when cells also lack the AP-1 heterotetrameric adaptorcomplex, encoded in part by APL2 (29). These data imply thatunder particular circumstances, some Gga functions could befulfilled by AP-1. Therefore, we also examined the function ofthe GGA2-hGGA3GAT chimeras in the absence of AP-1 to blockthis compensatory pathway. Each of the GGA2-hGGA3GAT chi-

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meras was introduced on a LEU2-based plasmid intogga1�,gga2�,apl2� triple mutant cells carrying a URA3-basedplasmid containing GGA2. Transformants were grown for sev-eral generations and then plated onto 5�-fluoroorotic acid todetermine whether loss of URA3-based GGA2 plasmid could betolerated (Fig. 3B). As expected, no cells could be recovered thatlacked GGA and APL2. However, apl2� cells containing GGA2-hGGA3GAT chimeras with or without a GAT domain capable ofbinding Ub were viable and showed no growth defect even at

higher temperatures. Furthermore, no vacuolar protein sortingdefect as measured by secretion of carboxypeptidase Y wasobserved in these strains (data not shown). Interestingly, apl2�cells carrying a gga2-�ARF allele incapable of binding Arf�GTPwere also viable and showed no carboxypeptidase Y sorting orgrowth defects (data not shown). These data indicate that mu-tations that inactivate the Ub binding regions of Gga are veryspecific for the trafficking of Gap1 but do not affect otherGGA-dependent functions.

FIG. 3. Elimination of both ubiquitin binding motifs results in ADCB-sensitive growth. A, cells containing the designated yeast GGA2alleles as their sole GGA gene were serially diluted and plated onto synthetic media containing ammonia as the nitrogen source and 75 �M ADCB.Chimeras using an HA epitope-tagged GGA2 were made in which the C-terminal three-helix bundle region of GGA2 (residues 226–322) wasreplaced with the corresponding region of either wild-type hGGA3 (residues 201–299) or mutant hGGA3 to make a series of GGA2-hGGAGAT

alleles. A schematic of these alleles is shown in the inset. The GGA2 and gga2-�GAT alleles were integrated into the genome; other alleles werecarried on low copy plasmids. GAT domain mutants with a single alanine substitution of Leu-227 (Site1) and/or Leu-280 (Site2) are designated asL�A. Mutants with double mutations in Site 1 (L227A,E230A) and/or Site2 (L280A,D284A) are designated as L�A, E/D �A. GAT domain mutantscarrying four alanine substitutions in Site1 and/or Site2 are designated as 4�A. B, triple mutant cells (gga1�,gga2�,apl2�) carrying a URA3-basedplasmid with wild-type GGA2 were transformed with a low copy LEU2-based plasmid carrying wild-type GGA2, APL2, or the indicated gga2 orGGA2-hGGA3GAT alleles. Cells were then plated onto 5�-fluoroorotic acid to select for cells that could tolerate loss of the URA3-based GGA2. C, cells(gga� end3�) carrying the gga2-�GAT allele or low copy plasmids expressing GGA2-hGGA3GAT without or with the indicated mutations in bothUb binding Sites were transformed with plasmids expressing Gap1�GFP or Fur4�GFP under the copper-inducible control of the CUP1 promoter.Cells were grown overnight in the absence of copper, pelleted, and resuspended in the presence of 200 �M copper. For Gap1�GFP sorting, cells weregrown in YPD for 4 h prior to viewing. For Fur4�GFP sorting, cells were grown in 50 �g/ml excess uracil for 7 h prior to viewing. D, wild-type hGGA3GAT domain and the Sites1,2 mutant GAT domain (L227A,E230A,L280A,D284A) were incubated with GST, GST�ARFGTP, or GST�ARFGDP. Beadswere washed and immunoblotted with anti-V5 antibodies recognizing an N-terminal V5 epitope on each GAT domain.

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DISCUSSION

Using both GST�Ub binding experiments and NMR spectros-copy with recombinant proteins, we found that the three-helixbundle in the C-terminal portion of the hGGA3 GAT domainpossesses two similar Ub binding motifs located in separate�-helices. These motifs bind a similar surface of Ub and canwork independently to mediate Ub-dependent sorting of theGap1 transporter. Sequence analysis suggests that these twomotifs are also present in yeast Gga2 and are composed of acentral surface-exposed hydrophobic residue flanked by acidicresidues on the same face of the �-helix. We focused our effortson testing the role of the leucine (Leu-227 or Leu-280) and theproximal acidic residue (Glu-230 and Asp-284). We did not testthe more N-terminal acidic residues (Glu-219 or Asp-273),which are conserved in the two motifs in hGGA3 and Tom1GAT domain. Thus, all the rules that define this Ub bindingmotif have yet to be established. Interestingly, the hGGA3 Ubbinding motifs partly resemble UIM domains. Both are �-hel-ical, and both contain an exposed hydrophobic region flanked atleast on one side with acidic residues. Also, in general terms,the same surface of Ub appears to be engaged in the binding ofUIM and GAT domains. However, there are likely to be impor-tant differences. For instance, Arg-42 and Ile-44, and Leu-8,respectively, interact with the acidic residues and the hydro-phobic middle of the UIM domain. These residues are impor-tant for binding UIMs and undergo significant HSQC spectralchanges when bound to UIMs (30–32). Ubiquitin mutated atIle-44 has been shown to abrogate GAT binding. However,there are conflicting reports on the binding ability of Ub mu-tated at Leu-8 (16, 20). Furthermore, we found only smallchemical shift changes for Ile-44 or Arg-42 in our NMR exper-iments. Clearly the best strategy for characterizing these in-terfaces is to solve either a crystal structure or solution struc-ture for each Site independently as well as the wild-type GATdomain bound to Ub. NMR experiments focused on this prob-lem are currently underway. Using the information at hand,however, we proposed a working model for how the GAT do-main binds Ub (Fig. 4). This model was constructed based onthe assumption that a central exposed leucine in both GAT Ubbinding sites would fit within the hydrophobic surface of Ubprovided by Leu-8, Ile-44, and Val-70, whereas the acidic res-idues in the GAT domain that flank the exposed leucine wouldbind to Lys-48 and Arg-72. In this model, the central leucine ofthe stronger binding Site1 is oriented more toward Leu-8 andVal-70, whereas that of Site2 is rotated more toward Ile-44.Interestingly, in further support of the model presented, sig-nificant chemical shift perturbations of Ile-44 in Ub were onlyobserved with hGGA3 GAT mutants in which only Site 2 wasintact. One possibility we considered is that the GAT domainmight be configured to bind favorably to Ub chains linked viaLys-63 because there is evidence that Lys-63-linked polyubiq-uitination may be required to mediate direct Golgi-to-vacuolesorting. In our model, a single GAT domain can bind two Ubmolecules. However, assuming that both Site1 and Site2 bindUb in similar orientations, the orientation of two Ubs bound toa single GAT would preclude a Lys-633Gly-76 linkage.

Our discovery of two relatively equivalent Ub binding motifswithin the GAT domain partly conflicts with previous studiesthat analyzed residues confined to Site2. Like us, Shiba et al.(16) showed that residues Leu-280 and Asp-284 of hGGA3 areimportant for Ub binding. Alteration of Leu-280 to Arg orAsp-284 to Gly blocked binding to Ub. However, even whenboth of these residues were changed to alanine, which in ouranalysis completely inactivates Site2 binding activity, we stillclearly observed binding of the GAT domain via Site1. Thisapparent discrepancy is most likely because of a greater sensi-

tivity for measuring binding in our studies. The L280R andD284G mutations identified by Shiba et al. were originallyidentified in a reverse yeast two-hybrid screen searching forpoint mutations that compromised Ub binding; the level ofsaturation in this screen was probably not sufficient to identifySite1. There are indications from previous studies that aremore consistent with the two-Site model proposed here. Forinstance, one interesting observation for many proteins thatcontain Ub binding modules such as CUE or UIM is that theythemselves can undergo ubiquitination (5, 33). The mechanismfor this monoubiquitination is not known, but it may be be-cause the ability to bind Ub allows these proteins to associatewith proteins undergoing ubiquitination by E3 ligases. Thus,Ub-binding proteins become ubiquitinated themselves as by-standers owing to their avidity for Ub. Regardless of the exactmechanism, the correlation between Ub binding and ubiquiti-nation is strong. It is therefore instructive that hGGA3 getsubiquitinated when overexpressed in cells and it is still ubiq-uitinated when Leu-280 or Asp-284 in Site2 are mutated, albeitto a lesser degree as predicted (34). The observation that thesemutant hGGA3 proteins are still monoubiquitinated indicatesthey still possess residual Ub binding activity.

A similar two-Site mechanism may operate for the Tom1GAT domain in which the two motifs are conserved (Fig. 1).Mutation of Leu-285 to Arg or Asp-289 to Gly, which corre-spond to Leu-280 and Asp-284 of Site2 in hGGA3, causes loss ofUb binding by Tom1 (17). However, the inability to measureresidual binding from Site1 could again be because it is belowthe threshold for detection in these studies. The Tom1-L1 pro-tein also known as Srcasm has a GAT domain highly similar tothat of Tom1. The putative position of Site2 fits well the con-sensus binding motif defined here and in other studies. How-ever, it lacks the equivalent acidic residue of Glu-230 present inSite1 of the hGGA3 GAT domain and instead possesses Ala-221. Interestingly, the Tom1-L1 GAT domain still bindsGST�Ub but binds Ub-agarose and ubiquitinated proteins from

FIG. 4. Model for ubquitin interaction with the GAT domain. A,the relevant helical portion encompassing Site1 and Site2 of the GATdomain docked with Ub. The hydrophobic patch (Leu-8, Ile-44, andVal-70) of Ub shown in yellow and basic residues of Ub (Lys-48 andArg-72) in blue are coordinated with the central leucine and flankingacidic residues of the two GAT binding Sites, respectively. The sidechains of Site1 (219, 227 230) and Site2 (273, 280, 284) are shown. B,the helical portion of Site1 with only Ub is shown where the surface ofUb is color-coded according to the chemical shift perturbations quanti-fied in Fig. 2 using the wild-type hGGA3 domain and 15N-Ub. C, thehelical portion of Site2 with only Ub where the surface of Ub is color-coded for acidic residues (red), basic residues (blue), and non-polarresidues (yellow). D, views of a single hGGA3 GAT domain (blue) withboth Ub binding Sites bound to Ub. Ub bound to Site1 is in orange; Site2is in red.

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cell lysates at a much reduced level relative to Tom1, possiblybecause it lacks Site1 (17).

Based on our data, we can present a more refined model forhow Ub binds the GGA GAT domains, which should facilitatebetter formulation of functional experiments in the future (Fig.4). Precise definition of the Ub binding requirements for GATdomains is becoming increasingly important because the func-tion of the GAT domain itself is more complex than previouslyappreciated. The GAT domain of Tom1 is not only sufficient forUb binding but also mediates binding with Toll-interactingprotein (Tollip), another endosomal protein that binds Ub via aCUE domain (17, 18). Different residues of the Tom1 GATdomain are used to bind these partners, but their binding iscompetitive, evoking a mechanism whereby ubiquitinated pro-teins may be passed sequentially from Tom1 to Tollip (17). ThehGGA3 GAT domain binds to Ub and also interacts withTSG101 in a yeast two-hybrid assay (20). The hGGA1 GATdomain also can interact with Ub and TSG101 as well asRabaptin-5, the latter of which can compete against Ub binding(21, 35). And although each of these GGA GAT interactingfactors appears to require a subset of different GAT residues forinteraction, mutations such as alanine substitution of Leu-277in hGGA1 or Leu-276 of hGGA3 result in decreased or com-pletely abrogated interaction of all three binding partners (21).This type of global effect on multiple partners complicates theinterpretation of previous in vivo experiments where traffick-ing of ubiquitinated proteins is followed in cells bearing ahGGA3 L276A mutant or yeast bearing Gga2 lacking the three-helix bundle GAT domain (12, 20). The trafficking effects onGap1 that we measured here by ADCB sensitivity did strictlycorrelate with Ub binding. No single point mutation causedcomplete loss of Ub binding, and mutation of only one Ubbinding site resulted in only minor ADCB sensitivity. Onlywhen Site1 and Site2 were mutated together did we see anincrease of ADCB sensitivity comparable with deletion of theentire C-terminal portion of the GAT domain. Furthermore,this effect was observed regardless of how many alanine sub-stitution mutations were used to inactivate both Sites. Theseresults strongly indicate that the effects we observed on Gga-dependent Gap1 and Fur4 sorting are strictly because of loss ofUb binding and not loss of other protein-protein interactions.Our failure to detect any other defects with these mutants,even in the absence of the AP-1 adaptor complex that maypartly compensate for GGA functions, strongly indicates thatUb binding of Ggas is a function strictly confined to sortingubiquitinated cargo and is not required for other GGA-depend-ent processes.

Acknowledgment—We thank Pat Scott at the University of Minne-sota Duluth for helpful comments and suggestions.

REFERENCES

1. Hicke, L., and Dunn, R. (2003) Annu. Rev. Cell Dev. Biol. 19, 141–1722. Shih, S. C., Sloper-Mould, K. E., and Hicke, L. (2000) EMBO J. 19, 187–1983. Booth, J. W., Kim, M. K., Jankowski, A., Schreiber, A. D., and Grinstein, S.

(2002) EMBO J. 21, 251–2584. Shih, S. C., Katzmann, D. J., Schnell, J. D., Sutanto, M., Emr, S. D., and Hicke,

L. (2002) Nat. Cell Biol. 4, 389–3935. Polo, S., Sigismund, S., Faretta, M., Guidi, M., Capua, M. R., Bossi, G., Chen,

H., De Camilli, P., and Di Fiore, P. P. (2002) Nature 416, 451–4556. Katzmann, D. J., Odorizzi, G., and Emr, S. D. (2002) Nat. Rev. Mol. Cell. Biol.

3, 893–9057. Alam, S. L., Sun, J., Payne, M., Welch, B. D., Blake, B. K., Davis, D. R., Meyer,

H. H., Emr, S. D., and Sundquist, W. I. (2004) EMBO J. 23, 1411–14218. Katzmann, D. J., Babst, M., and Emr, S. D. (2001) Cell 106, 145–1559. Bilodeau, P. S., Urbanowski, J. L., Winistorfer, S. C., and Piper, R. C. (2002)

Nat. Cell Biol. 4, 534–53910. Blondel, M. O., Morvan, J., Dupre, S., Urban-Grimal, D., Haguenauer-Tsapis,

R., and Volland, C. (2004) Mol. Biol. Cell 15, 883–89511. Helliwell, S. B., Losko, S., and Kaiser, C. A. (2001) J. Cell Biol. 153, 649–66212. 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., andPiper, R. C. (2004) Nat. Cell Biol. 6, 252–259

13. Bonifacino, J. S. (2004) Nat. Rev. Mol. Cell. Biol. 5, 23–3214. Wakasugi, M., Waguri, S., Kametaka, S., Tomiyama, Y., Kanamori, S., Shiba,

Y., Nakayama, K., and Uchiyama, Y. (2003) Biochem. Biophys. Res. Com-mun. 306, 687–692

15. Boman, A. L. (2001) J. Cell Sci. 114, 3413–341816. Shiba, Y., Katoh, Y., Shiba, T., Yoshino, K., Takatsu, H., Kobayashi, H., Shin,

H. W., Wakatsuki, S., and Nakayama, K. (2004) J. Biol. Chem. 279,7105–7111

17. Katoh, Y., Shiba, Y., Mitsuhashi, H., Yanagida, Y., Takatsu, H., and Na-kayama, K. (2004) J. Biol. Chem. 279, 24435–24443

18. Yamakami, M., Yoshimori, T., and Yokosawa, H. (2003) J. Biol. Chem. 278,52865–52872

19. Pizzirusso, M., and Chang, A. (2004) Mol. Biol. Cell 15, 2401–240920. Puertollano, R., and Bonifacino, J. S. (2004) Nat. Cell Biol. 6, 244–25121. Mattera, R., Puertollano, R., Smith, W. J., and Bonifacino, J. S. (2004) J. Biol.

Chem. 279, 31409–3141822. Boman, A. L., Salo, P. D., Hauglund, M. J., Strand, N. L., Rensink, S. J., and

Zhdankina, O. (2002) Mol. Biol. Cell 13, 3078–309523. Sivaraman, T., Arrington, C. B., and Robertson, A. D. (2001) Nat. Struct. Biol.

8, 331–33324. Sundd, M., Iverson, N., Ibarra-Molero, B., Sanchez-Ruiz, J. M., and Robertson,

A. D. (2002) Biochemistry 41, 7586–759625. Schneider, D. M., Dellwo, M. J., and Wand, A. J. (1992) Biochemistry 31,

3645–365226. Collins, B. M., Watson, P. J., and Owen, D. J. (2003) Dev. Cell 4, 321–33227. Roberg, K. J., Rowley, N., and Kaiser, C. A. (1997) J. Cell Biol. 137, 1469–148228. Soetens, O., De Craene, J. O., and Andre, B. (2001) J. Biol. Chem. 276,

43949–4395729. Black, M. W., and Pelham, H. R. (2000) J. Cell Biol. 151, 587–60030. Swanson, K. A., Kang, R. S., Stamenova, S. D., Hicke, L., and Radhakrishnan,

I. (2003) EMBO J. 22, 4597–460631. Shekhtman, A., Ghose, R., Wang, D., Cole, P. A., and Cowburn, D. (2001) J.

Mol. Biol. 314, 129–13832. Fisher, R. D., Wang, B., Alam, S. L., Higginson, D. S., Robinson, H., Sundquist,

W. I., and Hill, C. P. (2003) J. Biol. Chem. 278, 28976–2898433. Shih, S. C., Prag, G., Francis, S. A., Sutanto, M. A., Hurley, J. H., and Hicke,

L. (2003) EMBO J. 22, 1273–128134. Shiba, T., Kawasaki, M., Takatsu, H., Nogi, T., Matsugaki, N., Igarashi, N.,

Suzuki, M., Kato, R., Nakayama, K., and Wakatsuki, S. (2003) Nat. Struct.Biol. 10, 386–393

35. Mattera, R., Arighi, C. N., Lodge, R., Zerial, M., and Bonifacino, J. S. (2003)EMBO J. 22, 78–88

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William R. Kearney, Andrew D. Robertson and Robert C. PiperPatricia S. Bilodeau, Stanley C. Winistorfer, Margaret M. Allaman, Kavitha Surendhran,

Binding MotifsThe GAT Domains of Clathrin-associated GGA Proteins Have Two Ubiquitin

doi: 10.1074/jbc.M406654200 originally published online October 19, 20042004, 279:54808-54816.J. Biol. Chem. 

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