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Interactions between Conserved Domains within Homodimers in the BIG1, BIG2, and GBF1 Arf Guanine Nucleotide Exchange Factors *

Interactions between Conserved Domains within Homodimers in the BIG1, BIG2, and GBF1 Arf Guanine... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 39, pp. 28834 –28842, September 28, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Interactions between Conserved Domains within Homodimers in the BIG1, BIG2, and GBF1 Arf Guanine Nucleotide Exchange Factors Received for publication, July 5, 2007 Published, JBC Papers in Press, July 19, 2007, DOI 10.1074/jbc.M705525200 ‡1 ‡ ‡1 § Odile Ramaen , Alexandra Joubert , Philip Simister , Naı¨ma Belgareh-Touze´ , ‡2 ‡ ‡ Maria Conception Olivares-Sanchez , Jean-Christophe Zeeh , Sophie Chantalat , ‡ ‡ ‡ ‡3 Marie-Pierre Golinelli-Cohen , Catherine L. Jackson , Vale´rie Biou , and Jacqueline Cherfils From the Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and Institut Jacques Monod-CNRS, Universite´s Paris VI et VII, 75251 Paris, France Guanine nucleotide exchange factors carrying a Sec7 domain lated by guanine nucleotide exchange factors (ArfGEFs) carry- (ArfGEFs) activate the small GTP-binding protein Arf, a major ing a catalytic Sec7 domain (reviewed in Refs. 2 and 3). Evidence regulator of membrane remodeling and protein trafficking in is accumulating that ArfGEFs integrate upstream signals that eukaryotic cells. Only two of the seven subfamilies of ArfGEFs define the conditions of Arf activation. First, ArfGEFs localize (GBF and BIG) are found in all eukaryotes. In addition to the to specific trafficking organelles (4–9), which allows them to Sec7 domain, which catalyzes GDP/GTP exchange on Arf, the specify which subcellular site requires Arf activity. Second, GBF and BIG ArfGEFs have five common homology domains. binding partners involved in cell signaling, such as protein Very little is known about the functions of these noncatalytic kinase A, FK506-binding protein 13, and the AKAP-interacting domains, but it is likely that they serve to integrate upstream protein AMY-1, have been identified for the large Golgi-local- signals that define the conditions of Arf activation. Here we ized ArfGEFs (10–12). Finally, ArfGEFs may play a role in describe interactions between two conserved domains membrane recruitment of Arf effectors, such as coats, thus upstream of the Sec7 domain (DCB and HUS) that determine assembling downstream components of Arf signaling pathways the architecture of the N-terminal regions of the GBF and prior to Arf activation (5, 13). BIG ArfGEFs using a combination of biochemical, yeast two- An essential issue is to decipher how ArfGEFs implement hybrid, and cellular assays. Our data demonstrate a strong these functions and coordinate them with their biochemical interaction between DCB domains within GBF1, BIG1, and GDP/GTP exchange activity. To address this question, we BIG2 to maintain homodimers and an interaction between chose to focus on the large ArfGEFs, since (i) they are the only DCB and HUS domains within each homodimer. The DCB/ ArfGEFs found in all eukaryotes, and (ii) their multidomain HUS interaction is mediated by the HUS box, the most con- architecture may allow them to recapitulate the largest number served motif in large ArfGEFs after the Sec7 domain. In sup- of ArfGEF functions within a single polypeptide (14, 15). Large port of the in vitro data, we show that both the DCB and the ArfGEFs comprise two groups, which we refer to as the GBF HUS domains are necessary for GBF1 dimerization in mam- and BIG groups after their names in mammals. Both function in malian cells and that the DCB domain is essential for yeast maintaining organelle integrity and membrane traffic at the viability. We propose that the dimeric DCB-HUS structural Golgi or at endosomes (reviewed in Ref. 1). The best studied unit exists in all members of the GBF and BIG ArfGEF groups representatives are yeast Gea1p/Gea2p, Arabidopsis thaliana and in the related Mon2p family and probably serves an GNOM and mammalian GBF1 for the GBF group, and yeast important regulatory role in Arf activation. Sec7p and mammalian BIG1/BIG2 for the BIG group (reviewed in Refs. 1 and 2). We predicted earlier from a bioinformatics analysis that the GBF and BIG groups share a common archi- Small GTP-binding proteins of the Arf (ADP-ribosylation tecture, suggesting that both ArfGEF groups follow a common factor) family are major regulators of membrane traffic in the scenario for their activation of Arf (15). The predicted organi- exocytotic and endocytic pathways (reviewed in Ref. 1). They zation comprises two noncatalytic domains (dimerization and are activated by the exchange of GDP for GTP, which is stimu- cyclophilin-binding (DCB) and homology upstream of Sec7 (HUS)) in the N terminus of the Sec7 domain and three in its C terminus (HDS1, HDS2, and HDS3). The N-terminal DCB * This work was supported by a Human Frontiers in Science Program grant and a French Ministe`re de la Recherche ACI grant (to J. C.) and a CNRS ATIP domain (Fig. 1A) is the only domain to which a molecular func- grant (to V. B.). 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. The abbreviations used are: ArfGEF, guanine nucleotide exchange factor Supported by a grant from the CNRS. of Arf protein; GBF, Golgi-associated brefeldin A-resistant guanine Supported by a Marie-Curie grant from the European Community. nucleotide exchange factor; BIG, brefeldin A-inhibited guanine nucle- To whom correspondence should be addressed: Laboratoire d’Enzymologie otide exchange factor; DCB, dimerization/cyclophilin binding; HUS, et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur- homology upstream of Sec7; BD, binding domain; AD, activation Yvette Cedex, France. Tel.: 33-1-6982-3492; Fax: 33-1-6982-3129; E-mail: domain; GFP, green fluorescent protein; Tricine, N-[2-hydroxy- [email protected]. 1,1-bis(hydroxymethyl)ethyl]glycine. 28834 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 39 •SEPTEMBER 28, 2007 This is an Open Access article under the CC BY license. Interactions between BIG1, BIG2, and GBF1 Conserved Domains TABLE 1 tion has been assigned. It was originally identified in plant Liposome composition GNOM, where it was shown to be capable of dimerization in In all cases, 0.2% of the fluorescent lipid nitrobenzoxadizoldihexadecanoyl-phos- yeast two-hybrid and in vitro pull-down assays (16). Little is phatidylethanolamine was added as a tracer. PC, phosphatidylcholine; PG, phos- known about the other domains, except for an almost invariant phatidylglycerol; PIP2, phosphatidylinositol 4,5-bisphosphate. 5-residue motif in the HUS domain, the HUS box (15, 17) (Fig. 1B), which is essential for aspects of Golgi traffic in yeast (18). mol % mol % mol % mol % Here we combine biochemical, yeast two-hybrid, and cellular Soybean PC 95 65 92.5 62.5 Cholesterol 5 5 5 5 assays to analyze the domain architecture and interdomain Egg PG 0 30 0 30 interactions of the GBF and BIG groups of large ArfGEFs. Our Brain PIP2 0 0 2.5 2.5 data demonstrate the existence of two distinct interactions involving the DCB domain of the mammalian large ArfGEFs: Yeast Two-hybrid Assays—Plasmids expressing human homodimerization via a DCB/DCB interaction (generalizing BIG1 and BIG2 and Cricetulus griseus GBF1 are gifts from P. Melanc¸on (University of Alberta, Canada), the plasmid previous results from plants) and a novel DCB/HUS interaction expressing coxsackievirus 3A is a gift from F. van Kuppeveld depending on the HUS box. We propose that the DCB/DCB and DCB/HUS interactions define a common structure in all (Radboud University Nijmegen Medical Centre, The Neth- members of the BIG and GBF groups of ArfGEFs, which prob- erlands). All yeast two-hybrid constructs were cloned in the pAS and pACT2 vectors to create fusions with the Gal4- ably also exists in the related eukaryotic Mon2p family. DNA binding domain (BD) and Gal4 transcription activation EXPERIMENTAL PROCEDURES domain (AD), respectively. The Y190 (MATa, gal4-542, Expression of Recombinant DCB , DCB , and DCB- gal80-538, his3, trp1-901, ade2-101, ura3-52, leu2-3, 112, BIG1 BIG2 URA3::GAL1-LacZ, Lys2::GAL1-HIS3cyh ) and AH109 HUS-Sec7 —The DCB domain of human BIG1 (DCB , BIG1 BIG1 (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200 gal4, gal80, residues 2–224) was introduced into pET28a (Novagen) modi- fied to remove the thrombin cleavage site and to include alter- LYS2::GAL1 -GAL1 -HIS3, GAL2 -GAL2 -ADE2, UAS TATA UAS TATA native restriction sites (KpnI and AgeI). The E221K mutation URA3::MEL1 -MEL1 -lacZ) yeast strains were trans- UAS TATA formed with the different recombinant plasmids using the lith- was introduced into DCB by PCR using the QuikChange BIG1 ium acetate method (20). Y190 transformants autotrophic for site-directed mutagenesis kit (Stratagene). Both wild type and mutant DCB were expressed in the Rosetta(DE3)pLysS tryptophan and leucine were assayed for -galactosidase activ- BIG1 Escherichia coli strain (Merck KGaA). DCB was purified on ity using the filter technique (21). AH109 transformants were BIG1 tested for expression of HIS3 and ADE2 reporter genes. Stable aNi -nitrilotriacetic acid affinity column (GE Healthcare) fol- expression of each clone in pACT2 and pAS was confirmed by lowed by precipitation in ammonium sulfate to 70% saturation and gel filtration on a Superdex 75 column (GE Healthcare). Western blot using the Santa Cruz Biotechnology, Inc. (Santa The DCB domain of human BIG2 (DCB , residues 2–224) Cruz, CA) Gal4-AD and Gal4-BD monoclonal antibodies, BIG2 respectively. All experiments were performed at least three was cloned, expressed, and purified as described for DCB . BIG1 times. A construct spanning the DCB, HUS, and Sec7 domains of human BIG1 (DCB-HUS-Sec7 , residues 2–888) was intro- Biochemical Assays—Liposome binding experiments were BIG1 duced into pFastBac HTA vector (Invitrogen) using EcoRI and performed with liposomes prepared as described in Ref. 22 (Table 1). DCB (1 M) was incubated at room temperature KpnI restriction sites. Sf21 cells infected by baculoviruses har- BIG1 in 50 mM Hepes, pH 7.2, and 120 mM potassium acetate with boring this construct were used to express DCB-HUS-Sec7 . BIG1 The recombinant protein was purified on a Ni -nitrilotriace- sucrose-loaded vesicles (final lipid concentration, 1 mM)in tic acid affinity column followed by a desalting column and a gel small polycarbonate tubes. The samples were centrifuged at 360,000  g for 20 min, and the supernatants and the pellets filtration Superdex 200 column (GE Healthcare). Limited pro- were analyzed by SDS-PAGE with Sypro-orange staining. The teolysis was performed with 10 units of thrombin (Amersham Biosciences) per mg of protein at room temperature overnight. Sec7 domain of BIG1 was used as a negative control. Exchange DCB-HUS-Sec7 and its proteolysis products were analyzed reaction assays were performed by tryptophan fluorescence BIG1 kinetics using 17Arf1 as described in Ref. 23. The effect of by SDS-PAGE and Western blot. DCB on the exchange rate of Sec7 was analyzed by Biophysical Assays—Sedimentation velocity was measured at BIG1 BIG1 40,000  g for 24 h and analyzed with the SVEDBERG software comparing the results of experiments done in the absence or (available on the World Wide Web). Sedimentation equilib- presence of DCB (10 M). BIG1 Co-immunoprecipitation Assays—COS7 cells in 10-cm cul- rium experiments were carried out at 10,000, 15,000, and ture dishes were cotransfected with the plasmids pHA-GBF1 20,000  g for 46 h and analyzed with the Origin software (Beckman Coulter). CD scans were recorded between 185 and expressing human HA-GBF1 and either Venus-GBF1, Venus- 260 nm. Thermal denaturations were carried out in the temper- GBF1889, or YFP-GBF1-C expressing, respectively, human GBF1 and GBF1 deleted of the first 297 (DCB) and 710 amino ature range of 5–95 °C at a rate of 2 °C/min. Secondary struc- acids (DCB-HUS). After 20 h of expression, cells were ture composition was estimated with the CDDSTR software (19). The effect of protein concentration on its thermal dena- washed two times with 5 ml of cold phosphate-buffered saline turation was measured with 0.3, 0.75, and 3 M DCB and (137 mM NaCl, 2.7 mM KCl, 19 mM Na HPO , 1.8 mM KH PO ) BIG1 2 4 2 4 analyzed at 222 nm, a wavelength minimum that is character- istic of -helices. T. K. Niu and C. L. Jackson, unpublished data. SEPTEMBER 28, 2007• VOLUME 282 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 28835 Interactions between BIG1, BIG2, and GBF1 Conserved Domains and then disrupted in 0.5 ml of cold lysis buffer (50 mM Tris- domain of human BIGs qualifies as the bona fide homolog of the HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40). DCB domain of the GBF group member GNOM and refer to it After centrifugation at 4 °C, soluble cellular extracts were pre- as DCB hereafter. BIG cleared with 20 l of protein G-Sepharose 4 Fast Flow (GE In order to assess a possible dimer/monomer equilibrium, we Healthcare) at 4 °C for 30 min. Supernatants were incubated analyzed the CD thermal denaturation spectra of DCB (Fig. BIG1 with 3 g of anti-GFP antibodies (Roche Applied Science) for 2B). The model providing the best fit for the data was a two- 1.5 h at 4 °C. Then 30 l of protein G-Sepharose 4 Fast Flow was state transition between an all-helical structure and a random added, and the mixtures were incubated at 4 °C for 1.5 h. The coil denaturated state, without formation of a monomeric resin was washed two times with 1 ml of W100 buffer (50 mM intermediate. This and a denaturation temperature (69 °C) Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA), followed by two independent of the protein concentration and higher than the washes with 1 ml of phosphate-buffered saline. Proteins were average for proteins (around 55 °C) suggest that the dimer is then eluted by incubation with 60 l of SDS-PAGE sample stable with a dissociation constant below the concentration buffer for 5 min at 95 °C. Eluted proteins were separated on a 6% used in the experiment (100 nM). Analytical ultracentrifugation SDS-polyacrylamide gel and analyzed by Western immuno- confirmed the predominance of a dimeric species and the blotting using anti-HA antibodies as primary antibody (Sigma). absence of monomer. Plasmid Shuffle Assays in Yeast—The gea1-DCB construct To determine whether dimerization is a general feature of (coding for residues 233–1408 of Gea1p) was obtained by intro- DCB-like domains of the large ArfGEFs, we tested mammalian ducing a gap in the pAP22 (CEN, TRP1, GEA1) (gift of A. Pey- GBF1 and BIG2 DCB/DCB interactions using the yeast two- roche, Commissariat a` l’Energie Atomique, Saclay, France). hybrid system. We observed a strong interaction between DCB The PCR product extended to 1825 and 154 base pairs past the domains of hamster GBF1 (98% identity with human GBF1) 5 and 3 ends of the gap, respectively. The mutated fragment and between human BIG2 DCB domains, indicating that the and gapped plasmid were used to transform CJY52-10-2 yeast DCB domain mediates dimerization in all mammalian large cells that contained the pAP23 plasmid (CEN, URA3, GEA1) ArfGEFs (Fig. 3, sectors 1 and 12). We next looked for residues (24). Transformants were plated onto synthetic medium plates that contribute to the DCB/DCB interaction. Two highly con- 91 130 lacking tryptophan. Trp clones were grown at 30 °C in mini- served residues, Lys and Glu in DCB , are found in both GBF1 mal medium (YNB) containing 0.67% yeast nitrogen base with- large ArfGEF groups (Fig. 1A). Mutation of either residue to out amino acids (BD), supplemented with appropriate nutri- alanine in DCB abolished the interaction between mutant GBF1 ents and with 2% glucose. 5-Fluoroorotic acid monohydrate and wild-type DCB in the two-hybrid system (Fig. 3, sectors GBF1 (Toronto Research Chemicals) was added to a final concentra- 2 and 11). Thus, these residues are either part of the dimer tion of 0.1% to counterselect the URA3-containing cells. The interface or induce an abnormal structure in this interface. We presence of protein product of the gea1-DCB allele was con- then analyzed a mutation found in the C terminus of the DCB trolled by Western immunoblotting as follows. Total yeast cell domain of human BIG2 (Fig. 1A), which has been associated protein extracts were prepared using the NaOH/trichloroacetic with a congenital disease, autosomal recessive periventricular acid lysis technique (25). Proteins were separated by SDS- heterotopia with microencephaly (26). DCB carrying the BIG1 PAGE in 10% Tricine gels and were analyzed by immunoblot- equivalent mutation, E221K, was expressed in E. coli with a sol- ting. The primary antibody was either a polyclonal anti-Gea1p ubility similar to that of wild-type DCB . Analytical ultra- BIG1 E221K antibody or a monoclonal anti-Vat2p antibody directed against centrifugation experiments showed that DCB forms a BIG1 a subunit of the vacuolar ATPase (Invitrogen). dimer (data not shown). Thus, functions of DCB other BIG2 than dimerization are affected by this mutation in the auto- RESULTS somal recessive periventricular heterotopia with microen- Mammalian BIG and GBF ArfGEFs Have an N-terminal cephaly disorder. Dimerization DCB Domain—Using a bioinformatics approach, A Novel Interaction between the DCB and HUS Domains in we predicted previously that the DCB domain, originally iden- Large ArfGEFs—To determine whether interactions exist tified as a dimerization domain in the GBF group member between the different domains of the large ArfGEFs, we carried GNOM (16), is also present at the N terminus of large ArfGEFs out an extensive yeast two-hybrid analysis of domain-domain from the BIG group, where it should form an all-helical domain interactions for mammalian GBF1 and BIG1. All five noncata- (15). In order to address this question experimentally, we lytic domains in addition to the catalytic Sec7 domain were expressed in E. coli the N terminus of human BIG1, encompass- considered. For GBF1 (Table 2A) and BIG1 (Table 2B), only one ing the helical subdomain of highest sequence homology and interaction was detected in addition to the DCB/DCB interac- the more variable N-terminal subdomain (Fig. 1A), and purified tion described above. In both cases, a strong interaction it to homogeneity. The recombinant protein behaved as a 2  between the DCB and HUS domains of each ArfGEF was 26-kDa dimer (Fig. 2A), a molecular mass that was confirmed observed (Fig. 3, sectors 3 and 4). This interaction was inde- by analytical ultracentrifugation equilibrium sedimentation pendent of the variable DCB-HUS linker added on either the (54.3 kDa). A similar construct from human BIG2 was also DCB or HUS side (Table 2A). A strong interaction was also expressed and purified to homogeneity and, as for BIG1, eluted found between the DCB and HUS domains of human BIG2 (Fig. as a dimer on a gel filtration column (Fig. 2A). Deconvolution of 3, sector 5). CD spectra for both proteins was consistent with a mostly hel- The HUS box is an almost invariant N(Y/F)DC(D/N) motif, ical secondary structure. We thus conclude that the N-terminal which is predicted to lie between two -helices (Fig. 1B). Muta- 28836 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 39 •SEPTEMBER 28, 2007 Interactions between BIG1, BIG2, and GBF1 Conserved Domains FIGURE 1. Sequence alignments of DCB domains and HUS boxes. A, sequence alignment of DCB domains of the BIG, GBF, and Mon2p/Ysl1p/SF21 families. Predicted -helices are represented by helices. The black arrows indicate the conserved residues mutated in C. griseus GBF1. The gray arrow indicates the BIG2 glutamate mutated in autosomal recessive periventricular heterotopia with microencephaly and analyzed in this paper. B, sequence alignment of the HUS box (framed) in the HUS domain. The mutation analyzed in this study is shown by an arrow. C, sequence alignment of the putative HUS box (framed)inthe eukaryotic Mon2p/Ysl1p/SF21 family. The residue shown by an arrow is equivalent to that in B. tion of the central aspartate to alanine in this motif abolished involved only in the DCB/DCB interaction, whereas the Glu the DCB/HUS interaction in both GBF1 and BIG1 (Fig. 3, sec- residue is involved in both interactions. tors 18 and 19). Thus, the HUS box supports the DCB/HUS Next, we used the yeast two-hybrid system to analyze DCB/ interaction, which is the first molecular function to be associ- DCB and DCB/HUS interactions within GBF1 constructs span- ated with this motif. We then analyzed whether mutations that ning more than one domain. We first analyzed the ability of the impair the DCB/DCB interaction (see above) also affect the DCB domain alone to interact with larger ArfGEF fragments. DCB/HUS interaction. The E130A mutation in GBF1, but not DCB interacted with DCB-HUS , DCB-HUS-Sec7 , GBF1 GBF1 GBF1 the K91A mutation, abolished the DCB/HUS interaction (Fig. and full-length GBF1 (Fig. 3, sectors 7, 8, and 10). Although 3, sectors 16 and 17). These results indicate that residue Lys is these yeast two-hybrid interactions appeared to be weaker than SEPTEMBER 28, 2007• VOLUME 282 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 28837 Interactions between BIG1, BIG2, and GBF1 Conserved Domains FIGURE 3. Yeast two-hybrid analysis of DCB and HUS interactions in mammalian ArfGEFs. AH109 yeast cells were co-transformed with the expression plasmids for bait (BD) and prey (AD) fusion proteins and selected on double synthetic dropout (Trp/Leu) medium plates. The transformants were then selected for the expression of HIS3 and ADE2 genes by growing on synthetic dropout medium (Trp/Leu/His/Ade). K91A 1, DCB /DCB ; 2, DCB /DCB ; 3, DCB /HUS ; 4, DCB / GBF1 GBF1 GBF1 GBF1 GBF1 GBF1 BIG1 HUS ; 5, DCB /HUS ; 6, DCB /HUS-Sec7 ; 7, DCB /DCB- BIG1 BIG2 BIG2 GBF1 GBF1 GBF1 HUS ; 8, DCB /DCB-HUS-Sec7 ; 9, DCB /Sec7 ; 10, DCB / GBF1 GBF1 GBF1 GBF1 GBF1 GBF1 E130A GBF1; 11, DCB -DCB ; 12, DCB /DCB ; 13, GBF1/DCB-HUS- GBF1 GBF1 BIG2 BIG2 Sec7 ; 14, HUS /DCB-HUS-Sec7 ; 15, HUS /GBF1; 16, HUS / GBF1 GBF1 GBF1 GBF1 GBF1 K91A E130A D540A D570A DCB ; 17, HUS /DCB ; 18, HUS /DCB ; 19, HUS /DCB ; GBF1 GBF1 GBF1 GBF1 GBF1 BIG1 BIG1 20, HUS /Sec7 ; 21, DCB-HUS /3A; 22, HUS /3A; 23, DCB /3A. GBF1 GBF1 GBF1 GBF1 GBF1 TABLE 2 Summary of domain/domain interactions in mammalian ArfGEFs analyzed with the yeast two-hybrid assay Strong interactions are indicated in dark gray, and weaker interactions are shown in gray. Baits (BD) and preys (AD) are indicated in rows and columns, respectively. FIGURE 2. Biochemical characterization of recombinant proteins. A, elu- tion profiles of purified DCB , DCB , and DCB-HUS-Sec7 on a gel fil- BIG1 BIG2 BIG1 tration column. The elution volumes of calibration proteins are indicated. B, circular dichroism spectra of DCB (6 M) as a function of temperature. BIG1 CD spectra are taken every 5 °C. The isodichroic point lies at 200.5 nm (thin arrow), showing that unfolding occurs as a two-state helix-coil transition. the interaction between two DCB domains alone, they strongly suggest that the DCB/DCB interaction occurs also in the con- text of the full-length GBF1 protein. We then analyzed the DCB/HUS interaction in the context of multiple domains. A strong interaction was found between the DCB domain and a HUS-Sec7 construct (Fig. 3, sector 6). HUS also interacted GBF1 with DCB-HUS-Sec7 and full-length GBF1 although more GBF1 weakly than with DCB alone (Fig. 3, sectors 14 and 15). In GBF1 support for the dimerization of the N-terminal region taking place in larger constructs, recombinant DCB-HUS-Sec7 BIG1 eluted on a gel filtration column with a molecular weight con- sistent with a dimer (Fig. 2A). DCB or DCB/HUS cross-interactions between BIG1, BIG2, and No interactions between the noncatalytic domains other GBF1 in the yeast two-hybrid system (data not shown). than the DCB/DCB and DCB/HUS interactions were identified Functions of DCB/DCB and DCB/HUS Interactions in Arf- with the yeast two-hybrid assay. We also failed to detect an GEF Dimers—The DCB domain has features of a dimeric interaction of the noncatalytic domains with the Arf1 substrate helical bundle, which is a frequent arrangement in cytosolic (data not shown). In addition, we did not observe any DCB/ proteins involved in membrane recruitment, such as the mem- 28838 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 39 •SEPTEMBER 28, 2007 Interactions between BIG1, BIG2, and GBF1 Conserved Domains brane curvature-sensing BAR domain found in amphiphysins and Arfaptin/POR, an Arf effector (27). We thus investigated the binding of DCB to liposomes of various compositions BIG1 using a sedimentation assay (Table 1 and Fig. 4A). However, no such interaction could be observed regardless of the liposome composition, suggesting that the DCB homodimer does not have membrane-binding properties on its own. The Sec7 domain did not interact with the constructs tested in our yeast two-hybrid analysis (Table 2, A and B, and Fig. 3, sectors 9 and 20). To further analyze whether the N terminus could regulate the catalytic exchange activity of the Sec7 domain, we used recombinant proteins and a fluorescence kinetics assay. We first analyzed the effect of excess DCB on BIG1 the exchange rate of the Sec7 domain of human BIG1 (Sec7 ) using 17Arf1 as substrate. No inhibition or stimu- BIG1 lation of the exchange rate by DCB could be observed, BIG1 regardless of whether 17Arf1, Sec7 , or both had been pre- BIG1 incubated with DCB (Fig. 4B). We then analyzed the cata- BIG1 lytic activity of a BIG1 construct spanning the DCB-HUS-Sec7 domains using the same fluorescence assay. This construct was active at stimulating GDP/GTP exchange on17Arf1 (Fig. 4C), and it was inhibited by brefeldin A with a K of 23.9  7.2 M, which is similar to that measured for the Sec7 of BIG1 alone (23). To confirm that the DCB-HUS tandem had no effect on the catalytic activity, we took advantage of a unique thrombin cleavage site located at residue 622 between the HUS and Sec7 domains, which allowed us to generate free DCB-HUS and BIG1 Sec7 by limited proteolysis. Exchange rates measured with BIG1 a BIG1 peptide concentration of 0.5 M were in the same range for the uncleaved and cleaved fragments (0.073  0.005 and 0.098  0.012 s , respectively), suggesting that the DCB-HUS tandem does not have a simple one-to-one regulatory activity toward the Sec7 domain. The N terminus of large ArfGEFs interacts with several large ArfGEF protein partners (reviewed in Ref. 1). We thus investi- gated whether the DCB/HUS structure may be required for protein-protein interactions. To this end, we took advantage of the fact that the N terminus of GBF1 binds to 3A, a protein from enteroviruses that blocks host cell secretion by inhibiting GBF1 function (28). The cytosolic portion of 3A (residues 1–60) interacts with DCB-HUS and DCB-HUS-SEC7 in the GBF1 GBF1 yeast two-hybrid assay (Fig. 3, sector 21). In contrast, no inter- action was observed with individual DCB or HUS domains (Fig. 3, sectors 22 and 23). This is consistent with data showing that deletion of either the first 50 amino acids of GBF1 or deletion of FIGURE 4. Function of the DCB/DCB and DCB/HUS interactions. A, sedi- mentation analysis of DCB binding properties to liposomes. The lanes cor- the HUS domain and downstream sequences abolishes interac- BIG1 respond (in this order) to molecular weight markers, supernatant (S), and tion with the 3A protein in the mammalian two-hybrid system pellet (P) fractions of experiments with no liposomes (No lip) and liposomes (29). These results show that portions of both the DCB and 1– 4 (Table 1). The last lane represents the total amount of protein in the experiment (T). B, effect of DCB on the kinetics of Sec7 -stimulated BIG1 BIG1 HUS domains of GBF1 are required for binding to the viral 3A GDP/GTP exchange on 17Arf1 measured by tryptophan fluorescence. 1,no protein and suggest the possibility that an integral DCB-HUS DCB; 2, DCB was incubated with Sec7 domain for 5 min before 17Arf1- BIG1 GDP was added; 3, DCB was incubated with Arf1 for 5 min before the Sec7 structure is necessary for binding of the 3A protein. BIG1 domain was added. In all cases, GTP (100 M) was added 2 min after all pro- Dimerization of Large ArfGEFs in Vivo—The above analy- teins were mixed together. C, GDP/GTP exchange activity of DCB-HUS- sis suggests that the DCB domain supports the dimerization Sec7 on 17Arf1 measured at different GEF concentrations. BIG1 of large ArfGEFs and organizes a structure that can bind protein partners. We thus assessed the dimerization and We first analyzed the formation of human GBF1 dimers in function of this domain in cells for two large ArfGEFs of the mammalian cells by pull-down assays (Fig. 5A). We found out GBF group. that full-length GBF1 can easily be isolated as a dimer from SEPTEMBER 28, 2007• VOLUME 282 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 28839 Interactions between BIG1, BIG2, and GBF1 Conserved Domains FIGURE 5. In vivo assays. A, the DCB and HUS domains of human GBF1 are both involved in its dimerization in mammalian cells. HA-tagged GBF1 was coexpressed with either GFP, GFP-tagged GBF1, GFP-tagged GBF1DCB, or GFP-tagged GBF1(DCB-HUS). After incubation with anti-GFP antibodies fol- FIGURE 6. Models for the DCB/DCB and DCB/HUS interactions. A, dimer- lowed by an incubation with protein G-Sepharose, the resin was washed sev- ization by combined DCB/DCB and DCB/HUS intermolecular interactions. eral times, and proteins were eluted by incubation with SDS-PAGE sample B, DCB/DCB dimerization with intramolecular DCB/HUS interaction. C, a pos- buffer. Eluted proteins were separated on a 6% SDS-polyacrylamide gel and sible regulatory switch of the HUS box from domain/domain interaction to analyzed by Western blotting using anti-HA antibodies. B, the DCB domain of solvent exposure and/or alternative interactions. D, tetramer formation by Gea1p is essential for yeast inability. Left, yeast extracts from gea1gea2 three-dimensional domain swapping. cells bearing a URA3 plasmid containing the GEA1 wild type gene (plasmid pAP23, lane 1), pAP23 and a TRP plasmid containing the GEA1 wild-type gene (pAP22, lane 2), pAP23 and pAP22 containing the gea1-DCB1 gene (lane 3), or yeast extracts from a wild-type strain (lane 4) were separated on SDS-poly- domains, in which the DCB domain interacts with itself and acrylamide gels and analyzed by western immunoblotting using an anti- with the HUS domain. The DCB/HUS interaction requires the Gea1p antibody. An anti-Vat2p antibody was used as a loading control. Right, highly conserved HUS box, a five-amino acid motif found in all yeast cells deleted for the GEA1 and GEA2 genes and bearing a URA3 plasmid containing the GEA1 wild-type gene were transformed with a plasmid con- members of the BIG and GBF groups of ArfGEFs. taining either the wild type GEA1 gene (gea2 GEA1 cells) or the GEA1 gene Because of its bipartite organization, the DCB-HUS tandem deleted for the DCB domain (gea2 gea1-DCB cells). 10-Fold serial dilutions provides different ways for large ArfGEFs to form multimers. of yeast cultures were plated on media with or without 5-fluoroorotic acid monohydrate (5FOA) to counterselect the URA3-containing cells. One is through the DCB/DCB interaction, which is an obligate intermolecular interaction. Since the DCB domain forms a mammalian cells. Next, we examined dimer formation between strong homodimer in vitro, we propose that it supports consti- the full-length GBF1 and forms of GBF1 deleted of the DCB tutive homodimerization of large ArfGEFs. The existence of this interaction in native BIG and GBF ArfGEFs is supported by domain alone or of both the DCB and HUS domains. Clearly, its formation in a range of yeast two-hybrid GBF1 constructs, whereas deletion of the DCB domain alone reduced somewhat the formation of a dimer with full-length GBF1, both the DCB the dimerization of the recombinant BIG1 and BIG2 con- and HUS domains had to be deleted to nearly abolish dimer structs, and our in vivo data on GBF1. It is also consistent with the molecular weight of several large ArfGEFs of both the BIG formation. Thus, the DCB and the HUS domains are both and GBF groups as measured by size exclusion chromatogra- involved in the dimerization of GBF1. We then analyzed the effect of deleting the DCB domain of phy, including yeast Gea1p (30), human BIG1 and BIG2 (31, Gea1p, a member of the GBF group of large ArfGEFs in yeast, 32), and plant GNOM (16). All elute as large molecular weight complexes, which, given the uncertainty of this technique for using a plasmid shuffle strategy. The strain used contains the nonglobular proteins, is consistent with their association as wild-type GEA1 gene on a URA3 plasmid with both gea1 and gea2 deletions of the chromosomal copies of the genes (24). homodimers. The gea1-DCB allele was introduced into a low copy TRP In contrast, the DCB/HUS interaction can occur either between two monomers (intermolecular) (Fig. 6A) or within plasmid. The Gea1p-DCB protein was expressed and was not a single ArfGEF polypeptide (intramolecular) (Fig. 6B). An degraded (Fig. 5B, left). Clones failed to grow at 30 °C upon the loss of the wild-type GEA1 plasmid when the gea1-DCB plas- intermolecular DCB/HUS interaction would provide a sec- mid became the sole copy of the redundant GEA1 and GEA2 ond contribution to dimerization in addition to the DCB/ DCB interaction. This possibility is supported by our co- genes (Fig. 5B, right). This result indicates that the DCB domain immunoprecipitation results, which show that the DCB of Gea1p is essential for yeast viability. form of human GBF1 formed a dimer with full-length GBF1 DISCUSSION almost as efficiently as full-length GBF1 in mammalian cells, A Conserved DCB/DCB and DCB/HUS Structure in Eukary- whereas deletion of both DCB and HUS domains practically otic Large ArfGEFs and the Related Mon2p Family—In this eliminated dimerization with full-length GBF1. Interest- study, we investigated the domain/domain interactions within ingly, the HUS box has an unusual level of sequence conser- the BIG and GBF groups of large ArfGEFs, which we predicted vation and content of polar residues within a protein inter- previously to share a common architecture (15). Based on bio- face, pointing to a potential for the DCB/HUS interaction to chemical and yeast two-hybrid analyses of mammalian BIG1, open up and expose the HUS box (Fig. 6C). The HUS box BIG2, and GBF1, we establish that all three members share a could then carry out other functions, allowing in particular similar DCB-HUS organization upstream of their Sec7 the formation of ArfGEF tetramers through three-dimen- 28840 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 39 •SEPTEMBER 28, 2007 Interactions between BIG1, BIG2, and GBF1 Conserved Domains Gif-sur-Yvette, France) for performing the analytical ultracentrifuga- sional domain swapping (Fig. 6D). An interesting corollary is tion experiments; Simona Burlacu-Miron and Gil Craescu (CNRS/ that this could allow large ArfGEFs to form heterotetramers, Institut Curie, Orsay, France) for help with circular dichroism; Bruno which are more likely to form than heterodimers, given the Antonny (Institut de Pharmacologie Mole´culaire et Cellulaire, CNRS, stability of the homodimeric DCB/DCB interaction. BIG1 Valbonne, France) for help with the liposome assay; Sylvie Lazareg and BIG2 have been shown to co-immunoprecipitate in and Jean-Pierre le Caer (Institut de Chimie des Substances Naturel- human cells (32), which could thus be mediated by the for- les, CNRS, Gif-sur-Yvette, France) for performing mass spectrometry mation of heterotetramers containing one BIG1 homodimer measurements; and Rosine Haguenauer-Tsapis (Institut Jacques and one BIG2 homodimer. Monod-CNRS, Paris, France) for support. 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(2004) Science 303, 495–499 with the yeast two-hybrid assays; Fatima el Khadali (LEBS, CNRS, 28. Wessels, E., Duijsings, D., Niu, T. K., Neumann, S., Oorschot, V. M., de SEPTEMBER 28, 2007• VOLUME 282 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 28841 Interactions between BIG1, BIG2, and GBF1 Conserved Domains Lange, F., Lanke, K. H., Klumperman, J., Henke, A., Jackson, C. L., 33. Jochum, A., Jackson, D., Schwarz, H., Pipkorn, R., and Singer-Kruger, B. Melchers, W. J., and van Kuppeveld, F. J. (2006) Dev. Cell 11, 191–201 (2002) Mol. Cell Biol. 22, 4914–4928 29. Wessels, E., Duijsings, D., Lanke, K. H., Melchers, W. J., Jackson, C. L., and 34. Efe, J. A., Plattner, F., Hulo, N., Kressler, D., Emr, S. D., and Deloche, O. van Kuppeveld, F. J. (2007) J. Virol. 81, 5238–5245 (2005) J. Cell Sci. 118, 4751–4764 30. Peyroche, A., Paris, S., and Jackson, C. L. (1996) Nature 384, 479–481 35. Gillingham, A. K., Whyte, J. R., Panic, B., and Munro, S. (2006) J. Biol. 31. Morinaga, N., Tsai, S. C., Moss, J., and Vaughan, M. (1996) Proc. Natl. Chem. 281, 2273–2280 Acad. Sci. U. S. A. 93, 12856–12860 36. Monetta, P., Slavin, I., Romero, N., and Alvarez, C. (2007) Mol. Biol. Cell 32. Yamaji, R., Adamik, R., Takeda, K., Togawa, A., Pacheco-Rodriguez, G., 18, 2400–2410 Ferrans, V. J., Moss, J., and Vaughan, M. (2000) Proc. Natl. Acad. Sci. 37. Xu, K. F., Shen, X., Li, H., Pacheco-Rodriguez, G., Moss, J., and Vaughan, U. S. A. 97, 2567–2572 M. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 2784–2789 28842 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 39 •SEPTEMBER 28, 2007 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Interactions between Conserved Domains within Homodimers in the BIG1, BIG2, and GBF1 Arf Guanine Nucleotide Exchange Factors *

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 39, pp. 28834 –28842, September 28, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Interactions between Conserved Domains within Homodimers in the BIG1, BIG2, and GBF1 Arf Guanine Nucleotide Exchange Factors Received for publication, July 5, 2007 Published, JBC Papers in Press, July 19, 2007, DOI 10.1074/jbc.M705525200 ‡1 ‡ ‡1 § Odile Ramaen , Alexandra Joubert , Philip Simister , Naı¨ma Belgareh-Touze´ , ‡2 ‡ ‡ Maria Conception Olivares-Sanchez , Jean-Christophe Zeeh , Sophie Chantalat , ‡ ‡ ‡ ‡3 Marie-Pierre Golinelli-Cohen , Catherine L. Jackson , Vale´rie Biou , and Jacqueline Cherfils From the Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and Institut Jacques Monod-CNRS, Universite´s Paris VI et VII, 75251 Paris, France Guanine nucleotide exchange factors carrying a Sec7 domain lated by guanine nucleotide exchange factors (ArfGEFs) carry- (ArfGEFs) activate the small GTP-binding protein Arf, a major ing a catalytic Sec7 domain (reviewed in Refs. 2 and 3). Evidence regulator of membrane remodeling and protein trafficking in is accumulating that ArfGEFs integrate upstream signals that eukaryotic cells. Only two of the seven subfamilies of ArfGEFs define the conditions of Arf activation. First, ArfGEFs localize (GBF and BIG) are found in all eukaryotes. In addition to the to specific trafficking organelles (4–9), which allows them to Sec7 domain, which catalyzes GDP/GTP exchange on Arf, the specify which subcellular site requires Arf activity. Second, GBF and BIG ArfGEFs have five common homology domains. binding partners involved in cell signaling, such as protein Very little is known about the functions of these noncatalytic kinase A, FK506-binding protein 13, and the AKAP-interacting domains, but it is likely that they serve to integrate upstream protein AMY-1, have been identified for the large Golgi-local- signals that define the conditions of Arf activation. Here we ized ArfGEFs (10–12). Finally, ArfGEFs may play a role in describe interactions between two conserved domains membrane recruitment of Arf effectors, such as coats, thus upstream of the Sec7 domain (DCB and HUS) that determine assembling downstream components of Arf signaling pathways the architecture of the N-terminal regions of the GBF and prior to Arf activation (5, 13). BIG ArfGEFs using a combination of biochemical, yeast two- An essential issue is to decipher how ArfGEFs implement hybrid, and cellular assays. Our data demonstrate a strong these functions and coordinate them with their biochemical interaction between DCB domains within GBF1, BIG1, and GDP/GTP exchange activity. To address this question, we BIG2 to maintain homodimers and an interaction between chose to focus on the large ArfGEFs, since (i) they are the only DCB and HUS domains within each homodimer. The DCB/ ArfGEFs found in all eukaryotes, and (ii) their multidomain HUS interaction is mediated by the HUS box, the most con- architecture may allow them to recapitulate the largest number served motif in large ArfGEFs after the Sec7 domain. In sup- of ArfGEF functions within a single polypeptide (14, 15). Large port of the in vitro data, we show that both the DCB and the ArfGEFs comprise two groups, which we refer to as the GBF HUS domains are necessary for GBF1 dimerization in mam- and BIG groups after their names in mammals. Both function in malian cells and that the DCB domain is essential for yeast maintaining organelle integrity and membrane traffic at the viability. We propose that the dimeric DCB-HUS structural Golgi or at endosomes (reviewed in Ref. 1). The best studied unit exists in all members of the GBF and BIG ArfGEF groups representatives are yeast Gea1p/Gea2p, Arabidopsis thaliana and in the related Mon2p family and probably serves an GNOM and mammalian GBF1 for the GBF group, and yeast important regulatory role in Arf activation. Sec7p and mammalian BIG1/BIG2 for the BIG group (reviewed in Refs. 1 and 2). We predicted earlier from a bioinformatics analysis that the GBF and BIG groups share a common archi- Small GTP-binding proteins of the Arf (ADP-ribosylation tecture, suggesting that both ArfGEF groups follow a common factor) family are major regulators of membrane traffic in the scenario for their activation of Arf (15). The predicted organi- exocytotic and endocytic pathways (reviewed in Ref. 1). They zation comprises two noncatalytic domains (dimerization and are activated by the exchange of GDP for GTP, which is stimu- cyclophilin-binding (DCB) and homology upstream of Sec7 (HUS)) in the N terminus of the Sec7 domain and three in its C terminus (HDS1, HDS2, and HDS3). The N-terminal DCB * This work was supported by a Human Frontiers in Science Program grant and a French Ministe`re de la Recherche ACI grant (to J. C.) and a CNRS ATIP domain (Fig. 1A) is the only domain to which a molecular func- grant (to V. B.). 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. The abbreviations used are: ArfGEF, guanine nucleotide exchange factor Supported by a grant from the CNRS. of Arf protein; GBF, Golgi-associated brefeldin A-resistant guanine Supported by a Marie-Curie grant from the European Community. nucleotide exchange factor; BIG, brefeldin A-inhibited guanine nucle- To whom correspondence should be addressed: Laboratoire d’Enzymologie otide exchange factor; DCB, dimerization/cyclophilin binding; HUS, et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur- homology upstream of Sec7; BD, binding domain; AD, activation Yvette Cedex, France. Tel.: 33-1-6982-3492; Fax: 33-1-6982-3129; E-mail: domain; GFP, green fluorescent protein; Tricine, N-[2-hydroxy- [email protected]. 1,1-bis(hydroxymethyl)ethyl]glycine. 28834 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 39 •SEPTEMBER 28, 2007 This is an Open Access article under the CC BY license. Interactions between BIG1, BIG2, and GBF1 Conserved Domains TABLE 1 tion has been assigned. It was originally identified in plant Liposome composition GNOM, where it was shown to be capable of dimerization in In all cases, 0.2% of the fluorescent lipid nitrobenzoxadizoldihexadecanoyl-phos- yeast two-hybrid and in vitro pull-down assays (16). Little is phatidylethanolamine was added as a tracer. PC, phosphatidylcholine; PG, phos- known about the other domains, except for an almost invariant phatidylglycerol; PIP2, phosphatidylinositol 4,5-bisphosphate. 5-residue motif in the HUS domain, the HUS box (15, 17) (Fig. 1B), which is essential for aspects of Golgi traffic in yeast (18). mol % mol % mol % mol % Here we combine biochemical, yeast two-hybrid, and cellular Soybean PC 95 65 92.5 62.5 Cholesterol 5 5 5 5 assays to analyze the domain architecture and interdomain Egg PG 0 30 0 30 interactions of the GBF and BIG groups of large ArfGEFs. Our Brain PIP2 0 0 2.5 2.5 data demonstrate the existence of two distinct interactions involving the DCB domain of the mammalian large ArfGEFs: Yeast Two-hybrid Assays—Plasmids expressing human homodimerization via a DCB/DCB interaction (generalizing BIG1 and BIG2 and Cricetulus griseus GBF1 are gifts from P. Melanc¸on (University of Alberta, Canada), the plasmid previous results from plants) and a novel DCB/HUS interaction expressing coxsackievirus 3A is a gift from F. van Kuppeveld depending on the HUS box. We propose that the DCB/DCB and DCB/HUS interactions define a common structure in all (Radboud University Nijmegen Medical Centre, The Neth- members of the BIG and GBF groups of ArfGEFs, which prob- erlands). All yeast two-hybrid constructs were cloned in the pAS and pACT2 vectors to create fusions with the Gal4- ably also exists in the related eukaryotic Mon2p family. DNA binding domain (BD) and Gal4 transcription activation EXPERIMENTAL PROCEDURES domain (AD), respectively. The Y190 (MATa, gal4-542, Expression of Recombinant DCB , DCB , and DCB- gal80-538, his3, trp1-901, ade2-101, ura3-52, leu2-3, 112, BIG1 BIG2 URA3::GAL1-LacZ, Lys2::GAL1-HIS3cyh ) and AH109 HUS-Sec7 —The DCB domain of human BIG1 (DCB , BIG1 BIG1 (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200 gal4, gal80, residues 2–224) was introduced into pET28a (Novagen) modi- fied to remove the thrombin cleavage site and to include alter- LYS2::GAL1 -GAL1 -HIS3, GAL2 -GAL2 -ADE2, UAS TATA UAS TATA native restriction sites (KpnI and AgeI). The E221K mutation URA3::MEL1 -MEL1 -lacZ) yeast strains were trans- UAS TATA formed with the different recombinant plasmids using the lith- was introduced into DCB by PCR using the QuikChange BIG1 ium acetate method (20). Y190 transformants autotrophic for site-directed mutagenesis kit (Stratagene). Both wild type and mutant DCB were expressed in the Rosetta(DE3)pLysS tryptophan and leucine were assayed for -galactosidase activ- BIG1 Escherichia coli strain (Merck KGaA). DCB was purified on ity using the filter technique (21). AH109 transformants were BIG1 tested for expression of HIS3 and ADE2 reporter genes. Stable aNi -nitrilotriacetic acid affinity column (GE Healthcare) fol- expression of each clone in pACT2 and pAS was confirmed by lowed by precipitation in ammonium sulfate to 70% saturation and gel filtration on a Superdex 75 column (GE Healthcare). Western blot using the Santa Cruz Biotechnology, Inc. (Santa The DCB domain of human BIG2 (DCB , residues 2–224) Cruz, CA) Gal4-AD and Gal4-BD monoclonal antibodies, BIG2 respectively. All experiments were performed at least three was cloned, expressed, and purified as described for DCB . BIG1 times. A construct spanning the DCB, HUS, and Sec7 domains of human BIG1 (DCB-HUS-Sec7 , residues 2–888) was intro- Biochemical Assays—Liposome binding experiments were BIG1 duced into pFastBac HTA vector (Invitrogen) using EcoRI and performed with liposomes prepared as described in Ref. 22 (Table 1). DCB (1 M) was incubated at room temperature KpnI restriction sites. Sf21 cells infected by baculoviruses har- BIG1 in 50 mM Hepes, pH 7.2, and 120 mM potassium acetate with boring this construct were used to express DCB-HUS-Sec7 . BIG1 The recombinant protein was purified on a Ni -nitrilotriace- sucrose-loaded vesicles (final lipid concentration, 1 mM)in tic acid affinity column followed by a desalting column and a gel small polycarbonate tubes. The samples were centrifuged at 360,000  g for 20 min, and the supernatants and the pellets filtration Superdex 200 column (GE Healthcare). Limited pro- were analyzed by SDS-PAGE with Sypro-orange staining. The teolysis was performed with 10 units of thrombin (Amersham Biosciences) per mg of protein at room temperature overnight. Sec7 domain of BIG1 was used as a negative control. Exchange DCB-HUS-Sec7 and its proteolysis products were analyzed reaction assays were performed by tryptophan fluorescence BIG1 kinetics using 17Arf1 as described in Ref. 23. The effect of by SDS-PAGE and Western blot. DCB on the exchange rate of Sec7 was analyzed by Biophysical Assays—Sedimentation velocity was measured at BIG1 BIG1 40,000  g for 24 h and analyzed with the SVEDBERG software comparing the results of experiments done in the absence or (available on the World Wide Web). Sedimentation equilib- presence of DCB (10 M). BIG1 Co-immunoprecipitation Assays—COS7 cells in 10-cm cul- rium experiments were carried out at 10,000, 15,000, and ture dishes were cotransfected with the plasmids pHA-GBF1 20,000  g for 46 h and analyzed with the Origin software (Beckman Coulter). CD scans were recorded between 185 and expressing human HA-GBF1 and either Venus-GBF1, Venus- 260 nm. Thermal denaturations were carried out in the temper- GBF1889, or YFP-GBF1-C expressing, respectively, human GBF1 and GBF1 deleted of the first 297 (DCB) and 710 amino ature range of 5–95 °C at a rate of 2 °C/min. Secondary struc- acids (DCB-HUS). After 20 h of expression, cells were ture composition was estimated with the CDDSTR software (19). The effect of protein concentration on its thermal dena- washed two times with 5 ml of cold phosphate-buffered saline turation was measured with 0.3, 0.75, and 3 M DCB and (137 mM NaCl, 2.7 mM KCl, 19 mM Na HPO , 1.8 mM KH PO ) BIG1 2 4 2 4 analyzed at 222 nm, a wavelength minimum that is character- istic of -helices. T. K. Niu and C. L. Jackson, unpublished data. SEPTEMBER 28, 2007• VOLUME 282 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 28835 Interactions between BIG1, BIG2, and GBF1 Conserved Domains and then disrupted in 0.5 ml of cold lysis buffer (50 mM Tris- domain of human BIGs qualifies as the bona fide homolog of the HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40). DCB domain of the GBF group member GNOM and refer to it After centrifugation at 4 °C, soluble cellular extracts were pre- as DCB hereafter. BIG cleared with 20 l of protein G-Sepharose 4 Fast Flow (GE In order to assess a possible dimer/monomer equilibrium, we Healthcare) at 4 °C for 30 min. Supernatants were incubated analyzed the CD thermal denaturation spectra of DCB (Fig. BIG1 with 3 g of anti-GFP antibodies (Roche Applied Science) for 2B). The model providing the best fit for the data was a two- 1.5 h at 4 °C. Then 30 l of protein G-Sepharose 4 Fast Flow was state transition between an all-helical structure and a random added, and the mixtures were incubated at 4 °C for 1.5 h. The coil denaturated state, without formation of a monomeric resin was washed two times with 1 ml of W100 buffer (50 mM intermediate. This and a denaturation temperature (69 °C) Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA), followed by two independent of the protein concentration and higher than the washes with 1 ml of phosphate-buffered saline. Proteins were average for proteins (around 55 °C) suggest that the dimer is then eluted by incubation with 60 l of SDS-PAGE sample stable with a dissociation constant below the concentration buffer for 5 min at 95 °C. Eluted proteins were separated on a 6% used in the experiment (100 nM). Analytical ultracentrifugation SDS-polyacrylamide gel and analyzed by Western immuno- confirmed the predominance of a dimeric species and the blotting using anti-HA antibodies as primary antibody (Sigma). absence of monomer. Plasmid Shuffle Assays in Yeast—The gea1-DCB construct To determine whether dimerization is a general feature of (coding for residues 233–1408 of Gea1p) was obtained by intro- DCB-like domains of the large ArfGEFs, we tested mammalian ducing a gap in the pAP22 (CEN, TRP1, GEA1) (gift of A. Pey- GBF1 and BIG2 DCB/DCB interactions using the yeast two- roche, Commissariat a` l’Energie Atomique, Saclay, France). hybrid system. We observed a strong interaction between DCB The PCR product extended to 1825 and 154 base pairs past the domains of hamster GBF1 (98% identity with human GBF1) 5 and 3 ends of the gap, respectively. The mutated fragment and between human BIG2 DCB domains, indicating that the and gapped plasmid were used to transform CJY52-10-2 yeast DCB domain mediates dimerization in all mammalian large cells that contained the pAP23 plasmid (CEN, URA3, GEA1) ArfGEFs (Fig. 3, sectors 1 and 12). We next looked for residues (24). Transformants were plated onto synthetic medium plates that contribute to the DCB/DCB interaction. Two highly con- 91 130 lacking tryptophan. Trp clones were grown at 30 °C in mini- served residues, Lys and Glu in DCB , are found in both GBF1 mal medium (YNB) containing 0.67% yeast nitrogen base with- large ArfGEF groups (Fig. 1A). Mutation of either residue to out amino acids (BD), supplemented with appropriate nutri- alanine in DCB abolished the interaction between mutant GBF1 ents and with 2% glucose. 5-Fluoroorotic acid monohydrate and wild-type DCB in the two-hybrid system (Fig. 3, sectors GBF1 (Toronto Research Chemicals) was added to a final concentra- 2 and 11). Thus, these residues are either part of the dimer tion of 0.1% to counterselect the URA3-containing cells. The interface or induce an abnormal structure in this interface. We presence of protein product of the gea1-DCB allele was con- then analyzed a mutation found in the C terminus of the DCB trolled by Western immunoblotting as follows. Total yeast cell domain of human BIG2 (Fig. 1A), which has been associated protein extracts were prepared using the NaOH/trichloroacetic with a congenital disease, autosomal recessive periventricular acid lysis technique (25). Proteins were separated by SDS- heterotopia with microencephaly (26). DCB carrying the BIG1 PAGE in 10% Tricine gels and were analyzed by immunoblot- equivalent mutation, E221K, was expressed in E. coli with a sol- ting. The primary antibody was either a polyclonal anti-Gea1p ubility similar to that of wild-type DCB . Analytical ultra- BIG1 E221K antibody or a monoclonal anti-Vat2p antibody directed against centrifugation experiments showed that DCB forms a BIG1 a subunit of the vacuolar ATPase (Invitrogen). dimer (data not shown). Thus, functions of DCB other BIG2 than dimerization are affected by this mutation in the auto- RESULTS somal recessive periventricular heterotopia with microen- Mammalian BIG and GBF ArfGEFs Have an N-terminal cephaly disorder. Dimerization DCB Domain—Using a bioinformatics approach, A Novel Interaction between the DCB and HUS Domains in we predicted previously that the DCB domain, originally iden- Large ArfGEFs—To determine whether interactions exist tified as a dimerization domain in the GBF group member between the different domains of the large ArfGEFs, we carried GNOM (16), is also present at the N terminus of large ArfGEFs out an extensive yeast two-hybrid analysis of domain-domain from the BIG group, where it should form an all-helical domain interactions for mammalian GBF1 and BIG1. All five noncata- (15). In order to address this question experimentally, we lytic domains in addition to the catalytic Sec7 domain were expressed in E. coli the N terminus of human BIG1, encompass- considered. For GBF1 (Table 2A) and BIG1 (Table 2B), only one ing the helical subdomain of highest sequence homology and interaction was detected in addition to the DCB/DCB interac- the more variable N-terminal subdomain (Fig. 1A), and purified tion described above. In both cases, a strong interaction it to homogeneity. The recombinant protein behaved as a 2  between the DCB and HUS domains of each ArfGEF was 26-kDa dimer (Fig. 2A), a molecular mass that was confirmed observed (Fig. 3, sectors 3 and 4). This interaction was inde- by analytical ultracentrifugation equilibrium sedimentation pendent of the variable DCB-HUS linker added on either the (54.3 kDa). A similar construct from human BIG2 was also DCB or HUS side (Table 2A). A strong interaction was also expressed and purified to homogeneity and, as for BIG1, eluted found between the DCB and HUS domains of human BIG2 (Fig. as a dimer on a gel filtration column (Fig. 2A). Deconvolution of 3, sector 5). CD spectra for both proteins was consistent with a mostly hel- The HUS box is an almost invariant N(Y/F)DC(D/N) motif, ical secondary structure. We thus conclude that the N-terminal which is predicted to lie between two -helices (Fig. 1B). Muta- 28836 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 39 •SEPTEMBER 28, 2007 Interactions between BIG1, BIG2, and GBF1 Conserved Domains FIGURE 1. Sequence alignments of DCB domains and HUS boxes. A, sequence alignment of DCB domains of the BIG, GBF, and Mon2p/Ysl1p/SF21 families. Predicted -helices are represented by helices. The black arrows indicate the conserved residues mutated in C. griseus GBF1. The gray arrow indicates the BIG2 glutamate mutated in autosomal recessive periventricular heterotopia with microencephaly and analyzed in this paper. B, sequence alignment of the HUS box (framed) in the HUS domain. The mutation analyzed in this study is shown by an arrow. C, sequence alignment of the putative HUS box (framed)inthe eukaryotic Mon2p/Ysl1p/SF21 family. The residue shown by an arrow is equivalent to that in B. tion of the central aspartate to alanine in this motif abolished involved only in the DCB/DCB interaction, whereas the Glu the DCB/HUS interaction in both GBF1 and BIG1 (Fig. 3, sec- residue is involved in both interactions. tors 18 and 19). Thus, the HUS box supports the DCB/HUS Next, we used the yeast two-hybrid system to analyze DCB/ interaction, which is the first molecular function to be associ- DCB and DCB/HUS interactions within GBF1 constructs span- ated with this motif. We then analyzed whether mutations that ning more than one domain. We first analyzed the ability of the impair the DCB/DCB interaction (see above) also affect the DCB domain alone to interact with larger ArfGEF fragments. DCB/HUS interaction. The E130A mutation in GBF1, but not DCB interacted with DCB-HUS , DCB-HUS-Sec7 , GBF1 GBF1 GBF1 the K91A mutation, abolished the DCB/HUS interaction (Fig. and full-length GBF1 (Fig. 3, sectors 7, 8, and 10). Although 3, sectors 16 and 17). These results indicate that residue Lys is these yeast two-hybrid interactions appeared to be weaker than SEPTEMBER 28, 2007• VOLUME 282 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 28837 Interactions between BIG1, BIG2, and GBF1 Conserved Domains FIGURE 3. Yeast two-hybrid analysis of DCB and HUS interactions in mammalian ArfGEFs. AH109 yeast cells were co-transformed with the expression plasmids for bait (BD) and prey (AD) fusion proteins and selected on double synthetic dropout (Trp/Leu) medium plates. The transformants were then selected for the expression of HIS3 and ADE2 genes by growing on synthetic dropout medium (Trp/Leu/His/Ade). K91A 1, DCB /DCB ; 2, DCB /DCB ; 3, DCB /HUS ; 4, DCB / GBF1 GBF1 GBF1 GBF1 GBF1 GBF1 BIG1 HUS ; 5, DCB /HUS ; 6, DCB /HUS-Sec7 ; 7, DCB /DCB- BIG1 BIG2 BIG2 GBF1 GBF1 GBF1 HUS ; 8, DCB /DCB-HUS-Sec7 ; 9, DCB /Sec7 ; 10, DCB / GBF1 GBF1 GBF1 GBF1 GBF1 GBF1 E130A GBF1; 11, DCB -DCB ; 12, DCB /DCB ; 13, GBF1/DCB-HUS- GBF1 GBF1 BIG2 BIG2 Sec7 ; 14, HUS /DCB-HUS-Sec7 ; 15, HUS /GBF1; 16, HUS / GBF1 GBF1 GBF1 GBF1 GBF1 K91A E130A D540A D570A DCB ; 17, HUS /DCB ; 18, HUS /DCB ; 19, HUS /DCB ; GBF1 GBF1 GBF1 GBF1 GBF1 BIG1 BIG1 20, HUS /Sec7 ; 21, DCB-HUS /3A; 22, HUS /3A; 23, DCB /3A. GBF1 GBF1 GBF1 GBF1 GBF1 TABLE 2 Summary of domain/domain interactions in mammalian ArfGEFs analyzed with the yeast two-hybrid assay Strong interactions are indicated in dark gray, and weaker interactions are shown in gray. Baits (BD) and preys (AD) are indicated in rows and columns, respectively. FIGURE 2. Biochemical characterization of recombinant proteins. A, elu- tion profiles of purified DCB , DCB , and DCB-HUS-Sec7 on a gel fil- BIG1 BIG2 BIG1 tration column. The elution volumes of calibration proteins are indicated. B, circular dichroism spectra of DCB (6 M) as a function of temperature. BIG1 CD spectra are taken every 5 °C. The isodichroic point lies at 200.5 nm (thin arrow), showing that unfolding occurs as a two-state helix-coil transition. the interaction between two DCB domains alone, they strongly suggest that the DCB/DCB interaction occurs also in the con- text of the full-length GBF1 protein. We then analyzed the DCB/HUS interaction in the context of multiple domains. A strong interaction was found between the DCB domain and a HUS-Sec7 construct (Fig. 3, sector 6). HUS also interacted GBF1 with DCB-HUS-Sec7 and full-length GBF1 although more GBF1 weakly than with DCB alone (Fig. 3, sectors 14 and 15). In GBF1 support for the dimerization of the N-terminal region taking place in larger constructs, recombinant DCB-HUS-Sec7 BIG1 eluted on a gel filtration column with a molecular weight con- sistent with a dimer (Fig. 2A). DCB or DCB/HUS cross-interactions between BIG1, BIG2, and No interactions between the noncatalytic domains other GBF1 in the yeast two-hybrid system (data not shown). than the DCB/DCB and DCB/HUS interactions were identified Functions of DCB/DCB and DCB/HUS Interactions in Arf- with the yeast two-hybrid assay. We also failed to detect an GEF Dimers—The DCB domain has features of a dimeric interaction of the noncatalytic domains with the Arf1 substrate helical bundle, which is a frequent arrangement in cytosolic (data not shown). In addition, we did not observe any DCB/ proteins involved in membrane recruitment, such as the mem- 28838 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 39 •SEPTEMBER 28, 2007 Interactions between BIG1, BIG2, and GBF1 Conserved Domains brane curvature-sensing BAR domain found in amphiphysins and Arfaptin/POR, an Arf effector (27). We thus investigated the binding of DCB to liposomes of various compositions BIG1 using a sedimentation assay (Table 1 and Fig. 4A). However, no such interaction could be observed regardless of the liposome composition, suggesting that the DCB homodimer does not have membrane-binding properties on its own. The Sec7 domain did not interact with the constructs tested in our yeast two-hybrid analysis (Table 2, A and B, and Fig. 3, sectors 9 and 20). To further analyze whether the N terminus could regulate the catalytic exchange activity of the Sec7 domain, we used recombinant proteins and a fluorescence kinetics assay. We first analyzed the effect of excess DCB on BIG1 the exchange rate of the Sec7 domain of human BIG1 (Sec7 ) using 17Arf1 as substrate. No inhibition or stimu- BIG1 lation of the exchange rate by DCB could be observed, BIG1 regardless of whether 17Arf1, Sec7 , or both had been pre- BIG1 incubated with DCB (Fig. 4B). We then analyzed the cata- BIG1 lytic activity of a BIG1 construct spanning the DCB-HUS-Sec7 domains using the same fluorescence assay. This construct was active at stimulating GDP/GTP exchange on17Arf1 (Fig. 4C), and it was inhibited by brefeldin A with a K of 23.9  7.2 M, which is similar to that measured for the Sec7 of BIG1 alone (23). To confirm that the DCB-HUS tandem had no effect on the catalytic activity, we took advantage of a unique thrombin cleavage site located at residue 622 between the HUS and Sec7 domains, which allowed us to generate free DCB-HUS and BIG1 Sec7 by limited proteolysis. Exchange rates measured with BIG1 a BIG1 peptide concentration of 0.5 M were in the same range for the uncleaved and cleaved fragments (0.073  0.005 and 0.098  0.012 s , respectively), suggesting that the DCB-HUS tandem does not have a simple one-to-one regulatory activity toward the Sec7 domain. The N terminus of large ArfGEFs interacts with several large ArfGEF protein partners (reviewed in Ref. 1). We thus investi- gated whether the DCB/HUS structure may be required for protein-protein interactions. To this end, we took advantage of the fact that the N terminus of GBF1 binds to 3A, a protein from enteroviruses that blocks host cell secretion by inhibiting GBF1 function (28). The cytosolic portion of 3A (residues 1–60) interacts with DCB-HUS and DCB-HUS-SEC7 in the GBF1 GBF1 yeast two-hybrid assay (Fig. 3, sector 21). In contrast, no inter- action was observed with individual DCB or HUS domains (Fig. 3, sectors 22 and 23). This is consistent with data showing that deletion of either the first 50 amino acids of GBF1 or deletion of FIGURE 4. Function of the DCB/DCB and DCB/HUS interactions. A, sedi- mentation analysis of DCB binding properties to liposomes. The lanes cor- the HUS domain and downstream sequences abolishes interac- BIG1 respond (in this order) to molecular weight markers, supernatant (S), and tion with the 3A protein in the mammalian two-hybrid system pellet (P) fractions of experiments with no liposomes (No lip) and liposomes (29). These results show that portions of both the DCB and 1– 4 (Table 1). The last lane represents the total amount of protein in the experiment (T). B, effect of DCB on the kinetics of Sec7 -stimulated BIG1 BIG1 HUS domains of GBF1 are required for binding to the viral 3A GDP/GTP exchange on 17Arf1 measured by tryptophan fluorescence. 1,no protein and suggest the possibility that an integral DCB-HUS DCB; 2, DCB was incubated with Sec7 domain for 5 min before 17Arf1- BIG1 GDP was added; 3, DCB was incubated with Arf1 for 5 min before the Sec7 structure is necessary for binding of the 3A protein. BIG1 domain was added. In all cases, GTP (100 M) was added 2 min after all pro- Dimerization of Large ArfGEFs in Vivo—The above analy- teins were mixed together. C, GDP/GTP exchange activity of DCB-HUS- sis suggests that the DCB domain supports the dimerization Sec7 on 17Arf1 measured at different GEF concentrations. BIG1 of large ArfGEFs and organizes a structure that can bind protein partners. We thus assessed the dimerization and We first analyzed the formation of human GBF1 dimers in function of this domain in cells for two large ArfGEFs of the mammalian cells by pull-down assays (Fig. 5A). We found out GBF group. that full-length GBF1 can easily be isolated as a dimer from SEPTEMBER 28, 2007• VOLUME 282 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 28839 Interactions between BIG1, BIG2, and GBF1 Conserved Domains FIGURE 5. In vivo assays. A, the DCB and HUS domains of human GBF1 are both involved in its dimerization in mammalian cells. HA-tagged GBF1 was coexpressed with either GFP, GFP-tagged GBF1, GFP-tagged GBF1DCB, or GFP-tagged GBF1(DCB-HUS). After incubation with anti-GFP antibodies fol- FIGURE 6. Models for the DCB/DCB and DCB/HUS interactions. A, dimer- lowed by an incubation with protein G-Sepharose, the resin was washed sev- ization by combined DCB/DCB and DCB/HUS intermolecular interactions. eral times, and proteins were eluted by incubation with SDS-PAGE sample B, DCB/DCB dimerization with intramolecular DCB/HUS interaction. C, a pos- buffer. Eluted proteins were separated on a 6% SDS-polyacrylamide gel and sible regulatory switch of the HUS box from domain/domain interaction to analyzed by Western blotting using anti-HA antibodies. B, the DCB domain of solvent exposure and/or alternative interactions. D, tetramer formation by Gea1p is essential for yeast inability. Left, yeast extracts from gea1gea2 three-dimensional domain swapping. cells bearing a URA3 plasmid containing the GEA1 wild type gene (plasmid pAP23, lane 1), pAP23 and a TRP plasmid containing the GEA1 wild-type gene (pAP22, lane 2), pAP23 and pAP22 containing the gea1-DCB1 gene (lane 3), or yeast extracts from a wild-type strain (lane 4) were separated on SDS-poly- domains, in which the DCB domain interacts with itself and acrylamide gels and analyzed by western immunoblotting using an anti- with the HUS domain. The DCB/HUS interaction requires the Gea1p antibody. An anti-Vat2p antibody was used as a loading control. Right, highly conserved HUS box, a five-amino acid motif found in all yeast cells deleted for the GEA1 and GEA2 genes and bearing a URA3 plasmid containing the GEA1 wild-type gene were transformed with a plasmid con- members of the BIG and GBF groups of ArfGEFs. taining either the wild type GEA1 gene (gea2 GEA1 cells) or the GEA1 gene Because of its bipartite organization, the DCB-HUS tandem deleted for the DCB domain (gea2 gea1-DCB cells). 10-Fold serial dilutions provides different ways for large ArfGEFs to form multimers. of yeast cultures were plated on media with or without 5-fluoroorotic acid monohydrate (5FOA) to counterselect the URA3-containing cells. One is through the DCB/DCB interaction, which is an obligate intermolecular interaction. Since the DCB domain forms a mammalian cells. Next, we examined dimer formation between strong homodimer in vitro, we propose that it supports consti- the full-length GBF1 and forms of GBF1 deleted of the DCB tutive homodimerization of large ArfGEFs. The existence of this interaction in native BIG and GBF ArfGEFs is supported by domain alone or of both the DCB and HUS domains. Clearly, its formation in a range of yeast two-hybrid GBF1 constructs, whereas deletion of the DCB domain alone reduced somewhat the formation of a dimer with full-length GBF1, both the DCB the dimerization of the recombinant BIG1 and BIG2 con- and HUS domains had to be deleted to nearly abolish dimer structs, and our in vivo data on GBF1. It is also consistent with the molecular weight of several large ArfGEFs of both the BIG formation. Thus, the DCB and the HUS domains are both and GBF groups as measured by size exclusion chromatogra- involved in the dimerization of GBF1. We then analyzed the effect of deleting the DCB domain of phy, including yeast Gea1p (30), human BIG1 and BIG2 (31, Gea1p, a member of the GBF group of large ArfGEFs in yeast, 32), and plant GNOM (16). All elute as large molecular weight complexes, which, given the uncertainty of this technique for using a plasmid shuffle strategy. The strain used contains the nonglobular proteins, is consistent with their association as wild-type GEA1 gene on a URA3 plasmid with both gea1 and gea2 deletions of the chromosomal copies of the genes (24). homodimers. The gea1-DCB allele was introduced into a low copy TRP In contrast, the DCB/HUS interaction can occur either between two monomers (intermolecular) (Fig. 6A) or within plasmid. The Gea1p-DCB protein was expressed and was not a single ArfGEF polypeptide (intramolecular) (Fig. 6B). An degraded (Fig. 5B, left). Clones failed to grow at 30 °C upon the loss of the wild-type GEA1 plasmid when the gea1-DCB plas- intermolecular DCB/HUS interaction would provide a sec- mid became the sole copy of the redundant GEA1 and GEA2 ond contribution to dimerization in addition to the DCB/ DCB interaction. This possibility is supported by our co- genes (Fig. 5B, right). This result indicates that the DCB domain immunoprecipitation results, which show that the DCB of Gea1p is essential for yeast viability. form of human GBF1 formed a dimer with full-length GBF1 DISCUSSION almost as efficiently as full-length GBF1 in mammalian cells, A Conserved DCB/DCB and DCB/HUS Structure in Eukary- whereas deletion of both DCB and HUS domains practically otic Large ArfGEFs and the Related Mon2p Family—In this eliminated dimerization with full-length GBF1. Interest- study, we investigated the domain/domain interactions within ingly, the HUS box has an unusual level of sequence conser- the BIG and GBF groups of large ArfGEFs, which we predicted vation and content of polar residues within a protein inter- previously to share a common architecture (15). Based on bio- face, pointing to a potential for the DCB/HUS interaction to chemical and yeast two-hybrid analyses of mammalian BIG1, open up and expose the HUS box (Fig. 6C). The HUS box BIG2, and GBF1, we establish that all three members share a could then carry out other functions, allowing in particular similar DCB-HUS organization upstream of their Sec7 the formation of ArfGEF tetramers through three-dimen- 28840 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 39 •SEPTEMBER 28, 2007 Interactions between BIG1, BIG2, and GBF1 Conserved Domains Gif-sur-Yvette, France) for performing the analytical ultracentrifuga- sional domain swapping (Fig. 6D). An interesting corollary is tion experiments; Simona Burlacu-Miron and Gil Craescu (CNRS/ that this could allow large ArfGEFs to form heterotetramers, Institut Curie, Orsay, France) for help with circular dichroism; Bruno which are more likely to form than heterodimers, given the Antonny (Institut de Pharmacologie Mole´culaire et Cellulaire, CNRS, stability of the homodimeric DCB/DCB interaction. BIG1 Valbonne, France) for help with the liposome assay; Sylvie Lazareg and BIG2 have been shown to co-immunoprecipitate in and Jean-Pierre le Caer (Institut de Chimie des Substances Naturel- human cells (32), which could thus be mediated by the for- les, CNRS, Gif-sur-Yvette, France) for performing mass spectrometry mation of heterotetramers containing one BIG1 homodimer measurements; and Rosine Haguenauer-Tsapis (Institut Jacques and one BIG2 homodimer. Monod-CNRS, Paris, France) for support. 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Biol. Chem. 281, 11805–11814 ble of alternative interactions. The DCB/HUS structure char- 24. Peyroche, A., Courbeyrette, R., Rambourg, A., and Jackson, C. L. (2001) acterized here should provide a rational framework to address J. Cell Sci. 114, 2241–2253 this issue in the BIG and GBF groups of ArfGEFS. 25. Volland, C., Urban-Grimal, D., Geraud, G., and Haguenauer-Tsapis, R. (1994) J. Biol. Chem. 269, 9833–9841 26. Sheen, V. L., Ganesh, V. S., Topcu, M., Sebire, G., Bodell, A., Hill, R. S., Acknowledgments—We thank Barbara Mouratou (Laboratoire Grant, P. E., Shugart, Y. Y., Imitola, J., Khoury, S. J., Guerrini, R., and d’Enzymologie et Biochimie Structurales (LEBS), CNRS, Gif-sur- Walsh, C. A. (2004) Nat. Genet. 36, 69–76 Yvette, France) for initiating the yeast two-hybrid analysis and Julie 27. Peter, B. J., Kent, H. M., Mills, I. G., Vallis, Y., Butler, P. J., Evans, P. R., and Leroux (LEBS, CNRS, Gif-sur-Yvette, France) for technical assistance McMahon, H. T. (2004) Science 303, 495–499 with the yeast two-hybrid assays; Fatima el Khadali (LEBS, CNRS, 28. Wessels, E., Duijsings, D., Niu, T. K., Neumann, S., Oorschot, V. M., de SEPTEMBER 28, 2007• VOLUME 282 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 28841 Interactions between BIG1, BIG2, and GBF1 Conserved Domains Lange, F., Lanke, K. H., Klumperman, J., Henke, A., Jackson, C. L., 33. Jochum, A., Jackson, D., Schwarz, H., Pipkorn, R., and Singer-Kruger, B. Melchers, W. J., and van Kuppeveld, F. J. (2006) Dev. Cell 11, 191–201 (2002) Mol. Cell Biol. 22, 4914–4928 29. Wessels, E., Duijsings, D., Lanke, K. H., Melchers, W. J., Jackson, C. L., and 34. Efe, J. A., Plattner, F., Hulo, N., Kressler, D., Emr, S. D., and Deloche, O. van Kuppeveld, F. J. (2007) J. Virol. 81, 5238–5245 (2005) J. Cell Sci. 118, 4751–4764 30. Peyroche, A., Paris, S., and Jackson, C. L. (1996) Nature 384, 479–481 35. Gillingham, A. K., Whyte, J. R., Panic, B., and Munro, S. (2006) J. Biol. 31. Morinaga, N., Tsai, S. C., Moss, J., and Vaughan, M. (1996) Proc. Natl. Chem. 281, 2273–2280 Acad. Sci. U. S. A. 93, 12856–12860 36. Monetta, P., Slavin, I., Romero, N., and Alvarez, C. (2007) Mol. Biol. Cell 32. Yamaji, R., Adamik, R., Takeda, K., Togawa, A., Pacheco-Rodriguez, G., 18, 2400–2410 Ferrans, V. J., Moss, J., and Vaughan, M. (2000) Proc. Natl. Acad. Sci. 37. Xu, K. F., Shen, X., Li, H., Pacheco-Rodriguez, G., Moss, J., and Vaughan, U. S. A. 97, 2567–2572 M. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 2784–2789 28842 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 39 •SEPTEMBER 28, 2007

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Sep 28, 2007

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