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Biochemical and Biophysical Research Communications 357 (2007) 661–667

Characterization of Rab45/RASEF containing EF-hand domainand a coiled-coil motif as a self-associating GTPase

Mami Shintani, Minoru Tada, Tetsuo Kobayashi, Hiroaki Kajiho,Kenji Kontani *, Toshiaki Katada

Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received 29 March 2007Available online 13 April 2007

Abstract

Rab-family GTPases function as key regulators for membrane traffic. Among them, Rab45/RASEF is an atypical GTPase in that itcontains a coiled-coil motif at the mid region and a distinct N-terminal EF-hand domain with C-terminal Rab-homology domain. Here,we provide the initial biochemical characterization and intracellular localization of human Rab45. Rab45 bound guanine nucleotide tri-and di-phosphates through the C-terminal Rab domain. Rab45 was capable of self-interacting, and the self-interaction required the midregion containing the coiled-coil motif. Rab45 expressed in HeLa cells was localized in a small patch in the perinuclear area of the cell,and the localization was regulated by the guanine nucleotide-bound states of Rab45. Interestingly, the mid region, together with Rabdomain, appeared to be essential for the characteristic perinuclear localization of Rab45, indicating that the self-interaction may beinvolved in the intracellular localization of Rab45.� 2007 Elsevier Inc. All rights reserved.

Keywords: Membrane traffic; Rab45; RASEF; Rab; Ras; Small GTPase; EF-hand; Coiled-coil; Oligomerization

Rab-family GTPases play pivotal roles in the regulationof membrane traffic [1,2]. Given that the presence of manydifferent intracellular compartments requires a high orderregulation of membrane traffic to ensure delivery andfusion of transport vesicles at the appropriate site, it isnot surprising that over 60 mammalian Rab proteins haveso far been identified. Some of Rab proteins have beenextensively analyzed and their roles in membrane trafficare relatively well understood; however, there are still manyRab proteins, of which exact functions remain to be eluci-dated. Characterization and functional analysis of theseRab proteins, together with identification of their regula-tors and effectors, would help to unravel a complex net-work of membrane traffic.

In this study, we investigated the biochemical propertiesand subcellular localization of a novel Rab-family protein,

0006-291X/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2007.03.206

* Corresponding author. Fax: +81 3 5841 4751.E-mail address: kontani@mol.f.u-tokyo.ac.jp (K. Kontani).

Rab45/RASEF. It has been reported that the expression ofRab45 is decreased in cutaneous malignant melanoma [3],however, the physiological role(s) of Rab45 is unknown.Rab45 has a unique structure consisting of a N-terminalEF-hand domain, a mid region containing a coiled-coilmotif, and a C-terminal Rab-homology domain (Rabdomain). We found that the Rab domain of Rab45 exhibitsguanine-nucleotide binding ability and that Rab45 is capa-ble of self-interacting through the mid region. Further-more, we found that Rab45 expressed in HeLa cells islocalized to a small patch in the perinuclear area of the celland that the mid region of Rab45 besides Rab domain isresponsible for the intracellular localization of Rab45.We discuss a possible role of the self-associating Rab45in membrane traffic.

Materials and methods

Cell culture and fluorescence microscopy. HeLa and HEK293T cellswere maintained as described previously [4] and transfected with plasmid

662 M. Shintani et al. / Biochemical and Biophysical Research Communications 357 (2007) 661–667

constructs using LipofectAMINE 2000 (Invitrogen). For fluorescencemicroscopy, the cells grown on a glass base dish were viewed with a CarlZeiss LSM-510 confocal microscope 20–24 h after transfection. SYTO59(Molecular Probes) was used to label cell nuclei.

Plasmid construction. A plasmid DNA containing human Rab45cDNA (GenBank Accession No. BC023566) was obtained from ATCC(cat No. 7515966). cDNAs of Rab45 mutants were made by PCR andsubcloned in modified pCMV5 plasmids (pFLAG-CMV5 and pMyc-CMV5), which place the FLAG- and Myc-tag at its amino terminus,respectively. For yeast two-hybrid assay, a cDNA fragment of Rab45corresponding to the amino acid sequence 98–533 was subcloned inpGBT9 as a bait plasmid, and cDNA fragments corresponding to theamino acid sequence 1–740, 98–533, 527–740, and 1–97 were subcloned inpGAD424 as prey plasmids. pEGFP-C2 (Clontech) and pDsRed-Mono-mer (Clontech) were used for expression of GFP and DsRed-Monomerfusion proteins, respectively. A cDNA fragment of the Rab domain ofRab45 (the amino acid sequence 527–740) was subcloned in pGEX-6P-1 toprepare Escherichia coli recombinant proteins.

Expression and purification of recombinant Rab45 proteins. HEK293Tcells cultured in a 100-mm dish for 2 days after transfection with 5 lg ofpFLAG-CMV5-Rab45 plasmids were lysed with 1 ml of an ice-cold buffer(40 mM Tris–HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1%(w/v) CHAPS, and 0.5 mM Pefabloc SC). After centrifugation, thesupernatants were precleared with Sepharose 4B resin, followed byimmunoprecipitation with Anti-FLAG M2 Affinity Gel (Sigma). Afterwashing the resin with an ice-cold wash buffer (40 mM Tris–HCl (pH 7.5),150 mM NaCl, 5 mM MgCl2, and 1 mM DTT, 0.7% (w/v) CHAPS),FLAG-tagged Rab45 was eluted from the resin with the wash buffercontaining 100 lg/ml of FLAG-peptide. Purification of GST-fusion pro-teins was performed as described previously [4]. The Rab domain ofRab45 was prepared using PreScission protease (GE Healthcare) as sug-gested by the manufacturer.

Nucleotide binding assays. [35S]GTPcS binding assay was performed asdescribed previously [4]. Purified proteins (0.7 pmol) were incubated at30 �C in a total volume of 40 ll of reaction mixture consisting of 40 mMTris–HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.2% (w/v) CHAPS, and 2.5 lM [35S]GTPcS (0.17 KBq/pmol). After incubationfor the indicated times, samples were diluted with 1 ml of an ice-cold washbuffer (20 mM Tris–HCl (pH 7.5), 20 mM MgCl2, and 100 mM NaCl) andfiltered through a nitrocellulose membrane (0.45-lm pore size, ADVAN-TEC). After extensive washing with the ice-cold wash buffer, radioactivityretained on the membrane was determined by a liquid scintillationcounter.

In vivo phosphate labeling and immunoprecipitation. Guanine nucleo-tides bound to the GTP-binding proteins were analyzed essentially asdescribed previously [4]. Briefly, transfected HeLa cells (�3 · 106 cells)were labeled for 4 h with 32Pi (1.85 MBq/dish) in phosphate-free DMEMand lysed in 1 ml of an ice-cold solubilizing buffer (40 mM Tris–HCl (pH7.5), 100 mM NaCl, 20 mM MgCl2, 1 mM DTT, 1% (w/v) Triton X-100,2 lg/ml aprotinin, and 0.5 mM Pefabloc SC). The precleared cell lysateswere incubated with the anti-FLAG M2 monoclonal antibody (2 lg) andprotein G–Sepharose. After extensive washing of the immunocomplexes,associated nucleotides were separated by thin-layer chromatography andanalyzed with a BAS-1800 image analyzer (Fuji Film).

Co-immunoprecipitation assay. HeLa cells cultured in a 60-mm dish for36 h after co-transfection with the pMyc- and pFLAG-CMV5-derivedplasmids were lysed with 350 ll of an ice-cold extraction buffer (40 mMTris–HCl, (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% (w/v)Triton X-100, and 0.5 mM Pefabloc SC). The precleared cell lysates wereincubated with anti-Myc polyclonal antibody (1 lg) and 20 ll of ProteinA–Sepharose. After washing the resin with a wash buffer (40 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 0.2% (w/v)Triton X-100), the immunocomplexes were solubilized with 40 ll of SDS–PAGE sample buffer and subjected to SDS–PAGE, followed by immu-noblotting with anti-FLAG M2 or anti-Myc 9E10 monoclonal antibody.

Yeast two-hybrid analysis. Yeast strain Y190 was co-transformed withpGBT9- and pGAD424-derived plasmids containing Rab45 cDNA frag-ments. Transformants were grown on a synthetic medium lacking leucine

and tryptophan and subjected to quantitative b-galactosidase assays using4-methylumbelliferyl-b-D-galactoside as a substrate [5].

Results

Biochemical characterization of Rab45 as a guanine-

nucleotide binding protein

To examine the biochemical properties of Rab45, weexpressed it in HEK293T cells as a FLAG-tagged proteinand purified it using Anti-FLAG M2 Affinity Gel.Fig. 1A shows that the purified Rab45 bound GTPcS ina time-dependent manner, with maximum binding occur-ring at about 20 min. The GTPcS-binding to Rab45 wasselectively competed by GTP or GDP, but not by ATP(not shown), indicating that Rab45 specifically binds guan-ine nucleoside tri- and di-phosphates. Besides the C-termi-nal Rab domain, Rab45 has an additional N-terminalregion composed of an EF-hand domain and a coiled-coilmotif. We thus investigated whether other regions besidesRab domain are critical for guanine-nucleotide binding.Fig. 1B shows that the purified Rab domain protein boundGTPcS in a similar time-dependent manner to thatobserved for FLAG-Rab45, demonstrating that Rabdomain is responsible for the guanine-nucleotide bindingproperties of Rab45.

We next examined the nucleotide forms associated withRab45 in living cells. To analyze the nucleotide forms ofRab45 in living cells, FLAG-Rab45 proteins expressedin HeLa cells were immunoprecipitated after in vivo

phosphate labeling of the cells. The guanine nucleotidesassociated with the immunocomplex were then analyzedby thin-layer chromatography. We found that wild-typeRab45 predominantly existed as a GDP-bound form asshown in Fig. 1C (WT in upper panel). Rab45/Q600Land Rab45/S555N are mutants with substitution of Glnto Leu at position 600 and Ser to Asn at position 555located in Rab domain, respectively. Fig. 1C shows thatRab45/Q600L was predominantly a GTP-bound form,while most of Rab45/S555N existed as a nucleotide-freeform, as expected from the previous studies of the equiva-lent mutants in other small GTPases [6]. Fig. 1D showsthat the Rab domain of Rab45 bound guanine nucleotidesand the effects of point mutations on the states of guanine-nucleotide binding were the same as that observed for full-length Rab45. These results suggest that the EF-handdomain and the mid region of Rab45 are not essentialfor guanine-nucleotide binding to Rab45, though we can-not rule out the possibility that the N-terminal regionbesides Rab domain is involved in the stoichiometry ofguanine-nucleotide binding to Rab45.

Self-interaction of Rab45 through a mid region containing

coiled-coil motif

In the domain architecture analysis of Rab45 usingSMART database (http://smart.embl-heidelberg.de/), we

GTP

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Fig. 1. Characterization of Rab45 as a guanine-nucleotide binding protein. (A,B) GTPcS-binding to the purified Rab45 proteins. The purified proteinswere incubated with 2.5 lM [35S]GTPcS for the indicated times, and [35S]GTPcS bound to the proteins was determined as described under Materials andmethods. Each value represents the means ± SD of three independent experiments. The insets in A and B show SDS–PAGE analysis of the purifiedFLAG-Rab45 and Rab domain proteins, respectively (Coomassie brilliant blue stain). (C,D) Analysis of the guanine nucleotides associated with Rab45and Rab domain proteins in living cells. HeLa cells expressing FLAG-tagged proteins listed at the top were metabolically labeled with 32Pi, and the FLAG-tagged proteins were immunoprecipitated using an anti-FLAG antibody. Guanine nucleotides associated with the FLAG-tagged proteins were separatedby thin-layer chromatography (upper panel). Expression of the transfected constructs was confirmed by Western blotting with an anti-FLAG antibody(bottom panel).

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found that Rab45 has a coiled-coil motif extending fromresidues 170 to 362. Coiled-coil is a structural motif thatmediates oligomerization of proteins, and a number ofcoiled-coil proteins have been shown to be engaged inmembrane traffic [7,8]. Thus, we sought to determinewhether Rab45 is capable of self-interacting. HeLa cellswere co-transfected with FLAG-Rab45 and Myc-Rab45expression vectors, and the cell lysates were subjected toimmunoprecipitation using an anti-Myc antibody followedby Western blotting of the immunocomplexes with an anti-FLAG antibody. Fig. 2B shows that FLAG-Rab45 wasdetected in the immunocomplexes containing Myc-Rab45/WT (WT in Fig. 2B), indicating that FLAG-Rab45 can interact with Myc-Rab45 either directly orindirectly. The interaction between FLAG-Rab45 andMyc-Rab45 (Rab45 self-interaction) was independent onthe guanine-nucleotide form of Rab45: FLAG-Rab45was equivalently co-immunoprecipitated with Myc-Rab45/Q600L or Myc-Rab45/S555N (Q600L and S555Nin Fig. 2B). To investigate which regions of Rab45 are

responsible for the Rab45 self-interaction, a series of dele-tion mutants of Myc-Rab45 was co-expressed with FLAG-Rab45. Fig. 2A illustrates a schematic representation of theRab45 mutants used in this study. Fig. 2B shows FLAG-Rab45 was co-immunoprecipitated with a deletion mutantlacking EF-hand domain or Rab domain (DEF and DRabin Fig. 2B). In contrast, FLAG-Rab45 was no longer co-immunoprecipitated with a deletion mutant lacking themid region (DMid in Fig. 2B), indicating that the midregion is critical for the Rab45-interaction. Indeed,FLAG-Rab45 was co-immunoprecipitated with the Myc-tagged mid region (Mid in Fig. 2B). It has been shown thatsome Rab proteins can interact with themselves [9,10],however, the Myc-tagged Rab domain of Rab45 did notinteract with FLAG-Rab45 in the co-immunoprecipitationanalysis (Rab in Fig. 2B).

We next investigate whether the mid region of Rab45can directly interact with itself using yeast two-hybridassay. We constructed a pGBT9 vector containing thecDNA fragment of the mid region of Rab45 as a bait.

WTΔEF

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Fig. 2. Analysis of the self-interaction of Rab45. (A) Schematic repre-sentation of Rab45 mutants used in this study. The regions correspondingto the amino acid 1–97, 98–533, and 527–740 are referred to as the EF-hand domain, the mid region, and the Rab domain of Rab45 in the text,respectively. (B) Rab45 can interact with itself in HeLa cells. HeLa cellswere co-transfected with two expression vectors of a combination ofpFLAG-CMV5-Rab45/WT and each of pMyc-CMV5-Rab45 mutants.The cell lysates prepared from the transfected cells were immunoprecip-itated with an anti-Myc polyclonal antibody. The immunocomplexes werethen subjected to Western blotting with the anti-Myc (top) or anti-FLAG(middle) antibody. The lysates were also subjected to Western blottingwith the anti-FLAG antibody (bottom). (C) Self-interaction analysis ofthe mid region of Rab45 in yeast two-hybrid system. cDNA fragments ofthe mid region of Rab45 and various forms of Rab45 were inserted intopGBT9 and pGAD424 vectors, respectively. Y190 cells were cotrans-formed with pGBT9- and pGAD424-derived vectors, and the transformedcolonies were subjected to b-galactosidase assay as described underMaterials and methods.

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cDNA fragments of the full-length, the mid region, theRab domain, and the EF-hand domain of Rab45 were sub-cloned in pGAD424 vector. Fig. 2C shows that the midregion of Rab45 specifically interacted with the full-lengthor the mid-region of Rab45, but not with the Rab domainor the EF-hand domain. These results indicate that the mid

region including coiled-coil motif can directly interact withitself, which may be responsible for the Rab45 self-interaction.

Analysis of the intracellular localization of Rab45 in HeLa

cells

We next examined the intracellular localization ofRab45 in HeLa cells. When GFP-tagged Rab45 was tran-siently expressed in HeLa cells, the fusion proteins werepredominantly observed in a small patch near the perinu-clear region of the cells (Fig. 3A). We sought to determinewhich region(s) of Rab45 is responsible for the characteris-tic localization. For this purpose, we transfected a set ofplasmid constructs of GFP-Rab45 mutants into HeLa cellsto observe their intracellular localization. Fig. 3B showsthat GFP-Rab45/DEF exhibited a small patch localizationsimilar to that observed for GFP-Rab45/WT (a in Fig. 3B).In contrast, GFP-Rab45/DRab was dispersed throughoutthe cytoplasm (b in Fig. 3B), indicating Rab domain isessential for the small patch localization of Rab45. Thelocalization pattern of GFP-Rab45/DMid was also differ-ent from that of GFP-Rab45/WT: GFP-Rab45/DMidwas observed in both cytosol and dispersed vesicular struc-tures near the perinuclear region (c in Fig. 3B). In addition,GFP-Rab45/Rab showed a similar localization pattern tothat observed for GFP-Rab45/DMid (d in Fig. 3B). Theseresults indicate that the mid region besides Rab domain isrequired for the small patch localization of Rab45. BothGFP-Rab45/Mid and GFP-Rab45/EF were dispersedthroughout the cells (e and f in Fig. 3B).

Generally, GTP-bound active Rab proteins can associ-ate with specific membrane compartments [2]. Indeed,GFP-Rab45/Q600L was localized to the perinuclear com-partment, while GFP-Rab45/S555N was dispersedthroughout the cells (g and h in Fig. 3B). These observa-tions implied that the perinuclear localization of Rab45 isregulated by its guanine nucleotide-bound states.

Interestingly, when GFP-Rab45/DRab was co-expressedwith DsRed-Monomer-Rab45/WT (a–c in Fig. 3C), it wasredistributed to a small patch near the nuclear region,where it was remarkably colocalized with Rab45/WT(compare b in Fig. 3C with b in Fig. 3B). In addition,GFP-Rab45/Mid was also colocalized with DsRed-Mono-mer-Rab45/WT in the perinuclear region when co-expressed (compare e in Fig. 3C with e in Fig. 3B), whileGFP-Rab45/EF was still dispersed throughout the cellseven in the presence of DsRed-Monomer-Rab45/WT(compare h in Fig. 3C with f in Fig. 3B). These results,together with the results shown in Fig. 2B and 2C, supportthe idea that Rab45 can interact with itself through the midregion.

Discussion

Several Rab GTPases and Rab effectors can form homo-dimers or multimers, and the self-interaction of these pro-

Fig. 3. Localization of wild-type and mutant Rab45 in HeLa cells. (A) HeLa cells were transiently transfected with pEGFP-Rab45/WT and stained withSYTO59 to mark the nuclei. The fluorescence signals of GFP (green) and SYTO59 (red) were recorded in a Carl Zeiss LSM-510 confocal microscopy (a)and merged with images taken using differential interference contrast microscopy (b). (B) Localization of Rab45 mutants in HeLa cells. HeLa cells weretransiently transfected with pEGFP vectors containing each of Rab45 mutants and observed using the confocal microscopy (a–h). (C) Rab45 mutantscontaining the mid region are redistributed to a perinuclear region by co-expression with wild-type Rab45. HeLa cells were transiently co-transfected withpDsRed-Monomer-Rab45/WT and pEGFP-Rab45/DRab (a–c), pEGFP-Rab45/Mid (d–f) or pEGFP-Rab45/EF (g–i). The distribution of thefluorescence proteins was recorded by the confocal microscopy. (c, f, and i) Merged images for a and b, d and e, and g and h, respectively. Bars, 10 lm.

M. Shintani et al. / Biochemical and Biophysical Research Communications 357 (2007) 661–667 665

teins appear to be important for their functions [9–21]. TheRab7-binding domain of RILP forms a coiled-coil homodi-mer, and the disruption of RILP dimerization abolish itsinteraction with Rab7 and late endosomal/lysosomal tar-geting [12], suggesting that the dimerization of RILP isessential for its function. ALS2 is a Rab5-binding protein

acting as a GEF (guanine nucleotide-exchange factor) forRab5 [17]. Homo-oligomerization of ALS2 is crucial forthe GEF activity and the ALS2-mediated endosomeenlargement [18]. A family of Rab11-interacting proteins(FIPs) has also been shown to have ability to self-interact[19–21]. Structural analysis of the heterotetrameric com-

666 M. Shintani et al. / Biochemical and Biophysical Research Communications 357 (2007) 661–667

plex of Rab11 with the C-terminus of FIP3 showed thatdimerization generates a binding surface for Rab11 extend-ing over both chains of the FIP3 homodimer [20,21]. In thisstudy, we have shown that Rab45 can interact with itselfthrough the mid region. Given that the mid region containsa coiled-coil motif, the self-interaction of Rab45 is likely tobe mediated by the coiled-coil motif. Further analysis ofthe Rab45 self-interaction using deletion and pointmutants is required to determine whether the coiled-coilmotif is responsible for the self-interaction of Rab45.

Coiled-coil proteins involved in membrane traffic oftenhave functionally discrete domains at their N- or C-termini[8]. For example, a large coiled-coil protein, Rabaptin5, hasa C-terminal Rab5-binding domain and a distinct N-termi-nal Rab4-binding domain [13]. Rab5 is involved in endocy-tosis, whereas Rab4 is important for recycling from earlyendosomes to the plasma membrane. Given that Rabap-tin5 is recruited to early endosomes in a Rab5-dependentmanner, Rabaptin5 appears to function to coordinatetwo pathways of endocytosis and recycling traffic at endo-somes [13]. As described above, Rab45 has a C-terminusRab domain and a distinct N-terminus EF-hand domain.We found that the Rab domain is involved in the perinu-clear localization of Rab45 probably through its interac-tion with a membrane compartment. It has been shownthat Rab proteins are localized to specific membrane com-partments of both the endocytic and exocytic pathways,and the characteristic localization of Rab proteins is depen-dent on the guanine nucleotide-bound forms [2]. Indeed,we found that Rab45 was localized to a small patch in aperinuclear region, and the localization was regulated bythe guanine-nucleotide forms of the Rab domain. Interest-ingly, in addition to the Rab domain, the mid region is alsoinvolved in the perinuclear localization of Rab45. Consid-ering that the mid region is important for the self-interac-tion of Rab45, the self-interaction of Rab45 may beinvolved in the characteristic perinuclear localization.Rab proteins have been shown to regulate a membranetraffic step that is reflected by their characteristic localiza-tion [2]. Our preliminary results showed that Rab45 wassignificantly co-localized with Rab11 in a perinuclearregion of HeLa cells (not shown). Future analysis of thepossible involvement of Rab45 in Rab11-mediated mem-brane may provide insight into the physiological functionof Rab45.

Functional significance of the EF-hand domain ofRab45 remains to be determined in the present study.EF-hand proteins undergo conformational changes uponbinding Ca2+, and the conformational changes are respon-sible for the interaction with target molecules [22]. Consid-ering that Ca2+ is involved in various aspects ofintracellular membrane transport including exocytosis[23], it is tempting to speculate that the EF-hand domainof Rab45 binds target molecule(s) in a Ca2+-concentrationdependent manner and regulates membrane traffic in coop-eration with Rab domain. Identification of the bindingpartner(s) for the EF-hand domain, together with that

for the Rab domain, will help to analyze the Rab45-medi-ated membrane traffic pathway.

Acknowledgments

This work was supported in part by research Grantsfrom the Ministry of Education, Culture, Sports, Scienceand Technology (MEXT) of Japan, Japan Society for thePromotion of Science (JSPS), the Astellas Foundation forResearch on Metabolic Disorders, and the MochidaMemorial Foundation for Medical and PharmaceuticalResearch.

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