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CELL STRUCTURE AND FUNCTION 36: 155–170 (2011)

© 2011 by Japan Society for Cell Biology

Genome-wide Investigation of the Rab Binding Activity of RUN Domains: Development of a Novel Tool that Specifically Traps GTP-Rab35

Mitsunori Fukuda*, Hotaka Kobayashi, Koutaro Ishibashi, and Norihiko Ohbayashi

Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and

Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi

980-8578, Japan

ABSTRACT. The RUN domain is a less conserved protein motif that consists of approximately 70 amino acids,

and because RUN domains are often found in proteins involved in the regulation of Rab small GTPases, the RUN

domain has been suggested to be involved in Rab-mediated membrane trafficking, possibly as a Rab-binding site.

However, since the Rab binding activity of most RUN domains has never been investigated, in this study we

performed a genome-wide analysis of the Rab binding activity of the RUN domains of 19 different RUN domain-

containing proteins by yeast two-hybrid assays with 60 different Rabs as bait. The results showed that only six

of them interact with specific Rab isoforms with different Rab binding specificity, i.e., DENND5A/B with Rab6A/

B, PLEKHM2 with Rab1A, RUFY2/3 with Rab33, and RUSC2 with Rab1/Rab35/Rab41. We also identified the

minimal functional Rab35-binding site of RUSC2 (amino acid residues 982–1199) and succeeded in developing a

novel GTP-Rab35-specific trapper, which we named RBD35 (Rab-binding domain specific for Rab35).

Recombinant RBD35 was found to trap GTP-Rab35 specifically both in vitro and in PC12 cells, and

overexpression of fluorescently tagged RBD35 in PC12 cells strongly inhibited nerve growth factor-dependent

neurite outgrowth.

Key words: Rab35/Rab effector domain/RUN domain/RUN and FYVE domain-containing (RUFY)/RUN and SH3 domain-containing 2 (RUSC2)

Introduction

The small GTPases of the Ras superfamily play a crucial

role in a variety of cellular events, including cell prolifera-

tion, cell morphological change, intracellular membrane

trafficking, and nucleocytoplasmic transport (reviewed in

Takai et al., 2001). In general, these small GTPases func-

tion as a molecular switch by cycling between two nucle-

otide (GTP/GDP)-bound states, and the GTP-bound active

form promotes specific cellular events through interaction

with a specific effector molecule. The Rab small GTPases

constitute the largest family of members of the Ras super-

family, and they are implicated in the control of intracellular

membrane trafficking (reviewed in Stenmark, 2009; Zerial

and McBride, 2001). Approximately 60 Rab members have

been reported in humans and mice (Bock et al., 2001; Itoh

et al., 2006; Pereira-Leal and Seabra, 2001), and each

member is thought to regulate a specific type or specific

step of membrane traffic. During the past decade, many

more Rab effector molecules have been identified in an

effort to improve our understanding the molecular mecha-

nism of Rab-mediated membrane trafficking (Fukuda, 2008;

Grosshans et al., 2006; Schwartz et al., 2007). Furthermore,

systematic screening for novel Rab effectors by using 60

different mammalian Rab isoforms as bait has recently been

performed, and several candidate Rab effector domains,

e.g., a coiled-coil (CC) domain, an ankyrin repeat (ANKR)

domain, and a phosphotyrosine binding (PTB) domain,

have been identified (Fukuda et al., 2008; Kanno et al.,

2010). However, we do not think that all Rab effector

*To whom correspondence should be addressed: Mitsunori Fukuda,Laboratory of Membrane Trafficking Mechanisms, Department of Devel-opmental Biology and Neurosciences, Graduate School of Life Sciences,Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.

Tel: +81–22–795–7731, Fax: +81–22–795–7733E-mail: nori@m.tohoku.ac.jp

Abbreviations: CA, constitutive active; CC, coiled-coil; CN, constitutivenegative; DENN, differentially expressed in normal and neoplastic; EGFP,enhanced green fluorescent protein; FYVE, Fab1, YOTB/ZK632.12, Vac1,and EEA1; GAP, GTPase-activating protein; GEF, guanine nucleotideexchange factor; GFP, green fluorescent protein; GST, glutathione S-transferase; HRP, horseradish peroxidase; mStr, monomeric Strawberry;NGF, nerve growth factor; RBD35, Rab-binding domain specific forRab35; RFP, red fluorescent protein; RUFY, RUN and FYVE domain-containing; RUN, RPIP8, UNC-14, and NESCA; RUSC, RUN and SH3domain-containing; TBC, Tre-2/Bub2/Cdc16.

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domains have been identified, because the public databases

contain several uncharacterized protein motifs that have

been implicated in the control of Rab-mediated membrane

trafficking.

The RUN domain is one such protein motif (Callebaut et

al., 2001) and was originally identified in RPIP8 (Rap2-

interacting protein 8)/RUNDC3A (Janoueix-Lerosey et al.,

1998), UNC-14 (Ogura et al., 1997), and NESCA (new

molecule containing SH3 at the carboxy-terminus)/RUSC1

(Matsuda et al., 2000). Since RUN domains are present in

several proteins that have been linked to the function of

small GTPases, e.g., Rap and Rab, they have been sug-

gested to be involved in Ras-like GTPase signaling in ani-

mals (Callebaut et al., 2001). As examples, RUNDC3A/

RPIP8 and RUFY3 (RUN and FYVE domain-containing 3)/

RPIPX/SINGAR1 (referred to as RUFY3 below) interact

with Rap2 through their N-terminal domain containing the

RUN domain (Janoueix-Lerosey et al., 1998; Kukimoto-

Niino et al., 2006). Rab6IP1 (Rab6-interacting protein 1)/

DENND5A, a DENN (differentially expressed in normal

and neoplastic) domain-containing protein implicated in

Rab regulation (Levivier et al., 2001; Yoshimura et al.,

2010), interacts with Rab6A through the first RUN domain

(Recacha et al., 2009), and IPORIN (interacting protein of

rab1)/RUSC2 (RUN and SH3 domain-containing 2) inter-

acts with Rab1 through the RUN domain (Bayer et al.,

2005). In addition, several RUN domain-containing pro-

teins (simply referred to as RUN proteins below) include

a TBC (Tre-2/Bub2/Cdc16) domain, which is a putative

Rab-GAP (GTPase-activating protein) domain (Bernards,

2003; Fukuda, 2011; Yang et al., 2007), and RUTBC3/

RABGAP5/DJ1042K10.2/CIP85 interacts with Rab5 iso-

forms through its TBC domain (Haas et al., 2005; Itoh et

al., 2006). Consequently, RUN domains have often been

regarded as a binding domain for small GTPases, especially

Rab and Rap, in the literature. Nevertheless, even though

determination of the Rab binding activity of RUN domains

would be an important step toward understanding the func-

tion of RUN proteins, the Rab binding activity of most RUN

domains has never been investigated. In addition, if any of

the RUN domains functioned as a specific Rab effector

domain (i.e., binding of specific GTP-Rab), it would be an

ideal tool for trapping specific GTP-Rab and blocking

specific Rab-mediated membrane trafficking event in cells

if overexpressed.

In this study we used yeast two-hybrid assays (Fukuda et

al., 2008; Itoh et al., 2006; Tamura et al., 2009) to systemat-

ically investigate the Rab binding activity of 19 RUN pro-

teins that are found in the human genome or mouse genome.

The results showed that only six of them, i.e., DENND5A/

B, PLEKHM2, RUFY2/3, and RUSC2, interact with spe-

cific Rab isoforms with different Rab binding specificity,

i.e., Rab6A/B, Rab1A, Rab33, and Rab1/Rab35/Rab41,

respectively. We also identified the minimal Rab35-binding

site of RUSC2 and developed a novel RUN-domain-based

GTP-Rab35-specific trapper, named RBD35 (Rab-binding

domain specific for Rab35). In addition, we showed that

expression of enhanced green fluorescent protein (EGFP)-

tagged RBD35 in PC12 cells strongly inhibited nerve

growth factor (NGF)-induced neurite outgrowth, in which

Rab35 has recently been shown to be involved (Chevallier

et al., 2009; Kanno et al., 2010).

Materials and Methods

Materials

Horseradish peroxidase (HRP)-conjugated anti-FLAG tag (M2)

mouse monoclonal antibody and anti-FLAG tag antibody-

conjugated agarose were obtained from Sigma-Aldrich Corp. (St.

Louis, MO). HRP-conjugated anti-T7 tag mouse monoclonal anti-

body and anti-T7 tag antibody-conjugated agarose were purchased

from Merck Biosciences Novagen (Darmstadt, Germany). Anti-

green fluorescent protein (GFP) rabbit antibody, HRP-conjugated

anti-GFP rabbit antibody, HRP-conjugated anti-red fluorescent

protein (RFP) rabbit antibody, and HRP-conjugated anti-Myc tag

rabbit antibody were from MBL (Nagoya, Japan). HRP-conjugated

anti-GST (glutathione S-transferase) antibody was from Santa

Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-Rab1A rabbit

polyclonal antibody and anti-Rab35 rabbit polyclonal antibody

were from Abnova (Taipei, Taiwan) and ProteinTech Group

(Chicago, IL), respectively. The specificity of each anti-Rab anti-

body was confirmed by using recombinant FLAG-tagged Rab1A/B,

Rab35, and Rab41 expressed in COS-7 cells (data not shown). All

other reagents used in this study were analytical grade or the high-

est grade commercially available.

Molecular cloning of cDNAs of human and mouse RUN proteins and plasmid construction

cDNAs encoding human and mouse RUN proteins (see Fig. 1 and

Table I for details) were amplified from Marathon-Ready human/

mouse whole brain, testis, and/or fetal brain cDNA (BD Bio-

sciences, San Jose, CA) by PCR with the following pairs of

oligonucleotides containing a restriction enzyme site (underlined:

BamHI, BglII, or SalI) and/or a stop codon (in bold), essentially as

described previously (Fukuda et al., 1999): 5'-GGATCCATCCA-

GGAAGCCAGGACTAT-3' (hDENND5A-RUN1-5' primer, sense)

and 5'-TCACCTCTCATCCACCTCAGGCT-3' (hDENND5A-RUN1-

3' primer, antisense); 5'-GGATCCCCATGCCGGACCCCGCCGCT-

3' (hDENND5A-RUN2-5' primer, sense) and 5'-TCAGATGTCG-

ATGCCCTTGA-3' (hDENND5A-RUN2-stop primer, antisense);

5'-GGATCCATGCAAGAGGCACGAAGTTT-3' (hDENND5B-

RUN1-5' primer, sense) and 5'-CTACTTTACTAGATCTTCAT-

CTG-3' (hDENND5B-RUN1-3' primer, antisense); 5'-GGATCC-

CAGTGTCGGACTCCACCCCA-3' (hDENND5B-RUN2-5' primer,

sense) and 5'-TTACACATCCACTCCTTTGA-3' (hDENND5B-

RUN2-stop primer, antisense); 5'-GGATCCAAACACGTATGGG-

TACGCAC-3' (hRUTBC1-RUN-5' primer, sense) and 5'-TCA-

RUN domain and small GTPase Rab

157

CTGGACCAGCTCATCAGCAG-3' (hRUTBC1-RUN-3' primer,

antisense); 5'-GGATCCGCGGCTGGCTTCCTACGCAG-3'

(hRUTBC2-RUN1-5' primer, sense) and 5'-CTACCGCACGTG-

GGAGCTGTGGA-3' (hRUTBC2-RUN2-3' primer, antisense); 5'-

GGATCCGAGGAGCTGCTCTACCGGGC-3' (hRUTBC3-RUN-5'

primer, sense) and 5'-TCACTCCTTCAGGGGCTGCTGCG-3'

(hRUTBC3-RUN-3' primer, antisense); 5'-GGATCCTTCGCGC-

AGGTGCAGTTCCG-3' (mRUNDC1-N1 primer, sense) and 5'-

TCAAAAGGCATCTTTGATGT-3' (mRUNDC1-stop primer,

antisense); 5'-GGATCCATGAGCGGATCACAGAACAA-3'

(hRUNDC2A-Met primer, sense) and 5'-TTACTTCTCGGGC-

GACTCCG-3' (hRUNDC2A-stop primer, antisense); 5'-AGATCT-

ATGGAAGCGAGCTTTGTCCA-3' (hRUNDC3A-Met primer,

sense) and 5'-TCAGCTGGGGCTCAGTGCTG-3' (hRUNDC3A-

stop primer, antisense); 5'-AGATCTATGGCCTCCCGGAGC-

CTGGG-3' (hRUNDC3B-Met primer, sense) and 5'-TCAG-

GATGGAGTTAGGCCTG-3' (hRUNDC3B-stop primer, antisense);

5'-AGATCTATGCTTTCAGTGGTGGAGAA-3' (hPLEKHM1-Met

primer, sense) and 5'-TCACTTATGGCCCGAGTGATGGA-3'

(hPLEKHM1-RUN-3' primer, antisense); 5'-AGATCTATG-

GAGCCGGGGGAGGTGAA-3' (hPLEKHM2-Met primer, sense)

and 5'-TCAGGAGGACACAGTCTCTGCCG-3' (hPLEKHM2-

RUN-3' primer, antisense); 5'-ACCTCCACCAACCTGGAGTG-3'

(hPLEKHM2-N1 primer, sense) and 5'-AAGTCGCAAAGCGG-

CGGCCT-3' (hPLEKHM2-C1 primer, antisense); 5'-CCTTTCG-

GACCGGCTCTCC-3' (hPLEKHM2-N2 primer, sense) and 5'-

Fig. 1. Schematic representation of human (h) and mouse (m) RUN domain-containing proteins. Amino acid numbers are given on both sides. Solid lines

indicate the constructs used for the yeast two-hybrid assays (see Fig. 2), and the name of each construct is shown above (or below) each line. CC, coiled-

coil; C1, protein kinase C conserved region 1 domain (Cys-rich domain); DENN, differentially expressed in normal and neoplastic cells; FYVE, Fab1,

YOTB/ZK632.12, Vac1, and EEA1; PH, pleckstrin homology; PLAT, polycystin-1, lipoxygenase, alpha-toxin; RUN, RPIP8, UNC-14, and NESCA; SH3,

Src homology domain 3; and TBC, Tre-2/Bub2/Cdc16. The DENN domain is composed of three subdomains, an upstream subdomain (u-DENN), a core

subdomain (DENN), and a downstream subdomain (d-DENN) (Levivier et al., 2001).

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M. Fukuda et al.

GCCTCACACTTCGATTCTTG-3' (hPLEKHM2-C2 primer, anti-

sense); 5'-GAGAAGCTGGCACTGGCCAA-3' (hPLEKHM2-N3

primer, sense) and 5'-TCAGCACCAGGGGTCTCGGG-3'

(hPLEKHM2-stop primer, antisense); 5'-GGATCCATGCAGAG-

CATCCTCTATCA-3' (hKIAA0226-Met primer, sense) and 5'-

TCACAGGTACCGACCACCTTCAT-3' (hKIAA0226-C1 primer,

antisense); 5'-GGATCCATGAAACTCAGCATTAAGGT-3'

(mRUFY1-RUN-5' primer, sense) and 5'-TCATTCATGCTCT-

CTGCCGCTGT-3' (mRUFY1-RUN-3' primer, antisense); 5'-

GGATCCAGCGGCAGAGAGCATGAA-3' (mRUFY1-C-5'

primer, sense) and 5'-TCACTTCTCGCTTCTTTC-3' (mRUFY1-

stop primer, antisense); 5'-GGATCCATGGCTACAAAGGAC-

CCC-3' (mRUFY2-Met primer, sense) and 5'-TCATGGCATGT-

TAGAGGA-3' (mRUFY2-stop primer, antisense); 5'-GGATCC-

ATGTCTGCCCTGACGCCT-3' (mRUFY3-Met primer, sense)

and 5'-TTAATGGTGCTTTGGGAT-3' (mRUFY3-stop primer,

antisense); 5'-AGATCTATGAGCTTCATATGCTCA-3' (mRUFY4-

RUN-5' primer, sense) and 5'-TCAGGTGTCCTGGATTTC-3'

(mRUFY4-stop primer, antisense); 5'-GGATCCATGGCTTCTA-

GCAGCACTGA-3' (mFYCO1-Met primer, sense) and 5'-

TCAAATGCTGTCCTCGACTGC-3' (mFYCO1-CC1-3' primer,

antisense); 5'-GCGGATCCATGGCAGAAGCCCAGAGT-3'

(mRUSC1-RUN-5' primer, sense) and 5'-GCGTCGACCTATCG-

GAGACTCTGAGCCAA-3' (mRUSC1-RUN-3' primer, antisense);

and 5'-GCAGATCTTCTCCCGATGGCAACTCG-3' (mRUSC2-

RUN-5' primer, sense) and 5'-GCGTCGACTCACAGACAGAA-

Table I. SUMMARY OF THE RAB BINDING ACTIVITY AND SPECIFICITY OF RUN DOMAIN-CONTAINING PROTEINS

Names of RUN proteins Regions used for

the two-hybrid assays

(amino acid numbers)

GTP-Rab binding activity

and specificitya

Official names Gene ID Other names

DENND5A 23258 RAB6IP1/KIAA1091 RUN1 (720–1075) Rab6A/B

RUN2 (1076–1287) —b

DENND5B 160518 RUN1 (739–1093) Rab6A/B

RUN2 (1094–1309) —

RUTBC1/SGSM2 9905 KIAA0397 RUN (128–203) —

RUTBC2/SGSM1 129049 KIAA1941 RUN12 (36–345) —

RUTBC3/SGSM3 27352 RABGAP5/DJ1042K10.2/CIP85 RUN (629–731) —

RUNDC1 217201 RUN (103–613) —

RUNDC2A 84127 Full (1–375) —

RUNDC3A 10900 RPIP8/RAP2IP Full (1–446) —

RUNDC3B 154661 RPIP9 Full (1–456) —

PLEKHM1 9842 AP162/KIAA0356 RUN (1–240) —

PLEKHM2 23207 SKIP/KIAA0842 RUN (1–280) Rab1A

KIAA0226 9711 RUBICON RUN (1–200) —

RUFY1 216724 RABIP4 RUN (124–303) —

C (298–712) Rab4A, Rab14c

RUFY2 70432 KIAA1537/RABIP4R C (192–606), Full (1–606) Rab33A/Bd>Rab4A/B, Rab6A/B

RUFY3 52822 SINGAR1/KIAA0871/RPIPX RUN (1–249) —

C (250–469), Full (1–469) Rab33A>Rab33Be

RUFY4 435626 C (85–563) —

FYCO1 17281 RUN (1–289)f —

RUSC1 72296 NESCA RUN (599–837) —

RUSC2 100213 IPORIN/KIAA0375 RUN (982–1351) Rab35>Rab1A>Rab1B, Rab41

Since the names of RUN proteins have been variously reported in the literature, the official names in the NCBI (National Center for Biotechnology

Information) database rather than the commonly used names have been used throughout the text for clarity.a Relative strength of the Rab binding activity was estimated on the basis of the growth rate of the yeast cells.b —, RUN domains that did not exhibit any Rab binding activity.c The Rab binding specificity of RUFY1 was not determined in this study. See Cormont et al. (2001) and Yamamoto et al. (2010) for details.d RUFY2-C weakly bound a constitutive negative form of Rab33A.e Rab33B binding activity of RUFY3 was evident only when RUFY3-C was used for the two-hybrid assay.f FYCO1-RUN domain was identified by the Blastp program, but not by the SMART program.

RUN domain and small GTPase Rab

159

CTCAGCGGG-3' (mRUSC2-RUN-3' primer, antisense) or 5'-

GCGTCGACTCAGTTTTGGCTGCTCCC-3' (mRUSC2-stop

primer, antisense). cDNAs encoding the mouse DENND1A/con-

necdenn (Allaire et al., 2010; Marat and McPherson, 2010) were

similarly amplified from Marathon-Ready mouse whole brain and

testis cDNA by PCR using the following pairs of oligonucleotides

with a BglII site (underlined) or with a stop codon (in bold): 5'-

AGATCTATGGGCTCCAGGATCAAGCA-3' (DENND1A-Met

primer, sense) and 5'-CTAACCTTCGCCGGAATTGAGAA-3'

(DENND1A-C1 primer antisense); 5'-ATGTTGCATCTGTACG-

CCAG-3' (DENND1A-N1 primer, sense) and 5'-TCTAGGCT-

GCTGAAGACATC-3' (DENND1A-C2 primer antisense); and 5'-

GACCGAGCTGCCAGCATTGA-3' (DENND1A-N2 primer, sense)

and 5'-TCACTCAAAGGTCTCCCACT-3' (DENND1A-stop primer,

antisense). Purified PCR products were directly inserted into the

pGEM-T Easy vector (Promega, Madison, WI) and verified with

an automated sequencer. cDNA encoding an open reading frame

of DENND1A was constructed on the pGEM-T Easy vector by

using appropriate restriction enzyme sites. cDNAs containing each

of the RUN domain-containing fragments (or DENND1A) were

excised from the pGEM-T Easy vector with appropriate restriction

enzymes and then subcloned into the pGAD-C1 vector (James et

al., 1996), pmStr-C1 vector, pEGFP-C1 vector (BD Biosciences),

and/or pEF-T7/Myc tag mammalian expression vectors modified

from pEF-BOS as described previously (Fukuda et al., 1999). The

pEF-Myc tag (MEQKLISEEDLNEGS; an N-terminal Myc tag

sequence is shown in bold) expression vector was constructed by

PCR essentially as described previously (Fukuda et al., 1999).

Deletion mutants of RUFY1 (i.e., RUN, C, CC1, and CC2+FYVE;

see Fig. 1), RUFY2 (i.e., RUN, C, CC1, and CC2+FYVE; see Fig.

1 and Fig. 5A), RUFY3 (i.e., RUN, C, RUN+CC1, CC1, and CC2;

see Fig. 4A), and RUSC2 (i.e., RUN, ΔC1-ΔC5, ΔN1-ΔN4, ΔN3/

C2, and ΔN4/C2; see Fig. 6A) were also constructed by conven-

tional PCR techniques, and the resulting cDNAs were transferred

to the pGAD-C1 vector, pmStr-C1, and/or pEGFP-C1 vector as

described above. The sequences of the oligonucleotides used for

the construction of these deletion mutants are available from the

authors on request. A constitutive active (CA) mutant (Q67L) and

a constitutive negative (CN) mutant (S22N) of Rab35 were pre-

pared by site-directed mutagenesis (Itoh et al., 2006; Tamura et al.,

2009) and subcloned into the pEF-FLAG tag mammalian expres-

sion vectors as described previously (Fukuda et al., 1999). Other

expression plasmids, including pEF-FLAG-Rab1A, pEF-FLAG-

Rab1B, pEF-FLAG-Rab4A, pEF-FLAG-Rab33A, pEF-FLAG-

Rab35, pEF-FLAG-Rab41, and pEF-T7-TBC1D10C/mFLJ00332,

were also prepared as described previously (Fukuda, 2003; Itoh

and Fukuda, 2006).

Yeast two-hybrid assays

Yeast two-hybrid assays were performed by using pGBD-C1-

Rab(CA)/(CN) lacking the C-terminal geranylgeranylation site

(ΔCys) and pGAD-C1-RUN proteins/domains (in Fig. 2) as

described previously (Fukuda et al., 2008; Itoh et al., 2006;

Tamura et al., 2009). In Fig. 4, Fig. 5, and Fig. 6, only pGBD-C1-

Rab(CA)ΔCys constructs were used for two-hybrid assays. The

yeast strain, medium, culture conditions, and transformation proto-

col used were as described in James et al. (1996).

Cell cultures and transfections

COS-7 cells were maintained at 37°C in DMEM containing 10%

fetal bovine serum and antibiotics under a 5% CO2 atmosphere.

PC12 cells were cultured at 37°C on poly-L-lysine-coated or

collagen type IV-coated dishes in DMEM containing 10% horse

serum, 10% fetal bovine serum, and antibiotics with or without

NGF (100 ng/ml; Merck KGaA, Darmstadt, Germany) under a 5%

CO2 atmosphere. Plasmids were transfected into COS-7 cells (for

GST pull-down assays and co-immunoprecipitation assays) by

using Lipofectamine Plus or Lipofectamin LTX Plus (Invitrogen

Corp., Carlsbad, CA) according to the manufacturer’s instructions.

Plasmids were also transfected into PC12 cells (for immunofluo-

rescence analyses and neurite outgrowth assays) by using Lipo-

fectamine 2000 (Invitrogen Corp.) according to the manufacturer’s

instructions.

GST pull-down assays and co-immunoprecipitation assays in COS-7 cells

The RBD35 fragment was subcloned into the pGEX-4T-3 vector

(GE Healthcare Ltd., Little Chalfont, UK), and GST-RBD35 was

expressed in Escherichia coli BL21(DE3)pLysS and purified by

the standard protocol. Nucleotide exchange of Rab and GST pull-

down assays were performed as described previously (Fukuda and

Kanno, 2005; Kanno et al., 2010). Briefly, glutathione-Sepharose

beads (GE Healthcare Ltd.) coupled with 6–10 μg of GST-RBD35

were incubated for 1 h with COS-7 cell lysates containing FLAG-

Rab35, FLAG-Rab35(Q67L), FLAG-Rab35(S22N), FLAG-

Rab35+T7-DENND1A, or FLAG-Rab35+T7-TBC1D10C in the

presence of nothing, 1 mM GDP, or 0.5 mM GTPγS. After

washing the beads three times, proteins bound to the beads were

analyzed by 10% SDS-PAGE and then immunoblotted with HRP-

conjugated anti-FLAG tag antibody and HRP-conjugated anti-

GST antibody as described previously (Fukuda and Kanno, 2005).

Immunoreactive bands were visualized by enhanced chemilumi-

nescence (ECL; GE Healthcare Ltd.). GTP-Rab35 pull-down

assays from PC12 cell lysates were performed in a manner similar

to that described above.

Co-immunoprecipitation assays in COS-7 cells were performed

as described previously (Fukuda et al., 1999; Fukuda and Kanno,

2005). In brief, agarose beads coupled with nothing (Mock) or

FLAG-Rabs were incubated with the COS-7 cell lysates express-

ing T7/Myc-tagged Rab-binding proteins in the presence of 0.5

mM GTPγS. Alternatively, a complex between FLAG-Rab and

T7-Rab-binding protein in COS-7 cell lysates was immunoprecipi-

tated with anti-FLAG tag antibody-conjugated agarose beads in

the presence of 0.5 mM GTPγS (for Fig. 3C). After washing the

beads, proteins bound to the beads were analyzed by 10% SDS-

PAGE followed by immunoblotting with HRP-conjugated anti-

Myc tag antibody (1/5000 dilution), HRP-conjugated anti-T7 tag

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M. Fukuda et al.

Fig. 2. Rab binding specificity of RUN domain-containing proteins as revealed by yeast two-hybrid assays. Rab binding activity of the RUN domains of

DENND5A/B (A), RUTBC1-3 (B), RUNDC1-3 (C), PLEKHM1/2 and KIAA0226 (D), RUFY1-4 (E), FYCO1 (F), and RUSC1/2 (G). Yeast cells

containing pGBD plasmid expressing a constitutive active form (CA, which mimics the GTP-bound form) (Fukuda et al., 2008; Itoh et al., 2006) or a

constitutive negative form (CN, which mimics the GDP-bound form) (Tamura et al., 2009) of Rab (positions indicated in the left panels) and pGAD plasmid

expressing the RUN domain indicated were streaked on SC-AHLW medium and incubated at 30°C for one week. Positive patches are boxed. Note that only

six RUN proteins bound specific Rab isoforms.

RUN domain and small GTPase Rab

161

Fig. 3. Rab binding activity of RUN domain-containing proteins (PLEKHM2, RUFY2, RUFY3, and RUSC2) as revealed by co-immunoprecipitation

assays (A–C) and immunofluorescence analyses (D–G). (A) Interaction between PLEKHM2 (Full and RUN) and Rab1A, (B) between RUFY2/3 and

Rab4A/33A, and (C) between RUSC2-C (see Fig. 1) and Rab1A/35 in COS-7 cells. Co-immunoprecipitation assays in COS-7 cells were performed in the

presence of 0.5 mM GTPγS as described under Materials and Methods. After washing the beads, proteins bound to the beads were detected with anti-Myc

tag or anti-T7 tag antibody (middle panels), and anti-FLAG tag antibody (bottom panels). The open arrowheads and closed arrowheads indicate the

positions of Myc-PLEKHM2-Full and Myc-PLEKHM2-RUN, respectively, in A and of T7-RUFY2 and T7-RUFY3, respectively, in B. Input means 1/100

volume of the reaction mixture used for the immunoprecipitation (top panels). The positions of the molecular mass markers (Mr in kilodaltons) are shown

on the left. (D) Co-localization between EGFP-PLEKHM2 (Full and RUN) and mStr-Rab1A, (E) between mStr-RUFY2 and EGFP-Rab4A/33A, (F)

between mStr-RUFY3 and EGFP-Rab33A, and (G) between EGFP-RUSC2-C and mStr-Rab1A/35 in PC12 cells. PC12 cells co-expressing EGFP-Rab and

mStr-Rab-binding protein (or mStr-Rab and EGFP-Rab-binding protein) were analyzed with a confocal fluorescence microscope as described under

Materials and Methods. The arrowheads in the insets indicate the co-localization points. The small arrows in (G) indicate the co-localization points between

RUSC2-C and Rab35 at the tips of neurites. Insets show magnified views of the boxed area. Scale bars, 10 μm.

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M. Fukuda et al.

antibody (1/10,000 dilution) and/or HRP-conjugated anti-FLAG

tag antibody (1/10,000 dilution). Immunoreactive bands were

visualized by enhanced chemiluminescence. Associations between

EGFP-Rab33A and mStr (monomeric Strawberry)-RUFY2/3 trun-

cated mutants in COS-7 cells were similarly evaluated by immuno-

precipitation with 1 μl of anti-GFP antibody and 10 μl of anti-

rabbit IgG dynabeads (Invitrogen Corp.). The blots shown in this

paper are representative of at least two independent experiments.

Immunofluorescence analyses in PC12 cells

PC12 cells transiently expressing EGFP-Rab and mStr-tagged

Rab-binding protein (or mStr-Rab and EGFP-tagged Rab-binding

protein) were fixed in 4% paraformaldehyde in 0.1 M sodium

phosphate buffer for 20 min at room temperature. The cells were

examined for fluorescence with a confocal fluorescence micro-

scope (Fluoview FV1000; Olympus, Tokyo, Japan). The images

were processed with Adobe Photoshop software (CS5).

Neurite outgrowth of PC12 cells

The length of the neurites of NGF-differentiated PC12 cells was

measured essentially as described previously (Fukuda and

Mikoshiba, 2000). In brief, pEGFP-C1-RBD35, pEGFP-C1-

RUSC2-ΔC4, or pEGFP-C1 alone was transiently expressed in

PC12 cells by using Lipofectamine 2000, and the cells were

treated with 100 ng/ml NGF 24 hours after transfection. After

exposure to NGF for 36 hours, the cells were fixed, and images of

the cells were captured at random (n>100 in three different dishes

for each construct) with a confocal fluorescence microscope, and

the length of the neurites was measured with MetaMorph software

(Molecular Devices, Sunnyvale, CA).

Sequence analyses

RUN proteins in the human genome and mouse genome were ob-

tained by using the SMART (Simple Modular Architecture Re-

search Tool) program (available at http://smart.embl-heidelberg.de/)

or the Blastp program (available at http://blast.ncbi.nlm.nih.gov/).

Sequence alignment and depiction of the phylogenetic tree of the

RUN domains from humans and mice were performed by using the

ClustalW program (available at http://clustalw.ddbj.nig.ac.jp/top-

e.html) set at the default parameters.

Results

Rab binding activity and specificity of human and mouse RUN proteins

Nineteen RUN domain-containing proteins were identified

in the human (or mouse) genome by using the SMART pro-

gram or the Blastp program (Fig. 1 and Supplemental Fig.

S1), and we classified them into the following six groups

based on their domain structures and sequence similarities:

RUN protein containing a DENN/Rab-GEF (guanine nucle-

otide exchange factor) domain (DENND5A/B; green boxes

in Fig. 1), RUN proteins containing a TBC/Rab-GAP

domain (RUTBC1-3; blue boxes), RUN proteins containing

a pleckstrin homology (PH) domain (PLEKHM1/2 and

KIAA0226/RUBICON; purple boxes), RUN proteins con-

taining conserved CC domains (RUFY1-4 and FYCO1;

black boxes), RUN proteins containing a SH3 domain

(RUSC1/2; triangles), and others (RUNDC1-3). cDNA

fragments encoding each RUN protein (or RUN domain-

containing region) were amplified by PCR (solid lines in

Fig. 1) and constructed into the pGAD-C1 vector, a prey

vector for yeast two-hybrid assays. All of the possible inter-

actions between the 19 RUN proteins and 60 constitutive

active (CA)/negative (CN) mutants of mammalian Rabs

were investigated by using yeast two-hybrid panels (Fig. 2)

that we recently developed (Fukuda et al., 2008; Itoh et al.,

2006; Tamura et al., 2009). Although most of the RUN pro-

teins (or RUN domains) did not exhibit any Rab binding

activity (Fig. 2B and C), six of them clearly interacted with

a CA form of specific Rab isoforms, suggesting that these

RUN domains function as a Rab effector domain (summa-

rized in Table I, and white boxes in Fig. 2). The first RUN

domain of DENND5A (i.e., DENND5A-RUN1), but not

its second RUN domain (RUN2), interacted specifically

with the CA form of Rab6A and Rab6B, consistent with a

previous report (Recacha et al., 2009). DENND5B, a

DENND5A-related protein, also interacted specifically, but

weakly, with the CA form of Rab6A and Rab6B through the

RUN1 domain (Fig. 2A). The RUN domain of RUSC2/

IPORIN, originally described as a Rab1-binding domain

(Bayer et al., 2005), interacted with a CA form of Rab35

and Rab41, in addition to Rab1A/B (Fig. 2G), and based on

the growth rate of the yeast cells in the selection medium,

the RUSC2 RUN domain seemed to preferentially interact

with Rab35 rather than with Rab1A/B. Three additional

interactions, a RUFY2-Rab33A/B interaction, a RUFY3-

Rab33A interaction (Fig. 2E), and a PLEKHM2-RUN-

Rab1A interaction (Fig. 2D), were also identified, but this

last interaction seemed to be very weak (i.e., the growth rate

was very slow). These previously uncharacterized interac-

tions observed in yeast cells (Fig. 2) were confirmed in

mammalian cultured cells both by co-immunoprecipitation

assays in COS-7 cells (Fig. 3A-C) and by immunofluores-

cence analyses in PC12 cells (Fig. 3D-G). As an example,

consistent with the results of the yeast two-hybrid assays

(Fig. 2E), RUFY2 interacted with both Rab4A and Rab33A

in COS-7 cells (Fig. 3B, lanes 2 and 3) and co-localized

with both Rabs in PC12 cells (Fig. 3E), whereas RUFY3

interacted with Rab33A, but hardly at all with Rab4A (Fig.

3B, lanes 5 and 6), in COS-7 cells, and co-localized with

Rab33A, not with Rab4A (Fig. 3F). We then focused on

the three strong, previously uncharacterized interactions,

i.e., the RUFY2-Rab33A interaction, RUFY3 (originally

described as SINGAR1; single axon-related 1 (Mori et al.,

RUN domain and small GTPase Rab

163

2007))-Rab33A interaction, and RUSC2-Rab35 interaction,

and attempted to map the Rab-binding site of RUFY2/3

and RUSC2 in greater detail by deletion analysis.

Mapping of the domain responsible for Rab33 binding in RUFY2 and RUFY3

The RUFY family consists of four members (RUFY1-4)

both in humans and mice, and they share an N-terminal

single RUN domain and one or two CC domains in their C-

terminal portion (Fig. 1). In contrast to RUFY3, RUFY1,

originally described as RABIP4 (Rab4 interacting protein)

(Cormont et al., 2001; Mari et al., 2001), RUFY2, and

RUFY4 contain an additional FYVE (Fab1, YOTB/

ZK632.12, Vac1, and EEA1) domain at their C-terminal

end. Since RUFY1 has recently been shown to function as a

dual Rab effector (i.e, a dual effector for Rab4 and Rab14)

through its C-terminal region, not its N-terminal RUN

domain (Yamamoto et al., 2010), we first attempted to

determine whether the RUN domain of RUFY3 functions as

a Rab33A-binding domain by yeast two-hybrid assays with

five truncated mutants (Fig. 4A). Interestingly, however, the

results of the deletion analysis under our experimental con-

ditions indicated that Rab33A interacted with the first CC

domain (named CC1 below), not with the N-terminal RUN

domain (Fig. 4B). We further found that the CC1 domain of

RUFY3 is involved in the specific Rab33A binding activity

(Fig. 4C), although, in contrast to the full-length protein, the

CC1 domain of RUFY3 interacted with Rab33B weakly

(Fig. 2E). The interaction of Rab33A with the C-terminal

domain of RUFY3 was confirmed by two independent

approaches using mammalian cultured cells. In the first

approach, we showed by immunofluorescence analyses that

mStr-RUFY3-C clearly co-localized with EGFP-Rab33A in

PC12 cells, whereas no co-localization between mStr-

RUFY3-RUN and EGFP-Rab33A was observed in PC12

Fig. 4. Mapping of the site responsible for the Rab33A-binding site of RUFY3. (A) Schematic representation of the deletion mutants of RUFY3 used in

this study. RUN contains amino acid residues 1–249 of mouse RUFY3; C, amino acid residues 250–469; RUN+CC1, amino acid residues 1–329; CC1,

amino acid residues 250–329; and CC2, amino acid residues 330–469. (B) Specific interaction between the CC1 domain of RUFY3 and Rab33A as revealed

by yeast two-hybrid assays. (C) The CC1 domain of RUFY3 is responsible for specific interaction with Rab33. The yeast two-hybrid assays and description

of the data are described in the legend for Fig. 2. Positive patches are boxed.

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M. Fukuda et al.

cells (Supplemental Fig. S2C). In the second approach, we

showed by co-immunoprecipitation assays in COS-7 cells

that mStr-RUFY3-C actually interacted with EGFP-

Rab33A (Supplemental Fig. S2A, lane 4 in the middle

panel, open arrowhead), although, unexpectedly, interaction

between T7-RUFY3-C and FLAG-Rab33A was hardly

observed by co-immunoprecipitation assays in COS-7 cells

for an unknown reason (data not shown). Interestingly,

mStr-RUFY3-RUN alone also interacted with EGFP-Rab33A

by co-immunoprecipitation assays, although, unlike RUFY3-

C, a significant amount of the RUFY3 RUN domain non-

specifically bound to the control GFP beads (asterisk in the

middle panel of Supplemental Fig. S2A). However, since no

co-localization between mStr-RUFY3-RUN and EGFP-

Rab33A was observed in living cells (Supplemental Fig.

S2C), the interaction between them observed by the co-

immunoprecipitation assays may have been an artifact

caused by the adhesive nature of the RUFY3 RUN domain.

Alternatively, the RUFY3 RUN domain may function as a

second Rab33-binding site and contribute to the Rab33A

recognition in a whole protein. In any event, since the

RUFY3-C interacted with Rab33A by all three different

approaches described above (Fig. 4, and Supplemental Fig.

S2A and C), RUFY3 is likely to interact with Rab33A

mainly through the first coiled-coil domain.

Since RUFY2 also interacted with Rab33A/B (Fig. 2E),

we performed a deletion analysis (Fig. 5A) to investigate

whether the CC1 domain of RUFY2 functions as a Rab33-

binding site, the same as the RUFY3 CC1 domain does,

and, as expected, the RUFY2 CC1 domain alone interacted

Fig. 5. Mapping of the site responsible for the Rab33-binding site of RUFY2. (A) Schematic representation of the deletion mutants of RUFY2 used in this

study. RUFY2-C contains amino acid residues 192–606 of mouse RUFY2; CC1, amino acid residues 192–271; and CC2+FYVE, amino acid residues 272–

606. (B) Distinct domains of RUFY2 contribute to Rab33 binding and Rab4A/6A/6B binding as revealed by yeast two-hybrid assays. Note that the CC1

domain of RUFY2 is necessary and sufficient for specific interaction with Rab33, whereas CC2+FYVE interacts with Rab4A/6A. The yeast two-hybrid

assays and description of the data are described in the legend for Fig. 2. Positive patches are boxed. (C) Distinct Rab4A/Rab33A binding activity of RUFY1-

3. RUFY1 interacts with Rab4A through the region between CC2 and FYVE (Cormont et al., 2001; Yamamoto et al., 2010), whereas RUFY3 interacts with

Rab33A through the CC1 domain. By contrast, RUFY2 possesses both properties.

RUN domain and small GTPase Rab

165

with Rab33A/B by yeast two-hybrid assays (middle panel

of Fig. 5B). In contrast to the RUFY2-C construct, however,

the RUFY2 CC1 domain alone did not interact with Rab4A/

6A/6B, indicating that an additional domain of RUFY2

contributes to the binding of Rab4A/6A/6B. Actually, the

CC2+FYVE construct of RUFY2 was found to interact with

Rab4A/6A (right panel of Fig. 5B). These results, together

with the fact that RUFY1, which is structurally related to

RUFY2, interacts with Rab4A via the region between the

CC2 domain and the FYVE domain (Cormont et al., 2001;

Mari, et al., 2001; Yamamoto et al., 2010), indicated that

RUFY2 contains two Rab-binding sites, a Rab33-binding

site (i.e., CC1 domain) and a Rab4A/6A/6B-binding site,

the latter of which is presumably located between the CC2

domain and the FYVE domain (Fig. 5C). Thus, RUFY2 is

likely to function as a dual Rab effector, the same as

RUFY1 (Yamamoto et al., 2010), whereas RUFY3 func-

tions as a Rab33A-specific effector, because it lacks a

domain corresponding to the region between the CC2

domain and the FYVE domain of RUFY2. Since no co-

localization between mStr-RUFY2-RUN and EGFP-Rab33A

was observed in PC12 cells (Supplemental Fig. S2D) and

since mStr-RUFY2-C, but not mStr-RUFY2-RUN, inter-

acted with EGFP-Rab33A (Supplemental Fig. S2B, lane 4

in the middle panel, open arrowhead), we concluded that the

RUFY2 CC1 domain functions as the Rab33A-binding site,

the same as the RUFY3 CC1 domain does.

Determination of the minimal Rab35-binding site of RUSC2

We next attempted to identify the minimal Rab-binding site

of RUSC2 by a systematic deletion analysis (Fig. 6A),

because the original RUN fragment that we used for the

yeast two-hybrid assays (Fig. 2G) contained both N-terminal

and C-terminal flanking regions (i.e., more than 120 amino

acids flank the RUN domain on each side). As shown in

Fig. 6B, the N-terminal deletion constructs (ΔN1-ΔN4) had

no binding activity toward any of the four Rabs. The results

obtained with the C-terminal deletion constructs (ΔC1-

ΔC5), however, were somewhat complicated. Deletion of

the 32 C-terminal amino acids from the original RUN con-

struct (ΔC5) mostly abrogated Rab1A, Rab1B, and Rab41

binding activities, whereas C-terminal deletion of 72 or 112

amino acids (ΔC4 or ΔC3) completely abrogated all Rab

binding activity. To our surprise, however, additional C-

terminal deletion (deletion of 152 or 174 C-terminal amino

acids; ΔC2 or ΔC1) unexpectedly restored Rab35 binding

activity, but not Rab1A, Rab1B, or Rab41 binding activity,

suggesting that a region containing amino acids 1200-1279

has some inhibitory effect on the Rab35 binding activity of

RUSC2. Since the ΔC1 construct of RUSC2 exhibited very

weak Rab35 binding activity, we concluded that the ΔC2

construct of RUSC2 is the minimal functional Rab35-

binding site.

Development of a novel GTP-Rab35 trapper (RBD35) based on the RUSC2 RUN domain

A report of the identification of a specific effector domain

in the Rab research literature has often significantly

Fig. 6. Mapping of the minimal Rab35-binding site of RUSC2. (A)

Schematic representation of the deletion mutants of RUSC2 used in this

study. Amino acid numbers are shown on both sides of each construct. The

minimal high affinity Rab35-specific-binding site is indicated by a dotted

box and referred to as RBD35 (Rab-binding domain specific for Rab35) in

the text. Relative Rab binding activities (±, +, ++, or +++) are indicated at

the right of each construct based on the growth rate of the yeast cells (see

B). (B) Rab1A/B, Rab35, and Rab41 binding activities of the RUSC2

deletion mutants. Note that the RUN domain of RUSC2 alone did not

recognize Rab35 and that an additional N-terminal region adjacent to the

RUN domain was required for the Rab35 binding activity. The yeast two-

hybrid assays and description of the data are described in the legend for

Fig. 2.

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M. Fukuda et al.

advanced our understanding of the involvement of a spe-

cific Rab protein in membrane trafficking (Fukuda, 2010).

As an example, a Rab27-specific effector domain (i.e.,

synaptotagmin-like protein homology domain; SHD) can

trap and sense the level of endogenous GTP-Rab27A in

cells (i.e., GTP-Rab27 pull-down assay) (Imai et al., 2009;

Itoh and Fukuda, 2006), and the same construct can be used

to inhibit the function of endogenous Rab27 protein (e.g.,

melanosome transport in melanocytes and secretion by

secretory cells) (Holt et al., 2008; Kuroda et al., 2003)

by trapping GTP-Rab27 (i.e., dominant negative assay)

(Fukuda (2008) and references therein). We therefore at-

tempted to develop a RUN-domain-based GTP-Rab trapper.

Among the Rab-RUN protein interactions identified above

(Fig. 2), we focused on the ΔC2 construct of RUSC2, which

specifically and strongly interacted with Rab35 in yeast

cells (Fig. 6), because the Rab6-DENND5A-RUN1 interac-

tion has already been structurally investigated (Recacha et

al., 2009) and a CC-domain-based GTP-Rab33 trapper (i.e.,

the Atg16L1 CC domain) has recently been reported (Itoh et

al., 2008). In addition, considerable attention has recently

been directed toward Rab35, because it was recently shown

to be involved in a variety of cellular events, including

cytokinesis (Kouranti et al., 2006), endocytic recycling

(Patino-Lopez et al., 2008; Sato et al., 2008), actin remodel-

ing (Shim et al., 2010; Zhang et al., 2009), and neurite

outgrowth (Chevallier et al., 2009; Kanno et al., 2010).

However, only a very few Rab35-binding proteins have

been identified (Fukuda et al., 2008; Kanno et al., 2010;

Zhang et al., 2009), and the Rab binding specificity (or ef-

fector domain) of most of them has never been thoroughly

investigated. We therefore further characterized the Rab35

binding ability of the ΔC2 construct of RUSC2, which we

refer to as RBD35 (Rab-binding domain specific for Rab35)

below, by using mammalian cell cultures.

We did so by using beads coupled with GST-RBD35 to

investigate whether the RUSC2-ΔC2 construct could specif-

ically trap a GTP-bound form of FLAG-Rab35 in COS-7

cells. Consistent with the results of the yeast two-hybrid

assays (Fig. 6B), GST-RBD35 trapped FLAG-Rab35 only

in the presence of GTPγS, and not in the presence of GDP

(compare lanes 3 and 4 in the middle panel of Fig. 7A).

Similarly, GST-RBD35 trapped the CA form of Rab35 (i.e.,

Rab35(Q67L)), but not its CN form (i.e., Rab35(S22N))

(compare lanes 3 and 4 in the middle panel of Fig. 7B).

GST-RBD35 is likely to sense the level of GTP-Rab35 in

cells, because manipulation of the cellular level of GTP-

Rab35 either by expression of TBC1D10C, a specific GAP,

an inactivator for Rab35 (Patino-Lopez et al., 2008; Hsu et

al., 2010), or by expression of DENND1A/connecdenn, a

specific GEF, an activator for Rab35 (Allaire et al., 2010;

Marat and McPherson, 2010; Yoshimura et al., 2010),

altered the amount of GTP-Rab35 trapped by the beads

without altering the total level of Rab35 expression (Fig.

7C). Expression of TBC1D10C dramatically decreased the

level of GTP-Rab35 trapped by the beads (lane 2 in the

second panel from the top in Fig. 7C), whereas expression

of DENND1A dramatically increased the level of GTP-

Rab35 trapped by the beads (lane 3 in the second panel from

the top in Fig. 7C). Moreover, GST-RBD35 specifically

trapped recombinant Rab35, but did not trap Rab1A,

Rab1B, or Rab41, in COS-7 cells (lane 7 in the middle

panel in Fig. 7D), and it also trapped endogenous Rab35,

but not Rab1A, in PC12 cells (lane 4 in the middle panel in

Fig. 7E and F).

Effect of RBD35 on NGF-induced neurite outgrowth of PC12 cells

Lastly, we investigated the effect of RBD35 on NGF-induced

neurite outgrowth of PC12 cells, a process in which Rab35

has recently been shown to be involved (Chevallier et al.,

2009; Kanno et al., 2010). Expression of EGFP-tagged

RBD35 in PC12 cells was found to strongly inhibit neurite

outgrowth of PC12 cells when compared to control EGFP-

expressing cells (Fig. 8A and B). We also tested the effect

of RUSC2-ΔC4, which lacked Rab35 binding activity by

yeast two-hybrid assays (Fig. 6), on neurite outgrowth of

PC12 cells. The result showed that expression of EGFP-

RUSC2-ΔC4 had little effect on neurite outgrowth in com-

parison with the EGFP-RBD35-expressing cells (compare

left and right shaded bars in Fig. 8B), although it exhibited a

weak inhibitory effect on neurite outgrowth in comparison

with the control EGFP-expressing cells. This weak inhibi-

tory effect of RUSC2-ΔC4 may be attributable to its degra-

dation product, whose molecular mass almost coincides

with that of RBD35 (data not shown). Moreover, the strong

inhibitory effect of RBD35 on neurite outgrowth of PC12

cells was almost completely abolished by co-expression of

Rab35 (Fig. 8C), indicating that RBD35 is likely to inhibit

NGF-induced neurite outgrowth of PC12 cells by trapping

endogenous Rab35. On the basis of these findings, together

with the fact that RBD35 specifically trapped GTP-Rab35

(Fig. 7), we concluded that EGFP-RBD35 functions as a

dominant negative construct by trapping endogenous Rab35

molecules in living cells and inhibits neurite outgrowth of

PC12 cells as a result.

Discussion

Because a RUN domain is often found in proteins that are

involved in the regulation of small GTPase Rab (e.g., Rab1,

Rab4, Rab5, Rab6, and Rab7) (Fig. 1) (Bayer et al., 2005;

Bernards, 2003; Callebaut et al., 2001; Cormont et al.,

2001; Fukuda, 2011; Haas et al., 2005; Mari et al., 2001;

Pankiv et al., 2010; Recacha et al., 2009; Tabata et al.,

2010; Yamamoto et al., 2010; Yang et al., 2007), RUN

domains have often been considered to be the binding site

of some Rabs in the literature, even though their Rab

RUN domain and small GTPase Rab

167

binding activity had never been thoroughly investigated. In

the present study we for the first time performed a genome-

wide analysis of the Rab binding activity of RUN domains

in humans and mice by yeast two-hybrid assays in which

panels of CA/CN mutants of 60 Rabs were used as bait

(Fig. 2). The results showed that only four of the 19 RUN

proteins interacted with specific Rab isoforms via their

RUN domain-containing region, i.e., PLEKHM2 with

Rab1A, DENND5A/B with Rab6A/B, and RUSC2 with

Rab1A/B, Rab35, and Rab41 (Fig. 2 and Supplemental

Fig. S1A). The RUN domain alone of even DENND5A

and RUSC2, however, was insufficient to recognize spe-

cific Rabs, i.e., a PLAT (polycystin-1, lipoxygenase, alpha-

toxin) domain adjacent to the RUN1 domain of DENND5A

and an N-terminal adjacent region of the RUN domain of

RUSC2 were also required for Rab6A binding activity

(Recacha et al., 2009) and Rab35 binding activity (Fig. 6),

respectively. These results indicated that RUN domains

alone do not function as general Rab-binding motifs and

that only a few RUN domains partly contribute to specific

Rab binding (i.e., the RUN domain is just part of the Rab-

Fig. 7. Characterization of RBD35, a specific trapper of GTP-Rab35.

(A) GTP-dependent binding of GST-RBD35 with FLAG-Rab35.

Glutathione-Sepharose beads coupled with GST alone (lanes 1 and 2) or

GST-RBD35 (lanes 3 and 4) were incubated with COS-7 cell lysates

expressing FLAG-Rab35 in the presence of 1 mM GDP (lanes 1 and 3) or

0.5 mM GTPγS (lanes 2 and 4). Proteins bound to the beads were analyzed

by 10% SDS-PAGE followed by immunoblotting with HRP-conjugated

anti-FLAG tag antibody (1/10,000 dilution; middle panel) and HRP-

conjugated anti-GST antibody (1/5000 dilution; bottom panel). Input

means 1/200 volume of the reaction mixture used for the pull down-assay

(top panel). (B) GTP-dependent binding of GST-RBD35 with FLAG-

Rab35(Q67L). Glutathione-Sepharose beads coupled with GST alone

(lanes 1 and 2) or GST-RBD35 (lanes 3 and 4) were incubated with COS-7

cell lysates expressing FLAG-Rab35(S22N) in the presence of 1 mM GDP

(CN; lanes 1 and 3) or FLAG-Rab35(Q67L) in the presence of 0.5 mM

GTPγS (CA; lanes 2 and 4). Proteins bound to the beads were analyzed by

10% SDS-PAGE followed by immunoblotting with HRP-conjugated anti-

FLAG tag antibody (1/10,000 dilution; middle panel) and HRP-conjugated

anti-GST antibody (1/5000 dilution; bottom panel). Input means 1/200

volume of the reaction mixture used for the pull down-assay (top panel).

Note that GST-RBD35 specifically trapped the GTP-bound form of Rab35

(lane 4 in A and B). (C) Effect of Rab35-GAP (TBC1D10C) and Rab35-

GEF (DENND1A) on the GTP-Rab35 level in cultured cells. COS-7 cells

were co-transfected with pEF-FLAG-Rab35 and control pEF-BOS vector

(Mock; lane 1), pEF-T7-TBC1D10C (lane 2, open arrowhead), or pEF-T7-

DENND1A (lane 3, closed arrowhead). COS-7 cell lysates were incubated

with glutathione-Sepharose beads coupled with GST-RBD35, and proteins

bound to the beads were analyzed by 10% SDS-PAGE followed by

immunoblotting with HRP-conjugated anti-FLAG tag antibody (1/10,000

dilution; second panel from the top) and HRP-conjugated anti-GST

antibody (1/5000 dilution; bottom panel). Input means 1/100 volume of the

reaction mixtures used for the GST pull-down assay (top panel and third

panel from the top). Note that the amount of GTP-Rab35 trapped by the

RBD35 beads was increased by co-expression of DENND1A and

decreased by co-expression of TBC1D10C. (D) Rab binding specificity of

RBD35. Glutathione-Sepharose beads coupled with GST alone (lanes 1–4)

or GST-RBD35 (lanes 5–8) were incubated with COS-7 cell lysates

expressing FLAG-Rab1A, FLAG-Rab1B, FLAG-Rab35, or FLAG-Rab41

in the presence of 0.5 mM GTPγS and 10 mM MgCl2. Proteins bound to

the beads were analyzed by 10% SDS-PAGE followed by immunoblotting

with HRP-conjugated anti-FLAG tag antibody (1/10,000 dilution; middle

panel) and HRP-conjugated anti-GST antibody (1/5000 dilution; bottom

panel). Input means 1/100 volume of the reaction mixtures used for the

GST pull-down assay (top panel). Note that GST-RBD35 specifically

trapped Rab35 (lane 7), but not Rab1A, Rab1B, or Rab41. (E) and (F)

GST-RBD35 was capable of trapping endogenous Rab35 molecules, but

not endogenous Rab1A molecules, in PC12 cells. PC12 cell lysates were

incubated with 2.5 mM EDTA and either 1 mM GDP (lanes 1 and 3) or 0.5

mM GTPγS (lanes 2 and 4) on ice for 30 min, and MgCl2 was then added to

a final concentration of 10 mM. Glutathione-Sepharose beads coupled with

GST alone (lanes 1 and 2) or GST-RBD35 (lanes 3 and 4) were incubated

for 2 hours with the above PC12 cell lysates. Proteins bound to the beads

were analyzed by 10% SDS-PAGE followed by immunoblotting with anti-

Rab35 antibody (1/200 dilution; second panel in E), anti-Rab1A antibody

(1/500 dilution; second panel in F), and HRP-conjugated anti-GST

antibody (1/5000 dilution; bottom panels). Input means 1/100 volume of

the reaction mixture used for the pull-down assay (top panels). The

positions of the molecular mass markers (Mr in kilodaltons) are shown on

the left.

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M. Fukuda et al.

binding site for DENND5A and RUSC2, and an additional

region adjacent to the RUN domain is required for Rab

binding activity).

Although RUSC2 was initially identified as a Rab1A/B-,

Rab35-, and Rab41-binding protein by yeast two-hybrid

assays (Fig. 2G), we succeeded in identifying the minimal

functional Rab35-specific-binding site of RUSC2 (amino

acid residues 982–1199) by a systematic deletion analysis

(Fig. 6). We then developed a novel RUN-domain-based

GTP-Rab35 trapper, which we named RBD35 (Fig. 7).

GST-RBD35 specifically trapped both recombinant and

endogenous Rab35 in a GTP-dependent manner (Fig. 7A,

B, and E), but it did not trap Rab1A at all (Fig. 7D and F)

despite the original RUSC2 construct being bound to

Rab1A (Fig. 2G). Because of the specific GTP-Rab35

binding ability of RBD35, overexpression of RBD35 with

an EGFP-tag in PC12 cells strongly inhibited NGF-induced

neurite outgrowth (Fig. 8), the same phenotype as overex-

pression of Rab35(CN) in PC12 cells (Chevallier et al.,

2009; Kanno et al., 2010). We therefore consider fluores-

cently labeled RBD35 to be an ideal tool for investigating

the function of Rab35 in membrane trafficking, and the

effect of RBD35 on other types of Rab35-dependent events,

including cytokinesis, receptor recycling, and actin remod-

eling, is now under investigation in our laboratory.

What function(s) do RUN domains have in common?

This is still an open question, because our findings clearly

rule out the possibility that Rab is a common ligand of all

RUN domains. Since some RUN domains have been shown

to bind Rap, another type of small GTPase (Janoueix-

Lerosey et al., 1998; Kukimoto-Niino et al., 2006; Yang et

al., 2007), Rap may be a common ligand of RUN domains.

However, we think that this possibility is also unlikely

because of the very low sequence conservation among the

Fig. 8. Overexpression of EGFP-tagged RBD35 in PC12 cells inhibited neurite outgrowth of PC12 cells. (A) Typical images of EGFP-RBD35-

expressing cells (middle panel), EGFP-RUSC2-ΔC4 (right panel), and EGFP-expressing control cells (left panel). Scale bars, 30 μm. (B) Effect of

expression of EGFP-RBD35, EGFP-RUSC2-ΔC4, and EGFP alone on neurite outgrowth of PC12 cells. Total neurite length values (mean and S.E.) of

EGFP-RBD35-expressing cells, EGFP-RUSC2-ΔC4-expressing cells, and control cells are shown. Note that expression of EGFP-RBD35 strongly inhibited

neurite outgrowth of PC12 cells (**, p<0.01, in comparison with the control EGFP-expressing cells or EGFP-RUSC2-ΔC4-expressing cells; Student’s

unpaired t-test). (C) Co-expression of mStr-Rab35 with EGFP-RBD35 in PC12 cells attenuated the inhibitory effect of RBD35 on neurite outgrowth of

PC12 cells (**, p<0.01, in comparison with the control EGFP-expressing cells or EGFP-RBD35+mStr-Rab35-expressing cells; Student’s unpaired t-test).

RUN domain and small GTPase Rab

169

known RUN domains (only one Tyr residue is invariant in

all RUN domains; asterisk in Supplemental Fig. S1B).

Actually, the Arg-168 of RUFY3/RPIPX in the positively

charged surface of the RUN domain, which has previously

been proposed to be involved in Rap2 binding (Kukimoto-

Niino et al., 2006), is not conserved in all other RUN

domains (# in Supplemental Fig. S1B). Moreover, the mini-

mal Rap2-binding site of RUFY3 has been shown to be 40

amino acids larger than the predicted RUN domain

(Kukimoto-Niino et al., 2006), indicating that the RUN

domain alone is insufficient to recognize Rap2. This obser-

vation is very similar to our own results showing that the

minimal Rab35-binding site of RUSC2 is larger than the

predicted RUN domain (Fig. 6). Since we basically used

larger fragments (or full-length proteins) than the predicted

RUN domains in the yeast two-hybrid assays, the lack of

Rab binding activity of most of the RUN proteins should

not be attributable to the truncation of the Rab-binding site.

Since RUN domains are highly diversified at the amino acid

level, it would be interesting to investigate whether the

RUN domain-containing region of RUN proteins functions

as a binding site of small GTPases other than Rab and Rap

in the future.

In summary, we systematically investigated the Rab

binding activity of 19 human/mouse RUN proteins and

identified four novel interactions, i.e., interactions between

PLEKHM2 and Rab1A, RUFY2 and Rab33A/B, RUFY3

and Rab33A, and RUSC2 and Rab35. We also developed

RBD35, a novel RUN-domain-based GTP-Rab35 trapper,

and demonstrated that overexpression of EGFP-RBD35

strongly inhibits Rab35-dependent neurite outgrowth of

PC12 cells. We therefore think that RBD35 will be a useful

tool that will make it easy to probe the specific involvement

of Rab35 in cellular phenomena and possibly shed light on

the molecular mechanism of Rab35-dependent membrane

trafficking.

Acknowledgments. We thank Megumi Aizawa, Miho Inaba, and Namiko

Kamiyama for technical assistance, and members of the Fukuda Labora-

tory for valuable discussions. This work was supported in part by Grants-

in-Aid for Scientific Research from the Ministry of Education, Culture,

Sports, Science and Technology (MEXT) of Japan (to N.O. and M.F.) and

by a grant from the Global COE Program (Basic & Translational Research

Center for Global Brain Science) of the MEXT of Japan (to N.O. and

M.F.). H.K. and K.I. were supported by the Japan Society for the Promo-

tion of Science.

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(Received for publication, January 13, 2011, accepted, June 30, 2011

and published online, July 7, 2011)