Molecular Biology of the Cell
Vol. 19, 971–983, March 2008
The Interaction of JRAB/MICAL-L2 with Rab8 and Rab13
Coordinates the Assembly of Tight Junctions and
Rie Yamamura,*†Noriyuki Nishimura,* Hiroyoshi Nakatsuji,* Seiji Arase,†
and Takuya Sasaki*
*Departments of Biochemistry and†Dermatology, Institute of Health Biosciences, The University of
Tokushima Graduate School, Tokushima 770-8503, Japan
Submitted June 11, 2007; Revised November 5, 2007; Accepted December 6, 2007
Monitoring Editor: Asma Nusrat
The assembly of tight junctions (TJs) and adherens junctions (AJs) is regulated by the transport of integral TJ and AJ
proteins to and/or from the plasma membrane (PM) and it is tightly coordinated in epithelial cells. We previously reported
that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) mediated
the endocytic recycling of an integral TJ protein occludin and the formation of functional TJs. Here, we investigated the
role of Rab13 and JRAB/MICAL-L2 in the transport of other integral TJ and AJ proteins claudin-1 and E-cadherin to the
PM by using a Ca2?-switch model. Although knockdown of Rab13 specifically suppressed claudin-1 and occludin but not
E-cadherin transport, knockdown of JRAB/MICAL-L2 and expression of its Rab13-binding domain (JRAB/MICAL-L2-C)
inhibited claudin-1, occludin, and E-cadherin transport. We then identified Rab8 as another JRAB/MICAL-L2-C-binding
protein. Knockdown of Rab8 inhibited the Rab13-independent transport of E-cadherin to the PM. Rab8 and Rab13
competed with each other for the binding to JRAB/MICAL-L2 and functionally associated with JRAB/MICAL-L2 at the
perinuclear recycling/storage compartments and PM, respectively. These results suggest that the interaction of JRAB/
MICAL-L2 with Rab8 and Rab13 coordinates the assembly of AJs and TJs.
Tight junctions (TJs) and adherens junctions (AJs) are lo-
cated at the apical end of the basolateral membrane of po-
larized epithelial cells. Epithelial polarization entails a com-
plex interplay between the coordinated assembly of TJs and
AJs, and several fundamental cellular processes. These in-
clude the activation of evolutionarily conserved polarity
protein complexes, the reorganization of actin and microtu-
bule cytoskeletons, the redistribution of organelles, and the
polarization of membrane traffic. During a series of pro-
cesses, epithelial cells first form spot-like primordial AJs at
the tips of the initial cell–cell contacts, and they develop
belt-like mature AJs. In parallel with the maturation of AJs,
TJs are formed at the apical side of AJs (Yap et al., 1997;
Vasioukhin et al., 2000; Nelson, 2003; Takai and Nakanishi,
2003). However, the molecular mechanism that coordinates
the assembly of TJs and AJs remains to be clarified.
While AJs principally initiate and maintain cell–cell con-
tacts, TJs seal the intercellular space and delineate the
boundaries between the apical and basolateral membranes.
Both TJs and AJs are composed of integral membrane pro-
teins and plaque proteins associated with the cytosolic side
of the plasma membrane. The principal integral TJ and AJ
proteins are claudins and E-cadherin, which form ho-
mophilic interactions with the same family of proteins on an
adjacent cell to form the characteristic structures of TJs and
AJs, respectively (Takeichi, 1995; Yap et al., 1997; Tsukita et
al., 2001). Additional integral TJ and AJ proteins include
occludin, junctional adhesion molecules, and nectins (Takai
and Nakanishi, 2003; Ebnet et al., 2004). These integral mem-
brane proteins are linked to a number of TJ and AJ plaque
proteins in the cytosol, including zonula occludens (ZO)
proteins (ZO-1, ZO-2, and ZO-3) and catenins, which in turn
bind the actin cytoskeleton. These TJ and AJ plaque proteins
have multiple protein binding motifs, and they form an
organizing platform for a variety of scaffolding, signaling,
and membrane trafficking proteins (Gonzalez-Mariscal et al.,
Accumulating evidence has revealed that the membrane
trafficking of integral TJ and AJ proteins plays a key role in
the assembly and maintenance of TJs and AJs (Ivanov et al.,
2005). Among a number of membrane trafficking proteins
identified, Rab family small G proteins are key regulators
(Takai et al., 2001; Zerial and McBride, 2001; Pfeffer and
Aivazian, 2004). More than 60 different Rab family members
have been identified in mammalian cells, and each member
recognizes distinct subsets of intracellular membranes. Rab
proteins cycle between the “inactive” guanosine diphos-
phate (GDP)-bound and “active” guanosine triphosphate
(GTP)-bound forms, and they also undergo a membrane
insertion and extraction cycle, allowing both spatial and
temporal control of their activity. GTP-bound Rab proteins
interact with specific effector proteins, and together they act
This article was published online ahead of print in MBC in Press
on December 19, 2007.
Address correspondence to: Takuya Sasaki (email@example.com.
Abbreviations used: AJ, adherens junction; CC, coiled-coil; PM,
plasma membrane; siRNA, small interfering RNA; TfR, transferrin
receptor; TJ, tight junction; ZO, zonula occludens.
© 2008 by The American Society for Cell Biology971
to translate the signal from one Rab protein to several di-
verse components of membrane trafficking. In epithelial
cells, Rab8 and Rab13 are identified as AJ and TJ plaque
proteins (Zahraoui et al., 1994; Lau and Mruk, 2003). While
Rab8 localizes to the trans-Golgi network, recycling endo-
somes, vesicular and tubular structures in the cytosol, mem-
brane protrusions, and plasma membrane (PM) in addition
to AJs (Huber et al., 1993b; Lau and Mruk, 2003; Ang et al.,
2004), Rab13 resides at perinuclear membrane structures,
vesicular structures in the cytosol, and PM in addition to TJs
(Zahraoui et al., 1994; Marzesco et al., 2002; Terai et al., 2006).
Rab8 interacts with Rab8ip/germinal center kinase (GCK),
JFC1/Slp1, and Optineurin in a GTP-dependent manner
(Ren et al., 1996; Hattula and Pera ¨nen, 2000; Hattula et al.,
2006), and it is implicated in multiple transport pathways,
including the epithelial-specific adaptor protein complex
AP-1B-dependent basolateral transport, the polarized mem-
brane traffic to the dendritic membrane, the actin-dependent
movement of melanosomes, the formation and destruction
of membrane protrusions, and the cell–cell adhesion (Huber
et al., 1993a; Ang et al., 2003; Powell and Temesvari, 2004;
Chabrillat et al., 2005; Hattula et al., 2006). Rab13 also binds
to protein kinase A (PKA), and it regulates the assembly of
functional TJs, neurite outgrowth, and neuronal regenera-
tion (Marzesco et al., 2002; Ko ¨hler et al., 2004; Di Giovanni et
al., 2005). However, the exact transport routes regulated by
Rab8 and Rab13 remain to be determined.
Claudins, occludin, and E-cadherin are transported to and
from the PM by multiple exocytic and endocytic pathways. For
their transport from the PM, three distinct endocytosis path-
ways—clathrin-dependent endocytosis, caveolin-dependent
endocytosis, and macropinocytosis—are identified in different
cellular contexts (Bryant and Stow, 2004; D’Souza-Schorey,
2005; Ivanov et al., 2005). Endocytosed claudins, occludin, and
E-cadherin are also detected in multiple sites, including early
endosome antigen (EEA)1-positive early endosomes, Rab11-
positive recycling endosomes, Rab7-positive late endosomes,
Rab13-positive vesicles, Syntaxin4-positive compartments, and
Syntaxin3-positive vacuolar apical compartments (Le et al.,
1999; Harhaj et al., 2002; Hopkins et al., 2003; Ivanov et al., 2004;
Matsuda et al., 2004; Balzac et al., 2005; Bruewer et al., 2005;
Morimoto et al., 2005; Utech et al., 2005). From these sites,
claudins, occludin, and E-cadherin can be transported to the
PM. Although a variety of regulatory molecules, including
actin and microtubule cytoskeletons, myosin II, Rac1, Cdc42,
ARF6, and Rab11 have been identified, the exact transport
routes of claudins, occludin, and E-cadherin to and from the
PM are not defined (Bryant and Stow, 2004; D’Souza-Schorey,
2005; Ivanov et al., 2005).
We previously reported that Rab13 regulated the endo-
cytic recycling of occludin, and we identified a junctional
Rab13-binding protein (JRAB)/molecule interacting with
CasL-like 2 (MICAL-L2) as a novel Rab13 effector protein
(Morimoto et al., 2005; Terai et al., 2006). Although JRAB/
MICAL-L2 was originally identified as a MICAL-related
cDNA (Terman et al., 2002), it also mediated the endocytic
recycling of occludin and regulated the formation of func-
tional TJs in epithelial cells (Terai et al., 2006). In the
present study, we investigated the role of Rab13 and
JRAB/MICAL-L2 in the transport of claudins and E-cad-
herin to the PM by using a well-established Ca2?-switch
model (Kartenbeck et al., 1991). Although Rab13 specifi-
cally mediated claudin but not E-cadherin transport,
JRAB/MICAL-L2 regulated both claudin and E-cadherin
transport. We then identified Rab8 as an additional JRAB/
MICAL-L2 effector protein that controlled the Rab13-in-
dependent transport of E-cadherin.
MATERIALS AND METHODS
pCI-neo-Myc-JRAB/MICAL-L2-C, pCI-neo-HA-Rab1A, pCI-neo-HA-Rab3B,
pCI-neo-HA-Rab5A, and pCI-neo-HA-Rab13 were described previously
(Yamamoto et al., 2003; Terai et al., 2006). Rab13 cDNA was cloned into the
pGEX-6P-1, pCI-neo-Myc, and pCI-neo-FLAG vectors. JRAB/MICAL-L2-CC
(amino acids 806-912) and JRAB/MICAL-L2-CT (amino acids 913-1009)
cDNAs were generated by polymerase chain reaction (PCR) by using pCI-
neo-Myc-JRAB/MICAL-L2-F as a template, and they were then cloned into
the pCI-neo-Myc vector. Mouse Rab8A cDNA was amplified by PCR by using
a yeast two-hybrid prey clone (pACT2-Rab8A) as a template, and then it was
cloned into pCI-neo-HA, pCI-neo-Myc, and pCI-neo-FLAG vectors. Canine
Rab4A, mouse Rab8B, mouse Rab10, and rat Rab11A cDNAs were isolated by
reverse transcription (RT)-PCR from Madin-Darby canine kidney (MDCK)
cells, NIH3T3 cells, MTD-1A cells, and rat brain, respectively, and then they
were cloned into the pCI-neo-HA vector. cDNAs for mouse MICAL-1
(Q8VDP3, 1048 amino acids), mouse MICAL-3 (Q8CJ19, 864 amino acids), and
mouse MICAL-L1 (Q8BGT6, 870 amino acids) were isolated by RT-PCR from
mouse lung, mouse brain, and MTD-1A cells, respectively. Mouse MICAL-2
(Q8BML1, 960 amino acids) cDNA was generated by PCR by using the RIKEN
FANTOM clone 5330438E18 (DNAFORM, Ibaraki, Japan) as a template.
MICAL-1, MICAL-2, MICAL-3, and MICAL-L1 cDNAs were cloned into the
pCI-neo-Myc and pCI-neo-HA vectors. All plasmids constructed in this study
were sequenced using an ABI Prism 3100 Genetic Analyzer (Applied Biosys-
tems, Foster City, CA).
Glutathione-S-transferase (GST) and N-terminal GST-tagged Rab13 (GST-
Rab13) proteins were expressed from the pGEX-6P-1 (GE Healthcare, Pisca-
taway, NJ) vector in Escherichia coli strain DH5? and purified by using
glutathione-Sepharose beads (GE Healthcare) according to the manufacturer’s
instructions. Two milligrams of GST or GST–Rab13 protein were immobilized
on HiTrap NHS-activated columns (GE Healthcare) according to the manu-
facturer’s instructions. Two female Wistar rats were immunized with 100 ?g
of GST–Rab13 protein twice at 4-wk intervals, after which whole blood from
the animals was collected. Crude immunoglobulin fractions were prepared by
ammonium sulfate precipitation and passed through a GST-immobilized
column to remove any anti-GST antibody. The anti-Rab13 polyclonal anti-
body was further purified on a GST–Rab13-immobilized column according to
the manufacturer’s instructions. The rat anti-JRAB/MICAL-L2 antibody was
described previously (Terai et al., 2006). The rat anti-occludin (MOC37) anti-
body was the kind gift from Dr. S. Tsukita (Kyoto University, Kyoto, Japan).
Rabbit anti-claudin-1 and rabbit anti-ZO-1 were purchased from Zymed
Laboratories (San Francisco, CA); rat anti-E-cadherin was from Takara (Otsu,
Japan); mouse anti-EEA1 and mouse anti-Rab8 were from BD Biosciences
(San Jose, CA); mouse anti-Golgi 58K, mouse anti-?-actin, and mouse anti-
FLAG (M2) were from Sigma-Aldrich (St. Louis, MO); mouse anti-mannose
6-phosphate receptor (M6PR) was from Affinity BioReagents (Golden, CO);
mouse anti-Myc (9E10) was from American Type Culture Collection (Manas-
sas, VA); mouse anti-hemagglutinin (HA) (12CA5) and rat anti-HA (3F10)
were from Roche Diagnostics (Mannheim, Germany); and rabbit anti-green
fluorescent protein (GFP) was from Invitrogen (Carlsbad, CA).
Cell Culture and Transfection
MDCK, MDCK I, and MTD-1A cells were kindly supplied by Dr. W. Birch-
meier (Max Delbrueck Center for Molecular Medicine, Berlin, Germany), Dr.
T. Tsukamoto (Kitano Hospital, Osaka, Japan), and Dr. S. Tsukita (Kyoto
University, Kyoto, Japan), respectively. Baby hamster kidney (BHK) and
NIH3T3 cells were obtained from American Type Culture Collection. MDCK,
MTD-1A, and BHK cells were cultured in DMEM with 10% fetal bovine
serum (FBS), and MDCK I cells were cultured in DMEM with 5% FBS. MDCK,
MDCK I, MTD-1A, and BHK cells were transfected using a Nucleofector
device (Amaxa, Ko ¨ln, Germany) or with Lipofectamine 2000 transfection
reagent (Invitrogen) according to the manufacturers’ instructions.
Recombinant Adenovirus Infection
The recombinant adenovirus expressing enhanced green fluorescent protein
(EGFP) and Myc-JRAB/MICAL-L2-C (Ad-EGFP and Ad-Myc-JRAB/MICAL-
L2-C) was described previously (Terai et al., 2006). MTD-1A cells were in-
fected with Ad-EGFP or Ad-Myc-JRAB/MICAL-L2-C at a multiplicity of
infection of 100.
The 21-mer small interfering RNA (siRNA) duplexes targeting canine Rab13
canine MICAL-1 (XM_539079), canine MICAL-L1 (XM_538381), and canine JRAB/
MICAL-L2 (XM_547017) and the control nonsilencing siRNA duplexes were ob-
tained from B-Bridge (Sunnyvale, CA), and they were transfected using a Nucleo-
fector device (Amaxa) according to the manufacturer’s instructions. The target
R. Yamamura et al.
Molecular Biology of the Cell972
sequences were as follows: canine Rab13 (#1, 5?-GAGGACAGCTTCAACAACA-3?;
#2, 5?-GACAATAACTACTGCATAT-3?; and #3, 5?-GCGCCTGCTTCTAGGGAAC-
3?), mouse JRAB/MICAL-L2 (#1, 5?-GGACAAACCCTGTGGTTCA-3?; #2, 5?-
GGACGGTTCAGGAGGCAAA-3?; and #3, 5?-GGCTGAAGCCTGTGGATAA-3?),
AGCTACA-3?; and #3, 5?-GGAATCAAGTTCATGGAGA-3?), canine MICAL-1 (5?-
GTGGTGAACCAGCGAGATA-3?), canine MICAL-L1 (5?-GAGAGAAGGTGCT-
GATGCA-3?), and canine JRAB/MICAL-L2 (5?-GCAGCAACATCGTGGACGT-3?).
Quantitative Real-Time RT-PCR
Total RNA from MDCK cells transfected with control RNA, MICAL-1 siRNA,
MICAL-L1 siRNA, and JRAB/MICAL-L2 siRNA duplexes were isolated
using BioRobot EZ1 with RNA Universal Tissue kit (QIAGEN, Valencia,
CA) and reverse transcribed using QuantiTect Reverse Transcription kit
(QIAGEN) according to the manufacturers’ instructions. Real-time PCR anal-
ysis was performed with an ABI 7500 Real-Time PCR System (Applied
Biosystems) by using FastStart Universal SYBR Green Master (Roche Diag-
nostics) according to the manufacturer’s specifications. Each sample was
analyzed in triplicates for each pair of primers. The relative expression of
MICAL-1, MICAL-L1, and JRAB/MICAL-L2 to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was calculated by the relative standard curve
method using Sequence Detection Software version 1.4 (Applied Biosystems).
Primer sequences were as follows: canine MICAL-1 (forward, 5?-ACCAG-
GAAGGAGCCCTAAAG-3? and reverse, 5?-CTCCTGGAAGCGGATAAGTG-
3?), canine MICAL-L1 (forward, 5?-CAAGATGTTGGAAGCCATGA-3? and
reverse, 5?-TAGCAGCTTCAGCACCTTCA-3?), canine JRAB/MICAL-L2 (for-
ward, 5?-CACCTCGTGCAGAGACACCT-3? and reverse: 5?-GTGCAGTGT-
GTTGGAGCACT-3?), and canine GAPDH (forward, 5?-TCAACGGATTTG-
GCCGTATTGG-3? and reverse: 5?-TGAAGGGGTCATTGATGGCG-3?).
Ca2?-switch assay was performed as described previously (Kartenbeck et al.,
1991). Briefly, MTD-1A or MDCK cells were grown in DMEM with 10% FBS
(normal Ca2?ion medium [NCM]) and sequentially incubated in Ca2?-free
minimal essential medium without FBS (low Ca2?ion medium [LCM]) for 1 h
and in LCM with 20 mM EGTA (for MTD-1A cells) or 5 mM EGTA (for
MDCK cells) for 2 h to remove extracellular Ca2?. Cells were then incubated
in NCM for varying periods and processed for immunofluorescence micros-
copy. Experiments in the presence of cycloheximide were performed by
incubating MDCK cells sequentially with LCM, LCM containing 5 mM EGTA
and 20 ?M cycloheximide, and NCM containing 20 ?M cycloheximide.
MDCK cells were cultured in serum-free DMEM for 1 h, incubated with 50
?g/ml Alexa 488-transferrin (Alexa-Tf; Invitrogen) for 1 h, and subjected to
either a Ca2?-switch assay in the continuous presence of 50 ?g/ml Alexa-Tf
or immunofluorescence microscopy.
MDCK and MTD-1A cells were grown on glass coverslips and fixed with one
of the following: ?20°C methanol (for anti-claudin-1 antibody) on ice for 5
min, 1% formaldehyde (for anti-occludin antibody) in phosphate-buffered
saline (PBS) at room temperature for 15 min, or 2% formaldehyde (for other
antibodies) in PBS at room temperature for 15 min. After permeabilization
with 0.2% Triton X-100 in PBS for 15 min and blocking with 5% goat serum in
PBS for 60 min, cells were incubated with primary antibodies for 60 min and
with Alexa 488-, or Alexa 594-conjugated secondary antibodies (Invitrogen) at
room temperature for 60 min. For triple-labeling, anti-FLAG (M2) antibody
was labeled with Pacific Blue by using a Zenon antibody labeling kit (Invitro-
gen). Fluorescent images were acquired using a Radiance 2000 confocal
laser-scanning microscope (Bio-Rad, Hercules, CA) or a C1plus confocal
laser-scanning microscope (Nikon, Tokyo, Japan).
Quantitation of Claudin-1, Occludin, and E-Cadherin
To quantify the mean length of claudin-1, occludin, and E-cadherin per cell,
the fields were randomly selected from fluorescent images to contain ?100
cells. Total length of claudin-1, occludin, and E-cadherin at cell–cell contact
sites was measured using Lumina Vision 2.4 program (Mitani, Fukui, Japan),
and the mean length per cell was calculated by total length/total cell number.
Statistical analysis was performed using Student’s t test.
Ratio of Colocalized Area
To calculate the ratio of colocalized area, the fields were selected from
fluorescent images to contain ?100 cells. The JRAB/MICAL-L2-E-cadherin
and JRAB/MICAL-L2-occludin colocalized area at perinuclear recycling/
storage compartment (PNC) and PM was measured using Lumina Vision 2.4
program (Mitani). Although the colocalized structures that positioned at
cell–cell contact sites were defined as PM, all other colocalized structures
were defined as PNC. Ratio was calculated by colocalized area at PNC/
colocalized area at PM.
Measurement of Transepithelial Electrical Resistance
MDCK I cells (8 ? 104) transfected with control RNA, MICAL-1 siRNA,
MICAL-L1 siRNA, or JRAB/MICAL-L2 siRNA were plated onto Transwell
filters (polycarbonate membranes with 12-mm diameter and 0.4-?m pore size;
Corning Life Sciences, Acton, MA) and grown in NCM for 72–96 h and subjected
to a Ca2?-switch assay in which the incubation in LCM with 5 mM EGTA was
shortened to 10 min. TER was measured directly in culture media at 6 and 12 h
after restoring Ca2?by using a Millicell-ERS epithelial voltohmmeter (Millipore,
blank filters and by multiplying the surface area of the filter.
JRAB/MICAL-L2-C was cloned into the yeast two-hybrid bait vector
pGBDU-C1 (James et al., 1996). A mouse 11-d-old embryo cDNA library in the
yeast two-hybrid prey vector pACT2 was purchased from Clontech (Palo
Alto, CA). The yeast strain PJ69-4A (MATa trp1-901 leu2-3112 ura3-52 his3-200
gal4? gal80? GAL2-ADE2 LYS2::GAL1-HIS3 met2::GAL7-lacZ) was sequen-
tially transformed with pGBDU-JRAB/MICAL-L2-C and the mouse 11-d-old
embryo cDNA library. Two-hybrid screening was performed and evaluated
as described previously (James et al., 1996).
BHK and MTD-1A cells were lysed with 25 mM Tris/HCl, pH 7.5, containing
0.5% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS),
125 mM NaCl, 1 mM MgCl2, 20 ?g/ml (4-amidinophenyl)-methanesulfonyl
fluoride, and 100 ?M guanosine 5?-O-(3-thio)triphosphate (GTP?S)/GDP at
4°C for 15 min. After removing a fraction of the lysates, the remaining lysates
were immunoprecipitated with the indicated antibodies bound to protein
G-Sepharose beads (GE Healthcare) and washed three times with 25 mM
Tris/HCl, pH 7.5, containing 0.1% CHAPS, 300 mM NaCl, 1 mM MgCl2, and
10 ?M GTP?S/GDP. The samples were prepared for Western blot analysis.
Proteins were separated by SDS-polyacrylamide gel electrophoresis, and pro-
teins were transferred to polyvinylidene difluoride membranes. Membrane
blocking and antibody dilutions were done in Block Ace (Dainippon Phar-
maceutical, Osaka, Japan). Blots were developed by chemiluminescence using
a horseradish peroxidase-coupled secondary antibody (Jackson ImmunoRe-
search Laboratories, West Grove, PA) and Immobilon Western Chemilumi-
nescent Horseradish Peroxidase Substrate (Millipore).
Rab13 Is Involved in the Transport of Claudin-1 and
Occludin, but not E-Cadherin, to the PM
We previously reported that Rab13 and JRAB/MICAL-L2 specif-
ically regulated the endocytic recycling of occludin and the as-
sembly of functional TJs (Terai et al., 2006). Because claudins but
not occludin are recognized as the primary integral TJ proteins
TJ and AJ proteins is observed in a variety of physiological and
pathological conditions (Ivanov et al., 2005), the question has nat-
urally arisen as to whether Rab13 and JRAB/MICAL-L2 also
present study, we have used a well established Ca2?-switch
model (Kartenbeck et al., 1991) to investigate the transport of
cells were first incubated in Ca2?-chelated medium to internalize
disassembly of cell–cell junctions. Subsequently, they were cul-
tured in physiological Ca2?medium to transport claudins, occlu-
din, and E-cadherin to the PM and to induce the synchronous
assembly of cell–cell junctions. The localizations of claudin-1, oc-
cludin, and E-cadherin were then analyzed by immunofluores-
cence microscopy at 0, 2, 4, 6, and 24 h after the restoration of
To examine the role of Rab13, we designed siRNAs
against the canine Rab13 sequence (XM_850035), and we
transfected them into MDCK cells. All Rab13 siRNAs sup-
Interaction of JRAB/MICAL-L2 with Rab8 and Rab13
Vol. 19, March 2008 973
pressed the expression of Rab13 protein in MDCK cells,
whereas a nonsilencing control RNA did not (Figure 1A).
The most effective Rab13-3 siRNA was used in the present
study. In MDCK cells transfected with control RNA or
Rab13 siRNA, claudin-1, occludin, and E-cadherin internal-
ized from the PM and accumulated intracellularly after Ca2?
removal, and each was then transported to the PM within 24 h
of Ca2?restoration (Supplemental Figure S1, A–C). However,
the kinetics of the transport of claudin-1 and occludin to the
PM seemed different. In accordance with previous reports us-
ing a GTP hydrolysis-defective mutant of Rab13 (Rab13 Q67L)
(Marzesco et al., 2002; Terai et al., 2006), the depletion of Rab13
slowed the kinetics of claudin-1 and occludin transport to the
PM at 2, 4, and 6 h after Ca2?restoration (Figure 1, B and C).
ing of and/or the appearance of continuous PM staining of
control cells at 2 and 4 h after Ca2?restoration (Figure 1D).
These results demonstrated that Rab13 was specifically re-
quired for the transport of claudin-1 and occludin, but not
E-cadherin, to the PM.
JRAB/MICAL-L2 Is Involved in the Transport of
Claudin-1, Occludin, and E-Cadherin to the PM
We next addressed the role of JRAB/MICAL-L2 in the trans-
port of claudin-1, occludin, and E-cadherin to the PM by using
a Ca2?-switch assay. To this end, we designed three siRNAs
targeting the mouse JRAB/MICAL-L2 sequence (AB182579).
Transfection of all three JRAB/MICAL-L2 siRNAs reduced
the expression of JRAB/MICAL-L2 relative to a nonsilenc-
ing control RNA in MTD-1A cells (Figure 2A). The JRAB/
MICAL-L2-3 siRNA suppressed the expression of JRAB/
MICAL-L2 most efficiently, so it was used in the present
study. Next, we investigated the transport of claudin-1, oc-
cludin, and E-cadherin to the PM during the Ca2?switch
in control and JRAB/MICAL-L2-depleted MTD-1A cells. In
contrast to Rab13-depleted cells, the knockdown of JRAB/
MICAL-L2 considerably delayed the disappearance of intra-
cellular staining and/or the appearance of continuous PM
staining of claudin-1, occludin, and E-cadherin (Figure 2,
B–D, and Supplemental Figure S2, A–C), indicating that
JRAB/MICAL-L2 is required for the transport of claudin-1,
occludin, and E-cadherin to the PM.
Expression of the Rab13-binding Domain of JRAB/
MICAL-L2 Inhibits the Transport of Claudin-1, Occludin,
and E-Cadherin to the PM
The above-mentioned results clearly demonstrated that both
Rab13 and JRAB/MICAL-L2 controlled the transport of
claudin-1 and occludin to the PM. However, it also revealed
that JRAB/MICAL-L2 possessed the Rab13-independent
din-1 and occludin, but not E-cadherin, to the PM. (A)
MDCK cells were transfected with control RNA or
Rab13 siRNA and subjected to Western blot analysis
by using anti-Rab13 and anti-?-actin antibodies. The
result shown is representative of three independent
experiments. (B–D) MDCK cells transfected with con-
trol RNA or Rab13 siRNA were subjected to a Ca2?-
switch assay and then immunostained with anti-clau-
din-1, anti-occludin, and anti-E-cadherin antibodies at
the indicated time after Ca2?restoration. Representa-
tive images of three independent experiments are
shown. Bars, 20 ?m. Claudin-1, occludin, and E-cad-
herin length is quantified and shown as the mean and
SEM. The asterisks denote a significant difference be-
tween control RNA and Rab13 siRNA (p ? 0.05).
Rab13 is involved in the transport of clau-
R. Yamamura et al.
Molecular Biology of the Cell974
function related to the transport of E-cadherin to the PM. To
explore its molecular mechanism, we first expressed the
Rab13-binding domain of JRAB/MICAL-L2 (JRAB/MICAL-
L2-C) in MTD-1A cells and examined its effect on the trans-
port of claudin-1, occludin, and E-cadherin to the PM by
using a Ca2?-switch assay (Figure 3A). Compared with
GFP-expressing MTD-1A cells, the appearance of continu-
ous PM staining of claudin-1, occludin, and E-cadherin was
substantially delayed in JRAB/MICAL-L2-C–expressing
cells (Figure 3, B–D, and Supplemental Figure S3, B–D).
Importantly, JRAB/MICAL-L2-C efficiently blocked the
Rab13-independent transport of E-cadherin to the PM.
Rab8 Is Identified as Another JRAB/MICAL-L2-binding
Protein that Mediates the Rab13-independent Transport of
E-Cadherin to the PM
Because JRAB/MICAL-L2-C inhibited the Rab13-indepen-
dent function of JRAB/MICAL-L2, it might interact with
other molecules in addition to Rab13. To test this possibility,
we screened a yeast two-hybrid library constructed from a
mouse 11-d-old embryo cDNA by using JRAB/MICAL-L2-C
as bait. From the 4.1 ? 106clones screened, we obtained two
independent clones encoding the mouse Rab8A sequence in
addition to nine independent clones encoding the mouse
Rab13 sequence. Both Rab8A prey clones encoded the full-
length mouse Rab8A sequence and specifically bound
JRAB/MICAL-L2-C (Figure 4A).
To confirm the yeast two-hybrid interaction between
Rab8A and JRAB/MICAL-L2 in intact cells, we next per-
formed coimmunoprecipitation experiments. When HA-
tagged Rab8A (HA-Rab8A) was cotransfected with Myc-
tagged JRAB/MICAL-L2 (Myc-JRAB/MICAL-L2-F) into
BHK cells, Myc-JRAB/MICAL-L2-F was specifically coim-
munoprecipitated with HA-Rab8A in the presence of
GTP?S, but not GDP (Figure 4B). Endogenous Rab8 was also
immunoprecipitated from MTD-1A cells with the anti-
JRAB/MICAL-L2 antibody (Supplemental Figure S4A). Be-
cause single Rab effector protein can interact with closely
related multiple Rab proteins (Fukuda, 2003), we then ex-
amined the interaction of JRAB/MICAL-L2 with other Rab
proteins. When Myc-JRAB/MICAL-L2 was coexpressed
with HA-Rab1A, HA-Rab3B, HA-Rab5A, HA-Rab8A, HA-
Rab8B, HA-Rab10, or HA-Rab13 in BHK cells, it coimmuno-
precipitated with HA-Rab8A, HA-Rab8B, and HA-Rab13,
but not with HA-Rab1A, HA-Rab3B, HA-Rab5A, and HA-
Rab10 (Figure 4C). We further investigated the interaction of
JRAB/MICAL-L2 with Rab proteins implicated in the regu-
lation of endocytic recycling. When HA-Rab4A, HA-Rab8A,
HA-Rab11A, and HA-Rab13 were expressed in MTD-1A
cells, endogenous JRAB/MICAL-L2 specifically interacted
transport of claudin-1, occludin, and E-cadherin to
the PM. (A) MTD-1A cells were transfected with
control RNA or JRAB/MICAL-L2 siRNA and sub-
jected to Western blot analysis by using anti-JRAB/
MICAL-L2 and anti-?-actin antibodies. The result
shown is representative of three independent exper-
iments. (B–D) MTD-1A cells transfected with control
RNA or JRAB/MICAL-L2 siRNA were subjected to
a Ca2?-switch assay and then immunostained with
anti-claudin-1, anti-occludin, and anti-E-cadherin
antibodies at the indicated time after Ca2?restora-
tion. Representative images of three independent
experiments are shown. Bars, 20 ?m. Claudin-1, oc-
cludin, and E-cadherin length is quantified and
shown as the mean and SEM. The asterisks denote a
significant difference between control RNA and
JRAB/MICAL-L2 siRNA (p ? 0.05).
JRAB/MICAL-L2 is involved in the
Interaction of JRAB/MICAL-L2 with Rab8 and Rab13
Vol. 19, March 2008975
with HA-Rab8A and HA-Rab13, but not with HA-Rab4A
and HA-Rab11A (Figure 4D). These results suggested that
JRAB/MICAL-L2 specifically interacted with Rab8 and
Rab13 in epithelial cells.
If JRAB/MICAL-L2 mediated the Rab13-independent
transport of E-cadherin to the PM and also interacted with
Rab8A, Rab8A was possibly involved. To test this possibil-
ity, we designed three siRNAs targeting the canine Rab8A
sequence (NM_001003152). Although all Rab8A siRNAs
suppressed the expression of Rab8A protein in MDCK cells
relative to a nonsilencing control RNA, the Rab8A-3 siRNA
was the most effective and used in the present study (Sup-
plemental Figure S4B). When we examined the transport of
E-cadherin to the PM during the Ca2?switch in control and
Rab8A-depleted MDCK cells, it was substantially delayed in
Rab8A-depleted cells relative to control cells (Figure 4E and
Supplemental Figure S4C).
MICAL Family Proteins Specifically Interacts with Rab
Because JRAB/MICAL-L2 is a member of MICAL family pro-
teins that is conserved from flies to mammals, with two MI-
CAL family genes (D-MICAL and D-MICAL-L) identified in
Drosophila and five (MICAL-1, MICAL-2, MICAL-3, MICAL-
L1, and JRAB/MICAL-L2) found in mammals (Figure 5A)
(Suzuki et al., 2002; Terman et al., 2002), we further investigated
the interaction of Rab8A and Rab13 with other MICAL family
proteins. HA-Rab8A or HA-Rab13 was coexpressed with Myc-
MICAL-1, Myc-MICAL-2, Myc-MICAL-3, Myc-MICAL-L1, or
Myc-JRAB/MICAL-L2 in BHK cells and analyzed by coimmu-
noprecipitation. Although HA-Rab8A precipitated with Myc-
MICAL-1, Myc-MICAL-L1, and Myc-JRAB/MICAL-L2 but
not with Myc-MICAL-2 and Myc-MICAL-3, HA-Rab13 prefer-
C). To explore the physiological significance of these interac-
tions, we next examined the role of MICAL-1, MICAL-L1, and
JRAB/MICAL-L2 on the development of TER, which is often
used to monitor the tightness of the seal created by functional
TJs. For this purpose, we designed siRNAs targeting canine
MICAL-1 (XM_539079), MICAL-L1 (XM_538381), and JRAB/
MICAL-L2 (XM_547017), and we monitored their effect by
quantitative real-time RT-PCR. MICAL-1, MICAL-L1, and
JRAB/MICAL-L2 siRNAs efficiently suppressed the mRNA
expression compared with a nonsilencing control RNA,
and were used in the present study (Figure 5D). When
cultured on permeable filters for 72–96 h, all MDCK I cells
transfected with control RNA, MICAL-1 siRNA, MICAL-L1
siRNA, and JRAB/MICAL-L2 siRNA showed the high TER
domain of JRAB/MICAL-L2 inhibits the
transport of claudin-1, occludin, and E-cad-
herin to the PM. (A) The Rab13-binding do-
main of JRAB/MICAL-L2. CH, calponin ho-
mology domain; LIM, LIM domain; and CC,
coiled-coil domain. (B–D) MTD-1A cells in-
fected with Ad-EGFP (GFP) or Ad-Myc-
JRAB/MICAL-L2-C (JRAB-C) were subjected
to a Ca2?-switch assay, and then they were
immunostained with anti-claudin-1, anti-occlu-
din, and anti-E-cadherin antibodies at the
indicated time after Ca2?restoration. Repre-
sentative images of three independent exper-
iments are shown. Bars, 20 ?m. Claudin-1,
occludin, and E-cadherin length is quantified
and shown as the mean and SEM. The aster-
isks denote a significant difference between
GFP and JRAB/MICAL-L2-C (p ? 0.05).
Expression of the Rab13-binding
R. Yamamura et al.
Molecular Biology of the Cell 976
value (?800 ?cm2). However, these cells differed in the
kinetics of TER development during a Ca2?-switch assay.
Consistent with our previous observations in MTD-1A cells
(Terai et al., 2006), the development of TER after 6 and 12 h
of Ca2?restoration was impaired in JRAB/MICAL-L2–de-
pleted MDCK I cells compared with control cells (Figure 5E).
Although we cannot formally exclude the involvement of
MICAL-1 and MICAL-L1 at present, these results suggest
that JRAB/MICAL-L2 is a key MICAL family member in the
assembly of functional TJs.
Rab8 Competes with Rab13 for the Binding of
If JRAB/MICAL-L2 physiologically interacted with both
Rab8 and Rab13, it could make either a tripartite complex
(Rab8-JRAB/MICAL-L2-Rab13) or two separate complexes
(Rab8-JRAB/MICAL-L2 and Rab13-JRAB/MICAL-L2). To
discriminate between these possibilities, we first tried to
identify the Rab8A- and Rab13-binding domains within
JRAB/MICAL-L2-C. For this purpose, we divided JRAB/
MICAL-L2-C (amino acids 806-1009) into two parts: JRAB/
MICAL-L2-CC, with the coiled-coil (CC) domain (amino
acids 806–912), and JRAB/MICAL-L2-CT, without the CC
domain (amino acids 913-1009) (Figure 6A). We then exam-
ined their binding to Rab8A and Rab13 by coimmunopre-
cipitation. Although Myc-JRAB/MICAL-L2-C efficiently in-
teracted with Rab8A, neither Myc-JRAB/MICAL-L2-CC
nor Myc-JRAB/MICAL-L2-CT bound Rab8A (Figure 6B).
Similarly, Myc-JRAB/MICAL-L2-C, but not Myc-JRAB/
Rab13 (Figure 6B).
The inability to differentiate the Rab8-binding domain from
the Rab13-binding domain prompted us to examine whether
Rab8 competed with Rab13 for the binding of JRAB/MICAL-
L2. To this end, we first attempted to produce Rab8 and Rab13
proteins in E. coli, but both proteins were only partially soluble,
and the purified proteins did not function in our in vitro
JRAB/MICAL-L2-binding assay. Therefore, we coexpressed a
constant amount of HA-tagged JRAB/MICAL-L2 (HA-JRAB/
MICAL-L2) and Myc-tagged Rab13 (Myc-Rab13) into BHK
cells together with increased amounts of Myc-tagged Rab8A
(Myc-Rab8A), and we assessed the interaction of JRAB/
MICAL-L2 with Rab13 and Rab8A by coimmunoprecipita-
tion. Without Rab8A expression, Rab13 was efficiently co-
immunoprecipitated with JRAB/MICAL-L2. However, the
coexpression of Rab8A inhibited the interaction of JRAB/
MICAL-L2 with Rab13 in a dose-dependent manner (Figure
6C). Next, we performed the reverse coimmunoprecipitation
JRAB/MICAL-L2-binding protein that medi-
ates the Rab13-independent transport of E-cad-
herin to the PM. (A) Yeast transformants carry-
ing the bait vector (pGBDU or pGBDU-JRAB/
MICAL-L2-C) and the prey vector (pACT2 or
pACT2-Rab8A) were spotted on synthetic com-
plete medium containing or lacking adenine to
score for ADE2 reporter activity, and then they
were incubated at 30°C for 3 d. (B) BHK cells
expressing Myc-JRAB/MICAL-L2-F with HA-
Rab8A were immunoprecipitated (IP) in the
presence of either GTP?S (GTP) or GDP with an
anti-HA antibody and subjected to Western blot
(WB) analysis by using anti-Myc and anti-HA
antibodies. The arrow indicates Myc-JRAB/
MICAL-L2-F, and the asterisk indicates a non-
specific band. (C) BHK cells expressing Myc-
JRAB/MICAL-L2 with HA-Rab1A, HA-Rab3B,
HA-Rab5A, HA-Rab8A, HA-Rab8B, HA-Rab10,
anti-HA antibody and subjected to Western blot
analysis by using anti-Myc and anti-HA antibod-
ies. The arrow indicates Myc-JRAB/MICAL-
L2-F, and the asterisk indicates the nonspecific
band. (D) MTD-1A cells expressing HA-Rab4A,
HA-Rab8A, HA-Rab11A, or HA-Rab13 were
immunoprecipitated with an anti-HA antibody
and subjected to Western blot analysis by using
anti-JRAB/MICAL-L2 and anti-HA antibodies.
The results shown in A–D are representative of
three independent experiments. (E) MDCK cells
transfected with control RNA or Rab8A siRNA
were subjected to a Ca2?-switch assay, and then
they were immunostained with an anti-E-cad-
herin antibody at the indicated time after Ca2?
restoration. Representative images of three in-
dependent experiments are shown. Bar, 20 ?m.
E-cadherin length is quantified and shown as
the mean and SEM. The asterisks denote a sig-
nificant difference between control RNA and
Rab8A siRNA (p ? 0.05).
Rab8A is identified as another
Interaction of JRAB/MICAL-L2 with Rab8 and Rab13
Vol. 19, March 2008977
to examine whether Rab13 competed with Rab8 for the
binding of JRAB/MICAL-L2. Like Rab8A, the increasing
amount of Rab13 expression efficiently displaced the JRAB/
MICAL-L2-bound Rab8A (Figure 6D). These results sug-
gested that Rab8 competed with Rab13 for the binding to
JRAB/MICAL-L2 and formed the distinct JRAB/MICAL-L2
complex from Rab13 within a single cell.
JRAB/MICAL-L2 Interacts with Rab8 and Rab13 at
Specific Intracellular Sites
We then analyzed the intracellular localization of Rab8,
Rab13, and JRAB/MICAL-L2 in MDCK cells. In agreement
with previous reports (Huber et al., 1993b; Marzesco et al.,
2002; Ang et al., 2004; Terai et al., 2006), Rab8A, Rab13, and
JRAB/MICAL-L2 were all detected at perinuclear mem-
brane structures, vesicular structures in the cytosol, and PM
JRAB/MICAL-L2 interacted with Rab8A and Rab13, FLAG-
tagged Rab8A (FLAG-Rab8A) or FLAG-tagged Rab13 (FLAG-
Rab13) was coexpressed with HA-JRAB/MICAL-L2 and cola-
beled with markers for the Golgi (Golgi 58K), the early
endosome (EEA1), the late endosome (M6PR), the recycling
endosome (Alexa-Tf), and the PM (ZO-1). Although FLAG-
Rab8A colocalized with HA-JRAB/MICAL-L2 at the sites la-
beled with the internalized Alexa-Tf, Golgi 58K, and ZO-1, a
closer spatial relationship between FLAG-Rab8A, HA-JRAB/
MICAL-L2, and the internalized Alexa-Tf was observed (Fig-
ure 7A). Similarly, FLAG-Rab13, HA-JRAB/MICAL-L2,
and ZO-1 showed a closer spatial relationship, albeit
FLAG-Rab13 also associated with HA-JRAB/MICAL-L2
at the internalized Alexa-Tf and Golgi 58K-positive sites
(Figure 7B). To further define the interaction sites of
JRAB/MICAL-L2 with Rab8A and Rab13, HA-JRAB/MI-
CAL-L2 was coexpressed with a GTP hydrolysis-defective
mutant of FLAG-Rab8A (FLAG-Rab8A Q67L) or FLAG-
Rab13 (FLAG-Rab13 Q67L) in MDCK cells. Although an
intracellular distribution pattern of FLAG-Rab8A Q67L
and FLAG-Rab13 Q67L was very similar to that of FLAG-
Rab8A and FLAG-Rab13, the distinct localization of
complexes was frequently emphasized in the presence of
FLAG-Rab8A Q67L and FLAG-Rab13 Q67L (Figure 7, C
Rab8-JRAB/MICAL-L2 and Rab13-JRAB/MICAL-L2
Complexes Are Involved in the Recycling of E-Cadherin
and Occludin to the PM
The existence of distinct Rab8-JRAB/MICAL-L2 and Rab13-
JRAB/MICAL-L2 complexes within a single cell prompted
us to examine the molecular mechanism of how two JRAB/
MICAL-L2 complexes control the transport of E-cadherin
and occludin to the PM. To define the transport routes taken
with Rab family proteins. (A) Structures of MICAL
family proteins. FAD, FAD-binding domain; CH, cal-
ponin homology domain; LIM, LIM domain; and CC,
coiled-coil domain. (B and C) BHK cells expressing
Myc-MICAL-1, Myc-MICAL-2, Myc-MICAL-3, Myc-
and subjected to WB analysis by using anti-Myc
and anti-HA antibodies. The arrows indicate Myc-
MICAL-1, Myc-MICAL-L1, and Myc-JRAB/MICAL-
L2, and the asterisks indicate nonspecific bands. The
results shown in B and C are representative of three
independent experiments. (D) Total RNA was pre-
pared from MDCK I cells transfected with control
RNA, MICAL-1 siRNA, MICAL-L1 siRNA, or JRAB/
MICAL-L2 siRNA, and the relative mRNA expression
of MICAL-1, MICAL-L1, and JRAB/MICAL-L2 to
GAPDH was determined by quantitative real-time
PCR analysis. Data were shown as the mean and SEM
for triplicate determinations. The mean expression of
MICAL-1, MICAL-L1, and JRAB/MICAL-L2 mRNA
in MDCK I cells transfected with control RNA was set
to 1. (E) MDCK I cells transfected with control RNA,
MICAL-1 siRNA, MICAL-L1 siRNA, and JRAB/
MICAL-L2 siRNA were subjected to Ca2?-switch as-
say. TER was measured at 6 and 12 h after Ca2?
restoration. The results shown are the mean and SEM
of three independent experiments.
R. Yamamura et al.
Molecular Biology of the Cell 978
by E-cadherin and occludin, we first performed a Ca2?-
switch assay in the presence of cycloheximide to stop de
novo protein synthesis and expose the role of the PNC.
When MDCK cells were subjected to a Ca2?-switch assay in
the presence of cycloheximide, E-cadherin and occludin
were internalized from the PM and accumulated in the PNC,
where the internalized Alexa-Tf was also detected, after
Ca2?removal (Figure 8A). Then, they were gradually trans-
ported from the PNC to the PM during 3 h of Ca2?restora-
tion and detected both in the PNC and PM at 1 and 1.5 h
after Ca2?restoration (Figure 8A). We next examined the
colocalization of Rab8-JRAB/MICAL-L2 and Rab13-JRAB/
MICAL-L2 complexes with E-cadherin and occludin at 1 h
after Ca2?restoration. When MDCK cells coexpressing Myc-
JRAB/MICAL-L2 with FLAG-Rab8A or FLAG-Rab13 were
examined at 1 h after Ca2?restoration, the FLAG-Rab8A-
Myc-JRAB/MICAL-L2 complex was colocalized with E-cad-
herin both at the PNC and PM, whereas the cytosolic vesic-
ular structures containing all of FLAG-Rab8A, Myc-JRAB/
MICAL-L2, and E-cadherin were barely evident (Figure 8B).
Similarly, the colocalization of FLAG-Rab13-Myc-JRAB/
MICAL-L2 complex with occludin was also detected both at
the PNC and PM, but hardly in the cytosolic vesicular struc-
tures, at 1 h after Ca2?restoration (Figure 8B).
To define the role of Rab8-JRAB/MICAL-L2 and Rab13-
JRAB/MICAL-L2 complexes in the recycling of E-cadherin
and occludin from the PNC to the PM, we depleted JRAB/
MICAL-L2, Rab8A, and Rab13 in MDCK cells, and we per-
formed a Ca2?-switch assay in the presence of cyclohexi-
mide. Compared with control MDCK cells, the recycling of
both E-cadherin and occludin was inhibited in JRAB/
MICAL-L2-depleted cells at 1 h after Ca2?restoration (Figure
of E-cadherin and occludin at 1 h after Ca2?restoration, re-
spectively (Figure 8, D and E). To get an insight into the
functional sites for the Rab8A-JRAB/MICAL-L2 and Rab13-
JRAB/MICAL-L2 complexes, we next measured the JRAB/
ized area at the PNC and PM in control, Rab8A-depleted, and
Rab13-depleted cells at 1 h after Ca2?restoration. Consistent
with the decrease in the mean E-cadherin and occludin length
(Figure 8, D and E), the JRAB/MICAL-L2-E-cadherin and
JRAB/MICAL-L2-occludin colocalized area at the PM was also
tively. However, the ratio of the JRAB/MICAL-L2-E-cadherin
colocalized area at the PNC to that at the PM was slightly
decreased in Rab8A-depleted cells (0.58 ? 0.39) compared
with control cells (0.61 ? 0.16) (Figure 8D), whereas that
of JRAB/MICAL-L2-occludin was increased in Rab13-de-
pleted cells (0.69 ? 0.35) compared with control cells
(0.21 ? 0.12) (Figure 8E). These opposite changes in the
ratio of colocalized area suggest the distinct roles of
complexes at the PNC and PM.
The formation and destruction of TJs and AJs are essential
for the physiological development of multicellular organ-
isms. Failure in their regulation is manifested in a variety of
diseases, such as tissue fibrosis and tumor invasion/metas-
tasis. To gain insight into the molecular mechanisms of TJ
and AJ assembly, we previously showed that Rab13 and
JRAB/MICAL-L2 mediated the endocytic recycling of occlu-
din and regulated the assembly of functional TJs (Morimoto
et al., 2005; Terai et al., 2006). Our present study revealed five
key findings. First, JRAB/MICAL-L2 regulated the transport
of both TJ and AJ proteins to the PM, whereas Rab13 spe-
cifically mediated that of TJ but not AJ proteins. Second,
JRAB/MICAL-L2 also interacted with Rab8, which medi-
ated the Rab13-independent transport of AJ protein to the
binding to JRAB/MICAL-L2. (A) Structures
of JRAB/MICAL-L2-C, JRAB/MICAL-L2-CC,
and JRAB/MICAL-L2-CT proteins. CH, cal-
ponin homology domain; LIM, LIM domain;
and CC, coiled-coil domain. (B) BHK cells
JRAB/MICAL-L2-CC, or Myc-JRAB/MICAL-
L2-CT with HA-Rab8A or HA-Rab13 were IP
with an anti-HA antibody and subjected to
WB analysis by using anti-Myc and anti-HA
antibodies. The arrow indicates Myc-JRAB/
MICAL-L2-C, and the asterisks indicate non-
specific bands. (C) BHK cells cotransfected
with pCI-neo-HA-JRAB/MICAL-L2 (?, 2.0
?g) and pCI-neo-Myc-Rab13 (?, 1.0 ?g) to-
gether with pCI-neo-Myc (?, 2.0 ?g), pCI-
neo-Myc ? pCI-neo-Myc-Rab8A (?, 1.5 ? 0.5
?g), or pCI-neo-Myc-Rab8A (??, 2.0 ?g)
were IP with an anti-HA antibody and sub-
jected to WB analysis by using anti-Rab13,
anti-Rab8, and anti-JRAB/MICAL-L2 anti-
bodies. (D) BHK cells cotransfected with pCI-
neo-HA-JRAB/MICAL-L2 (?, 2.0 ?g) and
pCI-neo-Myc-Rab8A (?, 1.0 ?g) together with
pCI-neo-Myc (?, 2.0 ?g), pCI-neo-Myc ? pCI-
neo-Myc-Rab13 (?, 1.5 ? 0.5 ?g), or pCI-neo-
Myc-Rab13 (??, 2.0 ?g) were IP and sub-
jected to WB analysis as described in C. The
results shown in B–D are representative of
three independent experiments.
Rab8 competes with Rab13 for the
Interaction of JRAB/MICAL-L2 with Rab8 and Rab13
Vol. 19, March 2008 979
PM. Third, Rab8 and Rab13 competed with each other to
bind JRAB/MICAL-L2. Fourth, JRAB/MICAL-L2 mainly
associated with Rab8 and Rab13 at the PNC and PM,
respectively. Fifth, depletion of Rab8 and Rab13 impaired
the colocalization of JRAB/MICAL-L2 with AJ protein
at the PNC and TJ protein at the PM, respectively. Col-
lectively, these results suggest that JRAB/MICAL-L2 co-
ordinates the assembly of AJ and TJ by forming the dis-
tinct Rab8-JRAB/MICAL-L2 and Rab13-JRAB/MICAL-L2
complexes (Figure 9).
A key finding in the present study is the identification
of JRAB/MICAL-L2 as a shared Rab effector protein that
forms mutually distinct complexes with Rab8 and Rab13.
To ensure the proper transport and maintain the identity
of intracellular membrane compartments, the action of
each Rab protein needs to be coordinated with other Rab
proteins (Markgraf et al., 2007). The Rab coupling is potentially
mediated by Rab-binding proteins that can interact with mul-
tiple Rab proteins. Currently, two types of these Rab-binding
proteins are identified. One type comprises Rab-binding pro-
teins that function as an effector protein for one Rab protein
and as a GTP exchange factor (GEF) for another Rab protein.
The identification of this type of Rab-binding proteins that
include Sec2 and the class C-VPS/HOPS complex leads to the
concept of a Rab cascade (Ortiz et al., 2002; Rink et al., 2005).
Another type is a divalent Rab effector protein that binds
simultaneously to two Rab proteins associated with compart-
ments in dynamic continuity. Rabaptin5, Rabenosyn5, and
Rabip4? are able to interact simultaneously with Rab4 and
Rab5, and they are likely involved in the coordination of the
endocytic recycling pathway and the organization of Rab4 and
Rab5 domains on endosomal membranes (Vitale et al., 1998; de
Renzis et al., 2002; Fouraux et al., 2004). Although an increasing
number of Rab effector proteins are reported to interact with
closely related multiple Rab proteins (Fukuda, 2003), their
functional significance still remains to be determined. To our
knowledge, JRAB/MICAL-L2 is a novel type of Rab effector
protein that associate with closely related Rab proteins forming
mutually exclusive complexes.
In the present study, we found that JRAB/MICAL-L2
controlled the transport of claudins, occludin, and E-cad-
herin to the PM, and Rab8 regulated the Rab13-independent
transport of E-cadherin to the PM. Although knockdown of
Rab8 also impaired the transport of claudins and occludin
(Supplemental Figure S4, D and E), this could be caused by
the inhibition of E-cadherin transport as TJ protein transport
was depend on AJ protein transport in a Ca2?-switch model
(Yap et al., 1997; Takai and Nakanishi, 2003). The present
study adds JRAB/MICAL-L2 and Rab8 to a growing list of
regulatory molecules for E-cadherin trafficking (Bryant and
Stow, 2004; D’Souza-Schorey, 2005; Ivanov et al., 2005). Al-
though Rab8 is functionally linked to epithelial specific
clathrin adaptor complexes AP-1B and E-cadherin is initially
recognized as an AP-1B-independent basolateral cargo
(Miranda et al., 2001; Ang et al., 2003; Fo ¨lsch, 2005), a recent
observation that phosphatidylinositol-4-phosphate 5-kinase
JRAB Rab13 Q67L
JRAB Rab8 Q67LMerge
Rab8 and Rab13 at specific intracellular sites.
(A and B) MDCK cells coexpressing HA-
JRAB/MICAL-L2 with FLAG-Rab8A (A) or
FLAG-Rab13 (B) were triple labeled with an-
ti-FLAG antibody, anti-HA antibody, and or-
ganella markers (anti-EEA1 antibody, anti-
M6PR antibody, Alexa-Tf, anti-Golgi 58K
antibody, and anti-ZO-1 antibody). Magnified
images in inserts show the notable colocaliza-
tion with organella markers. Bars, 20 ?m. (C
and D) MDCK cells coexpressing HA-JRAB/
MICAL-L2 with FLAG-Rab8A Q67L (C) or
FLAG-Rab13 Q67L (D) were double labeled
with anti-FLAG and anti-HA antibodies. In the
merged images, Rab8A Q67L/Rab13 Q67L was
green and JRAB was red. The arrowheads in-
dicate the sites of prominent colocalization.
Bars, 20 ?m. The results shown in A–D are
representative of three independent experi-
JRAB/MICAL-L2 interacts with
R. Yamamura et al.
Molecular Biology of the Cell980
? modulates the basolateral transport of E-cadherin and
directly binds both E-cadherin and AP-1B supports our re-
sults (Ling et al., 2007). Furthermore, Rab8 is recently shown
to colocalize with Rab11 and ARF6, both of which are im-
plicated in the transport of E-cadherin, and it is functionally
linked to ARF6 (Palacios et al., 2001; Lock and Stow, 2005;
Hattula et al., 2006). Collectively, Rab8, Rab11, and ARF6
seem to coordinate the transport of E-cadherin to the PM.
Although the exact nature of this coordination remains elu-
sive, one possible explanation would be that Rab8, Rab11,
and ARF6 sequentially act through the same transport path-
way of E-cadherin. In this scenario, a cascade of Rab8,
Rab11, and ARF6 would be expected. Alternatively, Rab8,
Rab11, and ARF6 could simultaneously work on the distinct
transport pathways of E-cadherin. This case would predict
the existence of different populations of transport carriers
Then, the question of how JRAB/MICAL-L2 works as a
shared Rab8 and Rab13 effector protein on the transport of
E-cadherin and occludin to the PM has naturally arisen.
As JRAB/MICAL-L2 is directly or indirectly associated
with actin cytoskeletons (Terai et al., 2006), we speculate
the involvement of JRAB/MICAL-L2 in the membrane-
actin cytoskeleton interactions, and we can formulate sev-
eral models to explain the present results. Considering a
widespread concept that Rab and its effector proteins can
recruit myosin motors onto the transport carrier mem-
branes (Seabra and Coudrier, 2004), Rab8-JRAB/MI-
CAL-L2 and Rab13-JRAB/MICAL-L2 complexes could re-
cruit myosin motors onto the transport carrier membranes
containing E-cadherin and occludin, respectively. Al-
though we did not detect the colocalization of Rab8-
JRAB/MICAL-L2 and Rab13-JRAB/MICAL-L2 on the
transport carriers, we cannot formally exclude this model
at the moment. Alternatively, Rab8-JRAB/MICAL-L2 and
Rab13-JRAB/MICAL-L2 complexes could mediate the
membrane-actin cytoskeleton interactions at the PNC and
PM, respectively. According to this model, Rab8-JRAB/
MICAL-L2 complex would sort E-cadherin within the
PNC membranes or generate E-cadherin transport carrier
from the PNC, whereas Rab13-JRAB/MICAL-L2 complex
would tether/dock/fuse occludin transport carrier with
JRAB/MICAL-L2 complexes are involved in
the recycling of E-cadherin and occludin to the
PM. (A) MDCK cells labeled with Alexa-Tf
were subjected to a Ca2?-switch assay in the
presence of cycloheximide and then immuno-
stained with anti-E-cadherin or anti-occludin
antibody at the indicated time after Ca2?resto-
ration. The arrowheads indicate the overlap.
Bar, 20 ?m. (B) MDCK cells coexpressing HA-
JRAB/MICAL-L2 with FLAG-Rab8A or FLAG-
Rab13 were subjected to a Ca2?-switch assay in
the presence of cycloheximide, and then they
were triple labeled with anti-HA, anti-FLAG,
and anti-E-cadherin or anti-occludin antibodies
at 1 h after Ca2?restoration. The arrows and
arrowheads indicate the colocalization at the
PM and PNC, respectively. Bar, 20 ?m. (C–E)
MDCK cells transfected with control RNA or
JRAB/MICAL-L2 siRNA (C), pCI-neo-HA-
JRAB/MICAL-L2 with control RNA or Rab8A
siRNA (D), or pCI-neo-HA-JRAB/MICAL-L2
with control RNA or Rab13 siRNA (E) were
of cycloheximide and then immunostained
with anti-E-cadherin, anti-occludin, and anti-
HA antibodies at 1 h after Ca2?restoration.
E-cadherin or occludin length was quantified
and shown as the mean and SEM. The aster-
isks denote a significant difference (p ? 0.05).
The ratio of JRAB/MICAL-L2-E-cadherin or
JRAB/MICAL-L2-occludin colocalized area
at the PNC to that at the PM was quantified
and shown as the mean and SEM. The arrows
and arrowheads indicate the colocalization at
the PM and PNC, respectively. Bars, 20 ?m.
The images shown in A–E are representative
of three independent experiments.
Rab8-JRAB/MICAL-L2 and Rab13-
Interaction of JRAB/MICAL-L2 with Rab8 and Rab13
Vol. 19, March 2008981
the PM. JRAB/MICAL-L2 would directly mediate these
processes or scaffold the executing molecule(s) as it con-
tains multiple protein–protein interaction domains. The
further determination of the JRAB/MICAL-L2-interacting
molecules would be essential for this model.
Another important question is how JRAB/MICAL-L2
controls its interaction with Rab8 and Rab13. Although
the intracellular distribution of Rab8, Rab13, and JRAB/
MICAL-L2 were overlapped at the PNC and PM, the
Rab8-JRAB/MICAL-L2 and Rab13-JRAB/MICAL-L2 in-
teractions were preferentially detected at the PNC and
PM, respectively. Because Rab8 and Rab13 compete with
each other for the interaction with JRAB/MICAL-L2 (Fig-
ure 6, C and D), there must exist the additional mecha-
nism(s) to activate/stabilize the Rab8-JRAB/MICAL-L2
and Rab13-JRAB/MICAL-L2 interactions at the PNC
and PM, respectively. Alternatively, the Rab8-JRAB/
might be inactivated/prevented at the PM and PNC, re-
spectively. In support of stabilizing mechanism for the
Rab13-JRAB/MICAL-L2 interaction at the PM, JRAB/
MICAL-L2 also interacted with actinin-4 and made the
Rab13-JRAB/MICAL-L2-actinin-4 complex at TJs (our un-
and/or preventing mechanisms for the Rab8-JRAB/
MICAL-L2 and Rab13-JRAB/MICAL-L2 interactions are
currently under investigation.
In accordance with the involvement of JRAB/MICAL-L2
in TJ and AJ assembly, a series of recent studies begin to
reveal the potential role of MICAL family proteins in the
regulation of invasive growth (Comoglio and Trusolino,
2002). MICAL-1, MICAL-2, and MICAL-3 are shown to
function downstream of the Semaphorin 3 receptor Plexin A
during axon guidance (Terman et al., 2002). The MICAL-2
isoforms (MICAL-2 PVa and PVb) are implicated in the
progression of prostate cancer (Ashida et al., 2006). Consis-
tent with the obligatory demand for extensive membrane
trafficking and cytoskeletal rearrangement during invasive
growth, the interactions of MICAL family proteins with
Rab1, vimentin, and microtubules are also detected (Suzuki
et al., 2002; Weide et al., 2003; Fischer et al., 2005).
In summary, our results suggest that JRAB/MICAL-L2 rep-
resents a novel type of a shared Rab effector protein that forms
mutually distinct complexes with closely related Rab8 and
Rab13. JRAB/MICAL-L2 interacts with the GTP-bound forms
of Rab8 and Rab13 at the PNC and PM, respectively, and the
Rab8-JRAB/MICAL-L2 and Rab13-JRAB/MICAL-L2 com-
plexes coordinate the assembly of AJs and TJs through the
dependent claudins/occludin transport.
We thank Dr. W. Birchmeier for the MDCK cells, Dr. T. Tsukamoto for the
MDCK I cells, and Dr. S. Tsukita for the MTD-1A cells and the anti-occludin
(MOC37) antibody. This study was supported by Grants-in-Aid for Scientific
Research (18590271 to N.N. and 15079207, 18390089 to T.S.) from the Ministry
of Education, Culture, Sports, Science and Technology of Japan.
Ang, A., Fo ¨lsch, H., Koivisto, U., Pypaert, M., and Mellman, I. (2003). The
Rab8 GTPase selectively regulates AP-1B-dependent basolateral transport in
polarized Madin-Darby canine kidney cells. J. Cell Biol. 163, 339–350.
Ang, A., Taguchi, T., Francis, S., Fo ¨lsch, H., Murrells, L., Pypaert, M., Warren,
G., and Mellman, I. (2004). Recycling endosomes can serve as intermediates
during transport from the Golgi to the plasma membrane of MDCK cells.
J. Cell Biol. 167, 531–543.
Ashida, S. et al. (2006). Expression of novel molecules, MICAL2-PV (MICAL2
prostate cancer variants), increases with high Gleason score and prostate
cancer progression. Clin. Cancer Res. 12, 2767–2773.
Balzac, F., Avolio, M., Degani, S., Kaverina, I., Torti, M., Silengo, L., Small, J.,
and Retta, S. (2005). E-cadherin endocytosis regulates the activity of Rap 1, a
traffic light GTPase at the crossroads between cadherin and integrin function.
J. Cell Sci. 118, 4765–4783.
Bruewer, M., Utech, M., Ivanov, A., Hopkins, A., Parkos, C., and Nusrat, A.
(2005). Interferon-? induces internalization of epithelial tight junction proteins
via a macropinocytosis-like process. FASEB J. 19, 923–933.
Bryant, D., and Stow, J. (2004). The ins and outs of E-cadherin trafficking.
Trends Cell Biol. 14, 427–434.
Chabrillat, M., Wilhelm, C., Wasmeier, C., Sviderskaya, E., Louvard, D., and
Coudrier, E. (2005). Rab8 regulates the actin-based movement of melano-
somes. Mol. Biol. Cell 16, 1640–1650.
Comoglio, P., and Trusolino, L. (2002). Invasive growth: from development to
metastasis. J. Clin. Invest. 109, 857–862.
D’Souza-Schorey, C. (2005). Disassembling adherens junctions: breaking up is
hard to do. Trends Cell Biol. 15, 19–26.
de Renzis, S., So ¨nnichsen, B., and Zerial, M. (2002). Divalent Rab effectors
regulate the sub-compartmental organization and sorting of early endosomes.
Nat. Cell Biol. 4, 124–133.
Di Giovanni, S., De Biase, A., Yakovlev, A., Finn, T., Beers, J., Hoffman, E., and
Faden, A. (2005). In vivo and in vitro characterization of novel neuronal
plasticity factors identified following spinal cord injury. J. Biol. Chem. 280,
Ebnet, K., Suzuki, A., Ohno, S., and Vestweber, D. (2004). Junctional adhesion
molecules (JAMs): more molecules with dual functions? J. Cell Sci. 117, 19–29.
Fischer, J., Weide, T., and Barnekow, A. (2005). The MICAL proteins and rab
1, a possible link to the cytoskeleton? Biochem. Biophys. Res. Commun. 328,
Fo ¨lsch, H. (2005). The building blocks for basolateral vesicles in polarized
epithelial cells. Trends Cell Biol. 15, 222–228.
Fouraux, M., Deneka, M., Ivan, V., van der Heijden, A., Raymackers, J., van
Suylekom, D., van Venrooij, W., van der Sluijs, P., and Pruijn, G. (2004).
Rabip4? is an effector of rab5 and rab4 and regulates transport through early
endosomes. Mol. Biol. Cell 15, 611–624.
Fukuda, M. (2003). Distinct Rab binding specificity of Rim1, Rim2, rabphilin,
and Noc2. Identification of a critical determinant of Rab3A/Rab27A recogni-
tion by Rim2. J. Biol. Chem. 278, 15373–15380.
Gonzalez-Mariscal, L., Betanzos, A., Nava, P., and Jaramillo, B. (2003). Tight
junction proteins. Prog. Biophys. Mol. Biol. 81, 1–44.
MICAL-L2 and Rab13-JRAB/MICAL-L2 complexes in the assembly
of AJs and TJs. Whereas the Rab8-JRAB/MICAL-L2 complex re-
sided at the PNC mediates the recycling of E-cadherin to the PM
and the assembly of AJs, the Rab13-JRAB/MICAL-L2 complex re-
sided at the PM regulates the recycling of claudins and occludin to
the PM and the formation of TJs. N, nucleus.
Schematic model for the action of Rab8-JRAB/
R. Yamamura et al.
Molecular Biology of the Cell982
Harhaj, N., Barber, A., and Antonetti, D. (2002). Platelet-derived growth factor
mediates tight junction redistribution and increases permeability in MDCK
cells. J. Cell. Physiol. 193, 349–364.
Hattula, K., and Pera ¨nen, J. (2000). FIP-2, a coiled-coil protein, links Hunting-
tin to Rab8 and modulates cellular morphogenesis. Curr. Biol. 10, 1603–1606.
Hattula, K., Furuhjelm, J., Tikkanen, J., Tanhuanpaa, K., Laakkonen, P., and
Peranen, J. (2006). Characterization of the Rab8-specific membrane traffic
route linked to protrusion formation. J. Cell Sci. 119, 4866–4877.
Hopkins, A., Walsh, S., Verkade, P., Boquet, P., and Nusrat, A. (2003). Con-
stitutive activation of Rho proteins by CNF-1 influences tight junction struc-
ture and epithelial barrier function. J. Cell Sci. 116, 725–742.
Huber, L., de Hoop, M., Dupree, P., Zerial, M., Simons, K., and Dotti, C.
(1993a). Protein transport to the dendritic plasma membrane of cultured
neurons is regulated by rab8p. J. Cell Biol. 123, 47–55.
Huber, L., Pimplikar, S., Parton, R., Virta, H., Zerial, M., and Simons, K.
(1993b). Rab8, a small GTPase involved in vesicular traffic between the TGN
and the basolateral plasma membrane. J. Cell Biol. 123, 35–45.
Ivanov, A., Nusrat, A., and Parkos, C. (2004). Endocytosis of epithelial apical
junctional proteins by a clathrin-mediated pathway into a unique storage
compartment. Mol. Biol. Cell 15, 176–188.
Ivanov, A., Nusrat, A., and Parkos, C. (2005). Endocytosis of the apical
junctional complex: mechanisms and possible roles in regulation of epithelial
barriers. Bioessays 27, 356–365.
James, P., Halladay, J., and Craig, E. (1996). Genomic libraries and a host
strain designed for highly efficient two-hybrid selection in yeast. Genetics 144,
Kartenbeck, J., Schmelz, M., Franke, W., and Geiger, B. (1991). Endocytosis of
junctional cadherins in bovine kidney epithelial (MDBK) cells cultured in low
Ca2?ion medium. J. Cell Biol. 113, 881–892.
Ko ¨hler, K., Louvard, D., and Zahraoui, A. (2004). Rab13 regulates PKA
signaling during tight junction assembly. J. Cell Biol. 165, 175–180.
Lau, A., and Mruk, D. (2003). Rab8B GTPase and junction dynamics in the
testis. Endocrinology 144, 1549–1563.
Le, T., Yap, A., and Stow, J. (1999). Recycling of E-cadherin: a potential
mechanism for regulating cadherin dynamics. J. Cell Biol. 146, 219–232.
Ling, K., Bairstow, S., Carbonara, C., Turbin, D., Huntsman, D., and Anderson, R.
(2007). Type I? phosphatidylinositol phosphate kinase modulates adherens
junction and E-cadherin trafficking via a direct interaction with ?1B adaptin.
J. Cell Biol. 176, 343–353.
Lock, J., and Stow, J. (2005). Rab11 in recycling endosomes regulates the
sorting and basolateral transport of E-cadherin. Mol. Biol. Cell 16, 1744–1755.
Markgraf, D., Peplowska, K., and Ungermann, C. (2007). Rab cascades and
tethering factors in the endomembrane system. FEBS Lett. 581, 2125–2130.
Marzesco, A., Dunia, I., Pandjaitan, R., Recouvreur, M., Dauzonne, D.,
Benedetti, E., Louvard, D., and Zahraoui, A. (2002). The small GTPase Rab13
regulates assembly of functional tight junctions in epithelial cells. Mol. Biol.
Cell 13, 1819–1831.
Matsuda, M., Kubo, A., Furuse, M., and Tsukita, S. (2004). A peculiar inter-
nalization of claudins, tight junction-specific adhesion molecules, during the
intercellular movement of epithelial cells. J. Cell Sci. 117, 1247–1257.
Miranda, K., Khromykh, T., Christy, P., Le, T., Gottardi, C., Yap, A., Stow, J.,
and Teasdale, R. (2001). A dileucine motif targets E-cadherin to the basolateral
cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells.
J. Biol. Chem. 276, 22565–22572.
Morimoto, S., Nishimura, N., Terai, T., Manabe, S., Yamamoto, Y., Shinahara,
W., Miyake, H., Tashiro, S., Shimada, M., and Sasaki, T. (2005). Rab13 medi-
ates the continuous endocytic recycling of occludin to the cell surface. J. Biol.
Chem. 280, 2220–2228.
Nelson, W. (2003). Adaptation of core mechanisms to generate cell polarity.
Nature 422, 766–774.
Ortiz, D., Medkova, M., Walch-Solimena, C., and Novick, P. (2002). Ypt32
recruits the Sec4p guanine nucleotide exchange factor, Sec2p, to secretory
vesicles; evidence for a Rab cascade in yeast. J. Cell Biol. 157, 1005–1015.
Palacios, F., Price, L., Schweitzer, J., Collard, J., and D’Souza-Schorey, C.
(2001). An essential role for ARF6-regulated membrane traffic in adherens
junction turnover and epithelial cell migration. EMBO J. 20, 4973–4986.
Pfeffer, S., and Aivazian, D. (2004). Targeting Rab GTPases to distinct mem-
brane compartments. Nat. Rev. Mol. Cell Biol. 5, 886–896.
Powell, R., and Temesvari, L. (2004). Involvement of a Rab8-like protein of
Dictyostelium discoideum, Sas1, in the formation of membrane extensions,
secretion and adhesion during development. Microbiology 150, 2513–2525.
Ren, M., Zeng, J., De Lemos-Chiarandini, C., Rosenfeld, M., Adesnik, M., and
Sabatini, D. (1996). In its active form, the GTP-binding protein rab8 interacts
with a stress-activated protein kinase. Proc. Natl. Acad. Sci. USA 93, 5151–
Rink, J., Ghigo, E., Kalaidzidis, Y., and Zerial, M. (2005). Rab conversion as a
mechanism of progression from early to late endosomes. Cell 122, 735–749.
Seabra, M., and Coudrier, E. (2004). Rab GTPases and myosin motors in
organelle motility. Traffic 5, 393–399.
Suzuki, T., Nakamoto, T., Ogawa, S., Seo, S., Matsumura, T., Tachibana, K.,
Morimoto, C., and Hirai, H. (2002). MICAL, a novel CasL interacting mole-
cule, associates with vimentin. J. Biol. Chem. 277, 14933–14941.
Takai, Y., Sasaki, T., and Matozaki, T. (2001). Small GTP-binding proteins.
Physiol. Rev. 81, 153–208.
Takai, Y., and Nakanishi, H. (2003). Nectin and afadin: novel organizers of
intercellular junctions. J. Cell Sci. 116, 17–27.
Takeichi, M. (1995). Morphogenetic roles of classic cadherins. Curr. Opin. Cell
Biol. 7, 619–627.
Terai, T., Nishimura, N., Kanda, I., Yasui, N., and Sasaki, T. (2006). JRAB/
MICAL-L2 is a junctional Rab13-binding protein mediating the endocytic
recycling of occludin. Mol. Biol. Cell 17, 2465–2475.
Terman, J., Mao, T., Pasterkamp, R., Yu, H., and Kolodkin, A. (2002). MICALs,
a family of conserved flavoprotein oxidoreductases, function in plexin-medi-
ated axonal repulsion. Cell 109, 887–900.
Tsukita, S., Furuse, M., and Itoh, M. (2001). Multifunctional strands in tight
junctions. Nat. Rev. Mol. Cell Biol. 2, 285–293.
Utech, M., Ivanov, A., Samarin, S., Bruewer, M., Turner, J., Mrsny, R., Parkos,
C., and Nusrat, A. (2005). Mechanism of IFN-?-induced endocytosis of tight
junction proteins: myosin II-dependent vacuolarization of the apical plasma
membrane. Mol. Biol. Cell 16, 5040–5052.
Vasioukhin, V., Bauer, C., Yin, M., and Fuchs, E. (2000). Directed actin
polymerization is the driving force for epithelial cell-cell adhesion. Cell 100,
Vitale, G., Rybin, V., Christoforidis, S., Thornqvist, P., McCaffrey, M., Stenmark,
H., and Zerial, M. (1998). Distinct Rab-binding domains mediate the interaction
of Rabaptin-5 with GTP-bound Rab4 and Rab5. EMBO J. 17, 1941–1951.
Weide, T., Teuber, J., Bayer, M., and Barnekow, A. (2003). MICAL-1 isoforms,
novel rab1 interacting proteins. Biochem. Biophys. Res. Commun. 306, 79–86.
Yamamoto, Y., Nishimura, N., Morimoto, S., Kitamura, H., Manabe, S., Ka-
nayama, H., Kagawa, S., and Sasaki, T. (2003). Distinct roles of Rab3B and
Rab13 in the polarized transport of apical, basolateral, and tight junctional
membrane proteins to the plasma membrane. Biochem. Biophys. Res. Com-
mun. 308, 270–275.
Yap, A., Brieher, W., and Gumbiner, B. (1997). Molecular and functional
analysis of cadherin-based adherens junctions. Annu. Rev. Cell Dev. Biol. 13,
Zahraoui, A., Joberty, G., Arpin, M., Fontaine, J., Hellio, R., Tavitian, A., and
Louvard, D. (1994). A small rab GTPase is distributed in cytoplasmic vesicles
in non polarized cells but colocalizes with the tight junction marker ZO-1 in
polarized epithelial cells. J. Cell Biol. 124, 101–115.
Zerial, M., and McBride, H. (2001). Rab proteins as membrane organizers.
Nat. Rev. Mol. Cell Biol. 2, 107–117.
Interaction of JRAB/MICAL-L2 with Rab8 and Rab13
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