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Supplemental Data
XRab40 and XCullin5 form a ubiquitin ligase complex
essential for the noncanonical Wnt pathway
Rebecca Hui Kwan Lee, Hidekazu Iioka, Masato Ohashi, Shun-ichiro Iemura, Tohru
Natsume and Noriyuki Kinoshita
Figure S1
Figure S1. XCullin5, XElonginB, XElonginC and XMINK were expressed throughout the
Xenopus developmental stages.
Sequence alignments of (A) Xenopus Cullin5 (B) Xenopus ElonginB and (C) Xenopus ElonginC and
their human and Drosophila homologs. The identities in the deduced amino acid sequences between
human and Xenopus clones were 98% (Cullin5), 83% (ElonginB) and 100% (ElonginC). XCullin5N
mRNA was constructed with the deletion of the residues in blue. The order of amino acid sequence is
frog, human and fly. (D) Expression patterns of XRab40, XCul5, XElonginB, XElonginC were
analyzed by RT-PCR at various Xenopus developmental stages. (E) Xenopus MINK and its human and
Drosophila homologs. The order of amino acid sequence is frog, human and fly. (F) Schematic
illustration of XMINK, which is composed of a kinase (red) and a CNH (blue) domains. Xenopus
MINK shares 85% and 74% of the kinase and CNH domains, respectively, with its Drosophila
homolog. (G) Expression of XMINK was analyzed by RT-PCR analysis.
Figure S2
Figure S2. Specificities of XRab40, XCullin 5 and XMINK morpholinos.
(A) Specificity of XRab40 Mo. EGFP-XRab40 mRNA was coexpressed with mRFP in the animal
regions. (B) Mo target XRab40 mRNA was expressed together with mRFP and XRab40 Mo (2 pmol)
in the animal regions. (C) Specificity of XCullin5 Mo. EGFP-Mo sensitive XCul5 mRNA (500 pg) was
coexpressed with RFP-Mo resistant XCul5 mRNA (500 pg) with or without XCul5 Mo. (D) RFP-Mo
sensitive XCul5 mRNA (500 pg) was co-expressed with EGFP-Mo resistant XCul5 mRNA (500 pg)
with or without XCul5 Mo. For (C) and (D), the constructs of XCul5 were not generated in their fulllengths. (E) EGFP-Mo sensitive XMINK mRNA (400 pg) was co-expressed in the animal regions with
RFP-Mo resistant XMINK mRNA (400 pg) with or without XMINK Mo (3 pmol). (F) RFP-Mo
sensitive XMINK mRNA (400 pg) was co-expressed in the animal regions with EGFP-Mo resistant
XMINK mRNA (400 pg) with or without XMINK Mo. Scale bar = 50 m.
Figure S3
Figure S3. Endogenous expression and localization of Rab40 in Xenopus.
Endogenous Rab40 expression in Xenopus. (A) Either Rab40 mRNA or Rab40 Mo (4 pmol) was
injected in the animal cap cells. Equal loading was shown by blotting with anti-tubulin. (B) Endognous
localization of Rab40 in Xenopus. Animal cap cells were isolated and subjected to immunostaining
with Rab40 antibody. As a control, antibody against Rab40 was preabsorbed with its antigen for one
hour before staining (middle panel). XCul5 Mo was injected in the animal cap cells and subjected to
immunostaining using Rab40 antibody (right panel). Scale bar = 10 m.
Figure S4
Figure S4. Characterization of molecular mesodermal markers by XRab40 knockdown.
(A) Expression patterns of Xbra, chordin and gsc at the gastrulae stage, detected by in situ
hybridization. Xbra, Xenopus brachyury; gsc, goosecoid. (B) XRab40 Mo or XRab40 mRNA was
expressed in two dorsal blastomeres in the 4-cell stage embryos. The DMZ explants were isolated, and
the expressions of chordin, gsc, Xnr3, Xtwist and Xbra were analyzed by RT-PCR. Xnr3, Xenopus
nodal-related 3. (C) XRab40 does not interfere with Activin/Nodal signaling. Animal cap cells were
isolated and subjected to immunoblotting with anti-phospho-Smad2 or anti-GFP. (D) XRab40 Mo (4
pmol) was expressed together with or without embryonic FGF in the animal regions. The expression of
Xbra was analyzed by RT-PCR. (E) Localization of embryonic FGF (eFGF) and FGFR were not
affected by XRab40 Mo. Myc-eFGF was expressed with or without XRab40 Mo and subjected to
immunostaining (left panel). EGFP-FGFR was expressed with or without XRab40 Mo (right panel).
Scale bar = 50 m.
Figure S5
Figure 5. XRab40 is not localized at endosome, lysosome, ER and nuclear membrane.
EGFP-XRab40 was coinjected with RFP-tagged EEA1 (Early Endosome Antigen 1) with mRFP, RFPCD63 (Lysosome-Associated Membrane Glycoprotein 3) with mRFP, RFP-GP96 with RFP or RFPhLBR (human Lamin B Receptor) with RFP (top to bottom panels, respectively). (B) Numbers of
XRab40-EGFP vesicles overlapping with RFP-h1,4GT, EEA1 and CD63. Scale bar = 50 m.
Figure S6
Figure S6. The Specificities of XRab40 and XCul5 morpholinos on XRap2 localization, and
XRab40 and XCullin5 knockdowns do not affect the Golgi apparatus, endosome and ER
morphologies.
(A) Rescue of the mislocalization of XRap2 caused by XRab40 or XCul5 MOs. The localization of
EGFP-Rap2 is shown. MOs and mRNAs were coinjected in the animal regions (XRab40 and XCul5
MOs, 0.8 pmol each; XRab40 mRNA, 500 pg; XCul5 mRNA, 1 ng). (B) XCul5N but not other Cterminally truncated Cullins caused the mislocalization of XRap2. EGFP-XRap2 was coinjected with
C-terminally truncated Cullins (Cul1N, Cul2N, Cul3N, Cul4BN and Cul5N) mRNAs in the animal
regions. Scale bar = 50 m. (C) EGFP-h1,4GT, EEA1 and GP96 were expressed in animal regions
with or without XRab40 or
Figure S7
Figure S7. XRap2K117R shows a reduction in ubiquitination and increased cytoplasmic punctate
appearance.
(A) XRab40 GTPase fails to rescue the ubiquitination caused by XRab40 Mo. XRab40 GTPase,
C-GTPase or N-GTPase was coinjected with GST-fused XRap2, myc-tagged ubiquitin mRNA and
XRab40 Mo in animal regions. (B) EGFP-tagged XRap2 or -XRap2 K117R mRNAs were injected in
the animal cap cells. (C) XRap2K117R caused a reduction in ubiquitination. GST-fused XRap2 or
XRap2 K117R mRNAs were coinjected with myc-tagged ubiquitin mRNA in animal regions. Scale bar
= 20 m.
Figure S8
Figure S8. Retrieval of the vesicle localization and the plasma membrane translocation of Dsh in
embryos with XCul5 and XMINK knockdowns.
(A) EGFP-Dsh, Wnt11 and Fz were coinjected with XCul5 or XMINK morpholinos, and together with
or without the respective mRNAs in the animal cap cells (XCul5Mo, 2 pmol; XMINK Mo, 5 pmol;
XCul5 mRNA, 500 pg; XMINK mRNA, 1 ng). (B) EGFP-Dsh was coinjected with XCul5 or XMINK
morpholinos, and together with or without the respective mRNAs in the animal cap cells. Scale bar =
50 m.
Figure S9
Figure S9. XRap2 is required for gastrulation and XRab40 is involved in the noncanonical Wnt
pathway.
(A) XRap2 SN mRNA or (B) XRap2K117R mRNA caused gastrulation defects when dorsally injected
into the two blastomeres in the 4-cell stage embryos. Statistical data of gastrulation-defective
phenotypes caused by XRap2 SN and XRap2 K117R mRNA and rescue experiments coinjected with
XRap2 WT or XMINK mRNAs. (C) XRap2 SN or XRap2 K117R mRNA (2.5 ng each) was coinjected
with mEGFP into one of the two blastomeres in the 4-cell stage embryos. mRFP alone was injected
into the other dorsal blastomere. As a control, mEGFP was injected alone (left panel). (D) XRap2 SN
or XRap2 K117R mRNA was expressed in two dorsal blastomeres in the 4-cell stage embryos. The
DMZ explants were isolated, and the expressions of chordin, gsc, Xnr3 and Xbra were analyzed by RTPCR. gsc; goosecod; Xnr3, Xenopus nodal-related 3. (E) TOP-FLASH luciferase assay was carried
out in the animal cap cells (top panel) and either the ventral or dorsal side (bottom panel) of Xenopus
embryos. Error bars show the standard deviation. Scale bar = 50 m.
Supplemental Experimental Procedures
Plasmid construction
XRab40 cDNA fragments were amplified by PCR using the XL044o08 clone in the
NIBB Xenopus database (http://xenopus.nibb.ac.jp/) as a template. To express
XRab40 in Xenopus embryos and tissue culture cells, the fragments were cloned into
pCS2+ or pCS2+-based epitope tagging vectors. XRab40ΔSOCS lacks amino acid
#176-215. Ubiquitin, XCullin5, XElonginB and XElonginC cDNAs are derived from
NIBB database clones XL482g02, XL228c22ex, XL215d13 and XL264p24,
respectively. XCullin5N encodes amino acid #1-586. The cDNA fragments of
Xenopus MINK were amplified by PCR (OPEN Biosystems cat. #EXL10516667432) as a template and fused to pCS2+ or pCS2+-based epitope tagging vectors.
GenBank accession numbers of XRab40, XCullin5 and XMINK are AB262321,
AB262322 and AB262323, respectively. Dynamin cDNA is derived from the 5’
fragment (1.8 kb) of the NIBB database clone XL105e18 and the 3’ fragment (0.7 kb)
of the amplified cDNA from synthesized embryonic RNA. The fragments were fused
at the SphI site and then to the pCS2+ vector. The dominant negative dynamin was
amplified by PCR site mutagenesis bearing a K46A mutation. The Xenopus dynamin
is 89% similar with its human homolog. The cDNA fragments of human 1,4Galactosyltransferase were amplified by PCR using pEYFP-Golgi (Clontech cat.
#632358) as a template and fused to RFP. The cDNA fragments of human lamin B
receptor (BC020079; kind gift from from Dr. Haraguchi) were fused to the N-terminal
end of monomeric RFP. The Xenopus C-terminal fragment of EEA1 was amplified by
PCR using the XL064i18 clone and fused to the C-terminal end of mRFP. Xenopus
EEA1 is 61% identical to its human homolog. Xenopus CD63 cDNA fragments were
amplified by PCR using the XL327h02 clone in the NIBB Xenopus database and
fused to the N-terminal end of mRFP. Xenopus CD63 is 52% identical to its human
homolog. The 78-746 amino acid region of Xenopus GP96 (MGC68448) was
replaced with mRFP. 1-77 and 747-805 amino acid regions were fused to the N- and
C- terminal ends of mRFP, respectively. The sequence contains a cleavable signal (121 amino acid) and the ER retention motif VKDEL at the C terminus. The plasmids
harboring Xwnt11, Fz7 and Dsh were as previously described (Kinoshita et al., 2003).
Xwnt8 cDNA was amplified by PCR using Xenopus embryonic cDNA.
For mRNA microinjection, XRab40 Mo, 4 pmol; XCul5 Mo, 2 pmol;
XMINK Mo, 5 pmol; XCullins1N, 2N, 3N, 4BN and 5N, 1.5 ng; XRap2SN, 400 pg;
XRab40, XRab40 SOCS, GST-tagged Dsh, 1 ng; DN dynamin, 750 pg; myc-tagged
XCullin5, XElonginB/C and XMINK; GST-tagged XRab40 and deletion constructs
of XRab40, EGFP-tagged XRab40 and deletion constructs of XRab40, EGFP-tagged
XMINK, EGFP-tagged Fz, 500 pg; RFP-tagged 1,4-GT, 300 pg; XWnt11, Fz,
mEGFP, mRFP, myc-tagged ubiquitin, and XWnt11, GAL-DBD, GFP-tagged Dsh,
200 pg, XRap2 K117R, myc-tagged XRap2, GST or His-GST tagged XRap2 and
GFP, 100 pg; EGFP-tagged XRap2, 50pg; RFP-tagged XRap2, 50 pg; XWnt8, 10 pg;
Activin, 1 pg were used. The dosages are applied to all figures, unless otherwise
specified.
Morpholino oligos
Antisense morpholinos were obtained from Gene Tools Inc.
morpholino
oligo
sequences
were
as
follows:
GCTGCCCTGTGTGCCCATCCTCCCT-3’;
control
CACCGGGCTGCCCTGGGTGCCCATC-3’;
XCullin5
XRab40
Mo,
Mo,
Mo,
The
5’5’5’-
CGTCGCCATGTTCCCTCAACTTGTC-3’;
XMINK
Mo,
5’-AGCTGGTGG
TCTGATGCCATGATC-3’.
In situ hybridization and RT-PCR analysis
In situ hybridization in Xenopus was carried out as described in Harland (1991). The
detection of -galactosidase activity for tracing cell lineage was carried out as
described by Kurata and Ueno (2003). For RT-PCR analysis, RNA from Xenopus
embryos was prepared with Trizol (Life Technologies). cDNA was synthesized with
Reverse Transcriptase (#TRT-101, Toyobo). Sequences of the primers for Xbra and
Xnr3 were as described by Yamamoto et al. (2001) and for chordin, goosecoid, xtwist
and
ODC
as
described
on
E.
De
Robertis’
homepage
(http://www.hhmi.ucla.edu/derobertis/index.html).
Immunocytochemistry
Myc-tagged eFGF mRNA was injected into the animal pole of two-cell embryos. The
animal caps were dissected from stage 9-10 embryos and fixed with MEMFA,
followed by immunostaining with a fluorescence-labeled secondary antibody. The
localization was determined by laser-scanning confocal microscopy, using a Carl
Zeiss LSM510 microscope. The antibody for immunostaining was anti-myc (1:100;
Santa Cruz Biotechnology) and anti-Rab40.
Immunofluorescence staining in CHO cells
CHO cells were transiently transfected using Fugene 6 (Roche) according to the
manufacturer's instructions. Immunofluorescence staining for GM130 was done as
previously described (Miwako et al., 2001). Anti mouse monoclonal anti-GM130
(clone 35) was purchased from BD Transduction Laboratories. FITC-conjugated rat
anti-mouse IgG (Jackson ImmunoResearch) or TRITC-conjugated affinity-purified
donkey anti-mouse IgG (Chemicon) was used as the secondary antibody.
Images were obtained using an IX70 microscope (Olympus) equipped with an
UPlanApo x100 objective, and a cooled charge-coupled device camera (ORCA-AG,
Hamamatsu). Excitation, emission, and dichroic filters for multiband fluorescence
(DAPI, FITC, Texas Red set; Semrock) with excitation and emission filter wheels
(Hamamatsu) were used to select the appropriate spectra for imaging EGFP, FITC
(FITC channel), and mRFP1, TRITC (Texas Red channel). C-imaging software
(Compix) was used to control image acquisition.
JNK assay
mRNA encoding GAL4 (DBD)-tagged c-Jun (100 pg) was injected into two-cell
embryos. The animal caps were isolated at stage 10. Cell lysates were prepared as
mentioned above. Western blotting was performed using antibodies against GAL4
(DBD) (1:1000; #SC-510, Santa Cruz Biotechnology) and phospho-c-Jun (1:1000;
#9261S, Cell Signaling).
TOP-FLASH reporter assay
TOP-FLASH (50 pg) and pRL-TK (5 pg) DNAs (kind gifts from Prof. Randall Moon)
were injected either in the animal region, dorsal or ventral sides of the embryos
together with XRab40 Mo in two blastomeres of 4-cell embryos. Embryos were
collected at stage 11 and separated into three pools of five embryos each for assay in
triplicate and luciferase activity was assessed according to manufacturer’s instructions
with the Dual-Luciferase Reporter Assay System (Promega).
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