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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). References Argon Y, Simen BB (1999) GRP94, an ER chaperone with protein and peptide binding properties. Semin Cell Dev Biol 10: 495-505 Gaullier JM, Simonsen A, D'Arrigo A, Bremnes B, Stenmark H, Aasland R (1998) FYVE fingers bind PtdIns(3)P. Nature 394: 432-433 Haraguchi T, Koujin T, Segura-Totten M, Lee KK, Matsuoka Y, Yoneda Y, Wilson KL, Hiraoka Y (2001) BAF is required for emerin assembly into the reforming nuclear envelope. J Cell Sci 114: 4575-4585 Metzelaar MJ, Wijngaard PL, Peters PJ, Sixma JJ, Nieuwenhuis HK, Clevers, HC (1991) CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells. J Biol Chem 266: 3239-3245 Patki V, Lawe DC, Corvera S, Virbasius JV, Chawla A (1998) A functional PtdIns(3)P-binding motif. Nature 394: 433-434