Fig. 1
From: The HTLV-1 oncoprotein Tax is modified by the ubiquitin related modifier 1 (Urm1)
Urmylation of the HTLV-1 oncoprotein Tax strongly influences the subcellular localization of Tax. a Tax is targeted by urmylation upon co-expression of wild-type Tax and Myc-tagged Urm1 in HeLa cells. Mutagenesis of Tax, replacing all lysine residues (TaxK1-10R) or lysine 4-8 (TaxK4-8R) for arginines, completely abolished Urm1 conjugation to Tax. b Mutational analysis of the indicated lysine residues in Tax, expressed in HeLa cells, indicates that Urm1 targets the same lysine residues as ubiquitin and SUMO for conjugation, lysine residues 4-8. c Tax is urmylated in protein lysates derived from Drosophila melanogaster adult eyes expressing Myc-Tax and Flag-Urm1 under control of the GAL4/UAS system, using GMR-GAL4 as driver. – AB control represents a negative control, in which no antibody was added to the immunoprecipitation. d Duolink® in situ proximity ligation assay performed in HeLa cells expressing Tax and Myc-tagged Urm1, depicting that Tax-Urm1 protein complexes are primarily localized in the cytoplasmic compartment n = 25, P < 0.0001. e Co-expression of Myc-Urm1 (green) and Tax (red) in HeLa cells causes a shift in the subcellular localization of Tax, with a clear increase of cytoplasmic Tax, as compared to cells expressing Tax alone (two upper panels). The Urm1-dependent nuclear exclusion of Tax is abrogated in cells expressing the TaxK4-8R mutant, indicating that lysine 4-8 is required for Urm1-mediated regulation of Tax (two lower panels). f Also in ATL-derived HuT102 cells, interaction between endogenous Tax and endogenous Urm1 proteins is primarily encountered in the cytoplasm, n = 42, P < 0.0001. Representative images for the Duolink® experiments and immunohistochemical analysis in HeLa cells were acquired by confocal microscopy using either a Zeiss LSM 510 META confocal laser microscope or a Zeiss LSM 710 confocal microscope (Zeiss, Oberkochen, Germany) with a Plan Apochromat 63/1.4 numeric aperture oil-immersion objective using Zen 2009 (Carl Zeiss). High-resolution images were obtained with a deconvolution program (Autodeblur; Image Quant), using blind iterative algorithms