Figure 1.

UVRAG forms a complex with SMURF1 through the PPxY motif. (a) HEK293T cells were co-transfected with HA-UVRAG and MYC-SMURF1 constructs. 24 h later, cell lysates were used for co-immunoprecipitation with anti-HA or anti- MYC antibodies and WB analyses. (b) Endogenous SMURF1 and UVRAG proteins interact with each other in Huh7 cells. Upper panel, Huh7 cell lysates were incubated with protein A/G Sepharose conjugated with either control IgG or UVRAG antibody. The immunoprecipitates were analyzed using western blotting with indicated antibodies. Lower panel, a similar assay was performed as the upper panel but immunoprecipitated using anti-SMURF1 antibody. (c) Recombinant SMURF1 protein was incubated with GST-tagged UVRAG, BECN1 (positive control), or GST protein. GST affinity-isolation assay was performed with glutathione-agarose and blotted as indicated. (d) Bacterially expressed GST fusion proteins of wild-type (WT), C2 domain (C2), WW domain (WW), HECT domain (HECT) mutants of SMURF1 were bound to glutathione-Sepharose beads and incubated with cell lysates of HEK293T cells transfected with the His-UVRAG. Numbers represent the amino acid (aa) residues in human SMURF1. The interaction between UVRAG and SMURF1 domains is indicated by the plus signs (+). Bound His-UVRAG was subjected to WB analyses with an anti-His antibody. (e) Various deletion mutation constructs of UVRAG are shown schematically (PR: proline-rich domain; CCD: coiled coil domain). GST-UVRAG FL or fragments were incubated with His-SMURF1, and western blotting was performed to detect the interaction with an anti-His antibody. (f) Protein sequence alignment of UVRAG orthologues from the different species and the conserved PPxY motif was highlighted in red color. (g) MYC-UVRAG, the WW-binding-defective mutant UVRAG^Y523A, or the vector was cotransfected with HA-tagged SMURF1 into HEK293T cells. UVRAG or UVRAG^Y523A was immunoprecipitated with anti-MYC antibody. Coprecipitated SMURF1 was detected by immunoblotting with an anti-HA antibody. The expression levels of indicated proteins in the cell lysates are shown. (h) Bacterially expressed GST fusion proteins of UVRAG deletion mutants were bound to glutathione-Sepharose beads as indicated and incubated with cell lysates of HEK293T cells transfected with His-SMURF1. The sample was subjected to WB analyses with indicated antibody. (i) The subcellular colocalization of overexpressed Flag-UVRAG and MYC-SMURF1 were detected by immunofluorescence in HEK293T cells subjected to glucose deprivation. Nuclei were stained with DAPI. The insets show a high magnification of the selected areas. Scale bars: 10 µm. (j) The quantitation analysis of the co-localization of UVRAG and SMURF1 in Figure 1(i). The quantitative values are expressed as the mean ± SD (n = 100 cells) obtained from three independent experiments).