FIGURE 6.

AUP1 binds Ube2g2 via its G2BR domain in vitro. A, GST pulldown, AUP1 specifically binds to Ube2g2. GST alone or fusion proteins of GST with full-length Ube2g2 or its closest homologue, Ube2g1, were incubated with recombinant His₆-AUP1(221–410), comprising the soluble part of AUP1 (including G2BR domain). Complexes were precipitated with glutathione-Sepharose beads and analyzed by SDS-PAGE/Coomassie Brilliant Blue staining. The upper row shows the eluted GST fusion construct, and the lower row shows the co-eluted His₆-AUP1(221–410). Note: upper row is a composite of two pieces from the same gel, because the control GST differs in molecular weight from the two fusion proteins. B, GST pulldown, G2BR domain of AUP1 is sufficient to bind Ube2g2. Fusion proteins of GST with wild-type G2BR of AUP1 (G2BR-wt) or with mutated G2BR of AUP1 (G2BR-M1) were incubated with MBP-Ube2g2. Complexes were precipitated with glutathione-Sepharose beads, eluted with free GSH, and analyzed by SDS-PAGE/Coomassie Brilliant Blue staining. Note that the free MBP, which contaminates the MBP-Ube2g2 fusion protein, did not bind to the GST-G2BR fusions.