Figure 5.
POSH-mediated ubiquitination of the Ubl domain is required for Tpg-induced translocation. (A) Expression of dominant-negative POSH inhibits Herp redistribution. HeLa cells transiently coexpressing Herp-RFP and POSH (either wild type [Wt] or V14A) were visualized after the addition of either DMSO or Tpg. (B) HeLa cells expressing either Herp-Flag or HerpK15-78R were incubated with either DMSO or Tpg. Cells were subsequently immunostained in tandem; initially for Herp-Flag (with anti-Flag and Cy2-conjugated anti–mouse), and then blocked with an excess of unconjugated anti–mouse and for calnexin (with mouse anticalnexin and Cy3-cojugated anti–mouse). (C) Conjugation of Herp in vitro. Herp wild type and K15-78R mutant were incubated in the presence of either Ubc13/Uev1a (13) or UbcH5c (H5) in an in vitro ubiquitination reaction containing N-terminal biotin-Ub. After the incubation, Herp was pulled down with anti-Flag, and the Herp immune complexes were resolved by SDS-PAGE. Herp-Ub conjugates were then detected by Western blot analysis either with streptavidin (left) or with anti-Flag (right). The asterisk indicates a nonspecific immunoreactive protein band. (D) Activation of POSH by wild-type and mutant Herp. In vitro ubiquitination reactions were performed in the presence of increasing amounts of Herp as described in Fig. 2. POSH-Ub conjugates were detected by Western blot analysis with PT1.