Figure 2.

Irc22 interacts with Dsk2 but not Rad23 and Ddi1. (A) Interaction of Irc22 with Dsk2 in vivo. T7-Irc22 was co-expressed with GST-Dsk2, GST-Rad23, GST-Ddi1 or GST alone in wild-type yeast. The extracts (Lanes 1–6) and the GST-precipitates (Lanes 7–12) were immunoblotted as indicated. (B) The C-terminal UBA domain and the middle stretch of Dsk2 are required for its interaction with Irc22 in vivo. A series of truncated mutants of Dsk2 were constructed as indicated. T7-Irc22 was co-expressed with Dsk2 mutants in wild-type yeast, and the cell extracts (Lanes 1–9) were pulled down with glutathione-Sepharose 4B followed by immunoblotting with the indicated antibodies (Lanes 10-18). (C) Binding assay between Irc22 and UBL-UBA proteins in vitro. The binding of Irc22 to Dsk2, Rad23, Ddi1, or Pth2 (see Discussion) was tested in vitro. His₆-T7-Irc22 was mixed with GST-Dsk2, GST-Rad23, GST-Ddi1, GST-Pth2, or GST alone in lysis buffer and incubated with glutathione-Sepharose 4B. The bead-bound materials (Lanes 1–7) were immunoblotted with the indicated antibodies. (D) Summary of Irc22 interactions in vivo and in vitro. Binding of Irc22 to Dsk2, Rad23, and Ddi1 in vivo and in vitro are summarized. Interaction sites of Irc22 are shaded in the middle stretch and the UBA domain of Dsk2 drawing.