FIGURE 1.

The Rsp5 HECT domain binds directly to ubiquitin. A, a lysate prepared from yeast cells expressing HA-tagged Rps5 (LHY856) was incubated with ubiquitin-agarose beads (Ub) or agarose beads alone (Beads). Bound proteins were eluted, and Rsp5 was detected on an anti-HA immunoblot. B, schematic representation of Rsp5 indicating the position of its functional domains. Fragments tested for ubiquitin binding in C are shown. C, bacterial lysates from cells expressing the indicated GST-tagged Rsp5 domains were incubated with ubiquitin-agarose beads or agarose beads alone. Total lysates and bound proteins were analyzed by anti-GST immunoblotting.