FIGURE 1.

The Ubl domain of parkin binds the Pru domain of Rpn13. A, schematic representation of the E3 ligase parkin and proteasome-related ubiquitin receptors. Specific domains and motifs are depicted. UBD, ubiquitin-binding domain; UBL, ubiquitin-like domain; IBR, in between ring fingers. B, mapping of the parkin Ubl and Rpn13 binding regions. Yeast strain Y190 was transformed with parkin Ubl-pYTH9 vector and various fragments of hRpn13 in pACT2 vector as preys. C, overexpressed mRpn13 and parkin bind, as shown by co-immunoprecipitation in HEK293T lysates. Plasmids for hRpn2 and hUCH37 were used as positive binding controls. D, residues involved in Rpn13-Ub binding also mediate the Rpn13-parkin interaction. Lysates from HEK293T cells transiently transfected with full-length Myc-mRpn13 (aa 1–407) or its corresponding F76R mutant were used for GST pulldown assays with GST alone, GST Ub (positive control), and either WT or I44A mutant of GST-tagged Ubl domain of parkin. Empty vector pcDNA 3.1 was used as a negative control. IB, immunoblotting; TCL, total cell lysate.