Eighteen Rpn1 residues may be important for binding UBL domain proteins. (A) The utilized yeast two hybrid system allows for both positive (growth on -HIS, -URA, +3-AT) and negative counter selection (growth on 5FOA) of UBL- Rpn1391 to 642 interaction. (B) Rpn1391-642 was sufficient for binding UBA-UBL proteins in a yeast two-hybrid system. Yeast cells were co-transformed with plasmids expressing Gal4-DBD fused to Rpn1391 to 642 (Rpn1391 to 642DB) and Gal4-AD fused to either Rad23, Dsk2, Ddi1, Upb6, Ddi1ΔUBL or Rpn2. Protein-protein interaction is indicated by growth on 100 mM 3-AT and lack of growth on 0.2% 5FOA. (C) Representative rpn1 alleles found in the RY2H screen did not interact with Rad23 in the context of an Y2H experiment. (D) Sequence and secondary structure prediction alignments of yeast Rpn1 with mouse Rpn1 were made with MultiAlin http://multalin.toulouse.inra.fr/multalin/multalin.html using the model structure of Rpn1 . Identical residues (black) and similar residues (gray) are indicated. Mutations identified in the RY2H that disrupt the interaction of Rpn1391-642 with Rad23 and Dsk2 are indicated in red and rationally-designed mutations are indicated in blue. Mutant rpn1 alleles were plasmid shuffled into an rpn1Δ yeast strain and assayed for viability and proper 26S assembly. The positions of the identified mutations are indicated in the figure. A (-) indicates that assembly and viability were like wild type, a (+) indicates that we observed defects in proteasome stability (Figure S3) and (nd) indicates the strain was inviable. (E) The relative position of residues of interest from the RY2H screen and the rational sites chosen in (D) are shown on a model structure of Rpn1 proposed in reference 32. Residue A418 is not included in the model. The colors represent the residues indicated in the key.