Fig. 3.

The LRR domain interacts with Rpn1 of the 19S RP. a GSH beads to which recombinant yeast ^GSTRpn1, ^GSTRpn10, or GST was bound were incubated with yeast extracts from pac2Δ mutants expressing pGAL-GFP-PAC2 or pGAL-GFP-PAC2ΔUBL. The bead fraction was analyzed by Western blotting with anti-GFP and anti-GST antibodies; 10% is the volume of total cell extract used in the experiment. b Recombinant yeast Pac2 ^HisLRR domain produced in bacteria was bound to nickel beads and incubated with a bacterial lysate from cells that produced yeast ^GSTRpn1, ^GSTRpn10, or GST. The bead fraction was analyzed by Western blotting first with anti-GST and then with anti-His antibodies; 5% is of the bacterial cell lysate. c Recombinant yeast ^HISRpn10 produced in bacteria was incubated with yeast ^GSTPac2-LRR domain, full-length human TBCE, or subclones of the TBCE LRR (T-LRR), UbL (T-UbL), or CAP-Gly (CG) domains, or with control GST beads, all produced in bacteria. ^HISRpn10 that bound TBCE protein was detected with anti-His antibodies; the proteins on the beads are shown using anti-GST antibodies, and 10% is an aliquot of ^HISRpn10 bacterial lysate incubated with the beads