Figure 5.

Effects of MAD2 on the Ub-proteasome system in Δ90-arrested extracts. (A) Degradation of Ub-¹²⁵I-lysozyme conjugates in Δ90-arrested extract in the presence of MAD2 or subunit 5a (S5a) of the 26S proteasome. (B) Isopeptidase activity in Δ90-arrested extracts. Δ90 extract was incubated with either buffer (circles) or MAD2 (squares) for 10 min at 23°C. An aliquot (2 μl (○ and □) or 4 μl (• and ▪) was then added to 20 μl of Ub-¹²⁵I-lysozyme conjugates and 0.5 μl ATP-regenerating system to assay for isopeptidase activity as described under Materials and Methods. (C) Overall Ub-conjugating activity of Δ90-arrested extracts in the absence and presence of MAD2.