Figure 5.

Cdc53/Hrt1 comprise an NEM-resistant ubiquitin ligase module. (A) The proteins indicated were retrieved from baculovirus-infected insect cells on α-Py beads, and mixed with E1, Cdc34ΔC, ubiquitin, and ATP. Following a 60-min incubation at 20°C, reactions were evaluated for Cdc34ΔC autoubiquitination (top panel), levels of Hrt1 (middle panel), and levels of Cdc53^PyHA or ^PyHACdc4 (bottom panel) by SDS-PAGE following by immunoblotting. SCF^Cdc4 complexes (lanes 4,5) contained ^PyHACdc4 and untagged Cdc53. Thus, only ^PyHACdc4 is visible in these lanes in the α-HA blot (bottom panel; see asterisks). (B) Either Cdc53^PyHA/Hrt1 (lanes 1–3) or E1 enzyme (lane 4) was pretreated with the indicated amount of NEM for 10 min at 20°C. NEM was quenched with DTT, and complete reactions containing E1, Cdc34ΔC, ubiquitin, ATP, and α-Py bead-bound Cdc53^PyHA/Hrt1 were incubated for 60 min at 20°C. For the sample shown in lane 5, DTT was mixed with NEM prior to incubation with E1 to confirm the efficacy of the DTT quench. All reactions were subjected to SDS-PAGE followed by immunoblotting with α-Cdc34. (C) Same as B, except that complete reactions were first assembled and then treated with 5 mm NEM followed by 10 mm DTT (lanes 1,2) or vice versa (lanes 3,4). Afterwards, α-Py bead-bound Cdc53^PyHA/Hrt1 was recovered, washed, and supplemented with either buffer (lanes 1,3) or fresh E1, Cdc34ΔC, ubiquitin and ATP (lanes 2,4). The signal in lanes 1 and 3 represents Cdc34ΔC from the first stage incubation that was retained on the Cdc53^PyHA/Hrt1 matrix.