Figure 6.

The dependency of Spd1 degradation on Pcu4 and copurification of Pcu4 with the signalosome. (A) Ubiquitinated TAP-Spd1 forms are evident in mts3 mutant cells at both the permissive and restrictive temperatures. Wild-type, csn1-d, and mts3-1 mutant cells were used to express His₆-ubiquitin. Soluble extracts were prepared from each strain (mts3-1 cells were analyzed at both the permissive 25°C and restrictive 35°C temperatures) either with (+) or without (−) prior incubation in HU for 2.5 h. His₆-ubiquitin was purified on Ni²⁺ beads; the beads were boiled and the eluate was resolved by 12% SDS-PAGE and probed for the TAP-tag. nUb indicates the multiple-ubiquitinated forms of TAP-Spd1; Ub indicates the monoubiquitinated form. (B) Ubiquitinated TAP-Spd1 forms are evident in mts2 mutant cells, and these are lost in mts2 csn1-d double mutants. Wild-type, csn1-d, mts2, and two csn1-d mts2 double-mutant isolates were used to express His₆-ubiquitin. Soluble extracts were prepared and His₆-ubiquitin was purified on Ni²⁺ beads. The resulting beads were boiled, and the eluate was resolved by 8% SDS-PAGE and probed for the TAP-tag. As before, nUb indicates the multiple-ubiquitinated forms of TAP-Spd1; Ub indicates the monoubiquitinated form. A control aliquot of TAP-Spd1 extract is also run to show the size of unmodified TAP-Spd1 and demonstrate that nonubiquitinated Spd1 is not purified by IMAC chromatography. The star indicates a nonspecific band.