Figure 4. The deletion of Not4, but not the deletion of the deadenylase, stabilizes the proteasomal substrate CPY*.

A. CPY*-HA was expressed from an episome under control of a copper dependent promoter in wild-type (WT) and not4Δ cells. Cells were exponentially grown without induction to OD₆₀₀ of 0.6 (time 0). 0.1 mM CuSO₄ was added to the media and cells were collected at indicated time points (2, 4 and 11 h) and analyzed by SDS-PAGE and western blot with antibodies against HA, to see CPY*-HA levels, and against Egd2 as a loading control. The positions of CPY*-HA and ubiquitinated CPY*-HA (CPY*-HA-Ub) are indicated on the right. The molecular weight markers are indicated on the left. B. Stability of CPY*-HA was analyzed in wild-type, caf1Δ, ccr4Δ, and not4Δ cells. Cells were grown exponentially and treated (+CHX) or not (−CHX) with CHX. Samples were collected at indicated time points and analyzed as in A. Since CPY*-HA expression was different in mutant strains (see Fig. S2A) 2 times less material was loaded on the gel in the case of not4Δ samples compared to wild type, and 4 times less material was loaded on the gel in the case of the ccr4Δ and caf1Δ samples compared to wild type. C. Stability of CPY*-HA was analyzed in ubr1Δ, ubr2Δ, ltn1Δ, and san1Δ cells as in B. D. Stability of CPY*-HA was analyzed in cim3-1 and pre1-1 cells as in B.