Figure 7.

Low-level Msh2 variants with partial function are targeted by the Not4 ubiquitin ligase. (A) Deletion of NOT4 increases steady-state levels of Msh2 and Msh2 variants. The isogenic wild type (+) and not4Δ (–) expressing MSH2 or variants from a centromere-based plasmid (pCEN-MSH2-HA) were grown to exponential phase and processed for immunoblotting as described in Figure 1A. (B) Deleting NOT4 stabilizes Msh2 and Msh2 variants with partial function. Immunoblots were quantified and the levels of Msh2 or variants in not4Δ were presented as the relative level (%) to wild-type Msh2 level in NOT4. Data bars for Msh2 missense variants are colored according to the partial functioning including, partial mismatch repair function(MMR±) when expressed from a centromere-based plasmid, the ability to interact with Msh6 based on a yeast 2-hybrid assay, and the ability to function in mismatch repair function when overexpressed (OE rescue). The horizontal gray bar highlights the variants whose levels are increased to at least 50% of wild-type Msh2 levels. (C) Lower Wild-type Msh2 levels in strains lacking Msh6 is not a function of San1 or Not4 activity. Indicated cells were grown to exponential phase and processed for immunoblotting as described in Figure 1A. MSH6 was endogenously expressed from a centromere-based plasmid. PGK and Kar2 were used as a loading control. Band intensities of Msh2 were normalized to the loading controls using ImageJ and are shown below the immunoblot image as the percentage of the wild-type control (msh2Δ expressing MSH2 on a centromere-based plasmid). (D) Not4 specific turnover of Msh2 is not observed in the absence of Gcn5. Msh2 steady-state levels in the presence (+) or absence (–) of MSH6, NOT4 or GCN5. Cells were grown to exponential phase and processed for immunoblotting as described previously. The levels from duplicate experiments were quantified, normalized to Kar2 and graphed as the relative intensity of Msh2 (AU, arbitrary units). Error bars are standard error of the mean.