Figure 7.

Psh1 facilitates the association of Npl4 with mislocalized Cse4. (A) Ubiquitinated chromatin-bound Cse4 is reduced in a cdc48-3 psh1Δ strain. Solubilized chromatin lysate was prepared from cdc48-3 GAL-CSE4 or cdc48-3 psh1Δ GAL-CSE4 strains. Cultures were grown in sucrose/galactose (2%) for 4hrs at 25°C to induce expression of Cse4 and Ub pull-down assay was performed using chromatin lysates. Ub pull down samples and chromatin-bound Cse4 were analyzed using anti-HA (Cse4) antibody. Untagged Cse4 was used as a negative control. Asterisk shows nonmodified Cse4. (B) Levels of chromatin-bound Cse4 ubiquitination from A were quantified in arbitrary density units after normalization to chromatin-bound Cse4. Error bars represent the standard deviation of three biological repeats. Statistical significance was assessed by unpaired t-test. (C) Cse4 is mislocalized upon overexpression of Cse4 in a cdc48-3 mutant. ChIP-qPCR was performed on chromatin lysate from cdc48-3 strain transformed with vector (endogenous Cse4) or GAL-CSE4 (overexpressed Cse4). Equal volume of solubilized, crosslinked chromatin from each strain was used for ChIP with anti-HA (Cse4), anti-Myc (Npl4) and anti-GST. qPCR was performed for association with CEN3, pericentromeric R1 region of CEN3, and SAP4 and RDS1 promoter regions. Enrichment of Cse4 is shown as a fold change over endogenous Cse4. Error bars represent standard deviation of the mean of two independent experiments. (D) Chromatin-bound Npl4 was significantly reduced at the promoters of SAP4 and RDS1 in a cdc48-3 psh1Δ strain. ChIP-qPCR was performed on chromatin lysate from cdc48-3 or cdc48-3 psh1Δ strain transformed with GAL-CSE4. Equal volume of solubilized, crosslinked chromatin from each strain was used for ChIP with anti-HA (Cse4) and anti-Myc (Npl4). Occupancy of Npl4 normalized to that of Cse4 was measured. Error bars represent standard deviation of the mean of three independent experiments.