Figure 7.

Hir2 interacts with Cse4 and facilitates Psh1-mediated proteolysis of Cse4. (A) Hir2 interacts with Cse4 in vivo. Immuno-precipitation (IP) experiments were performed using a HIR2-TAP strain (OpenBiosystem) transformed with either GALCSE4HA (pMB1597) or vector control. Equal amounts of WCE prepared from strains grown in galactose medium for 2 hr were immunoprecipitated with rabbit IgG agarose to pull down Hir2-TAP. The presence of Cse4HA in the IP was detected by Western blot analysis using α-HA antibody. (B) Quantification of interaction of Cse4 and Hir2 in WT strain. Western blots from (A) were used for quantification, where interaction between Hir2-TAP and Cse4HA was quantitated as fold increase in Cse4HA ratio (IP/Input) of Hir2-TAP strain vs. non-TAP strain. Error bar represents SD from three independent experiments. P-value was calculated by Student’s t-test. (C) Reduced interaction of Cse4 and Psh1 in a hir2∆ strain. WT, Psh1-TAP (Open Biosystems) or isogenic hir2∆ strains transformed with either empty vector (pMB433) or GALCSE4HA (pMB1458) were grown logarithmically and Cse4 expression induced by growth in galactose-containing medium for 2 hr. IP experiments were performed as described in (A), and blots were probed with α-HA and α-TAP (CAB1001; Thermo Scientific) antibodies. (D) Quantitation of reduced interaction of Cse4 and Psh1 in hir2∆ strain. Western blots from (B) were quantified to determine the interaction between Psh1-TAP and Cse4HA. The ratios showing levels of Cse4HA over Psh1-TAP from co-IP samples were calculated and normalized to a value of 100 for the WT strain. Error bars represent SD from three independent experiments. (E) Overexpression of Psh1 suppresses the lethality caused by GALCSE4 in hir2∆ strains. Viability assays were performed with a hir2∆ strain containing GALCSE4HA (pMB1458) and PSH1 (From MoBY 2µ ORF library) or GALCSE4HA (pMB1458) and vector alone. A WT strain containing GALCSE4HA (pMB1458) was used as a control. About 1200 cells from each strain were plated on glucose- and galactose-containing medium. Viability is calculated as the ratio of colonies on galactose plates over glucose plates. Average ± SD from three independent experiments is shown. ns, not significant (P-value = 0.07); ** P-value <0.001, Students’ t-test. (F) Overexpression of Psh1 facilitates proteolysis of Cse4 in hir2∆ strains. Western blot analysis was performed using whole cell extracts from hir2∆ strains with GALCSE4HA (pMB1458) and 2μ-PSH1 or vector control. Expression of Cse4 was induced in galactose (2%)-containing medium for 2 hr at 30° and cells shifted to glucose medium and treated with CHX (10 μg/ml). Stability of Cse4 at various time points post-CHX treatment was determined by Western blot analysis probing with α-HA and α-Tub2 (loading control) antibodies. Two biological repeats were performed, with experimental variation within 10% from the mean. (G) Defects in chromosome segregation in hir2∆ strain. Loss of CEN containing plasmid (pRS416 CEN URA3) was measured in WT, psh1∆, hir2∆, and psh1∆ hir2∆ strains. Strains were grown in SC-URA media selecting for the plasmid pRS416 (denoted as generation G₀) followed by dilution and growth in nonselective YPD medium for 10 generations (denoted G₁₀). The frequency of plasmid retention was calculated as the ratio of number of colonies on SC-URA over YPD, where G₀ values for each strain were normalized to 100%. At least 1200 cells for each strain at G₀ and G₁₀ were plated and average frequency of plasmid loss ± SD is shown for three biological replicates. ns, not significant (P-value = 0.18); * P-value = 0.02; Students’ t-test.