Figure 6

RENT complexes containing phospho-site mutant Net1(7m) is refractory to disassembly by Polo-like kinase in vitro , but exhibit minor phenotype in vivo . (A) RENT complexes from isogenic NET1-myc9 (WY374) and net1(7m)-myc9 (WY347) cells were immunoprecipitated on 9E10 beads. Beads were divided into equal portions, and treated with indicated amounts of Plx1. Cdc14 retained on Net1-bound beads or released into the supernatant was fractionated by SDS-PAGE and detected by immunoblotting with anti-Cdc14 antibodies. (B) Asynchronous NET1-myc9 (+), net1(7m)-myc9 (7m), and net1(19m')-myc9 (19m') cultures were subjected to indirect immunofluorescence using anti-Cdc14 and anti-tubulin antibodies. Cells with long mitotic spindles (~10% of total) were examined further to calculate the percentage that displayed focal Cdc14 staining. (C) NET1-myc9 (+) and net1(7m)-myc9 (7m) cells were arrested in G1 with α factor, and released into YP + 2% glucose media (T = 0) at 25°C. Samples withdrawn and fixed at the indicated time points were double-labeled with anti-Cdc14 and anti-tubulin antibodies. The percentage of cells with long mitotic spindles (L.S.) and the percentage of cells with delocalized Cdc14 (14) were calculated and plotted independently. (D) Cells harboring the dbf2-1 mutation and either a wild-type or mutant net1 allele were grown in YPD at 25°C, arrested in G1 with α-factor (12 μg/ml), released into YPD prewarmed to 37°C, and incubated at 37°C thereafter. Cells collected at 70–110 minutes after α factor release were double-labeled with anti-Cdc14 and anti-tubulin antibodies. Spindle length was measured and localization of Cdc14 was determined to be in one of the three categories: 1. full release (black box): complete release of Cdc14 from the nucleolus into the nucleus; 2. partial release (gray box): Cdc14 was nuclear in one of the DAPI masses and nucleolar in the other DAPI mass in the same cell, or Cdc14 was nuclear but with stronger nucleolar staining; and 3. no release (clear box): Cdc14 was strictly nucleolar. In each of the three panels, more than 300 cells were counted. (E) Starting with 3000 cells, three-fold serial dilutions of NET1-myc9 (+), net1(7m)-myc9 (7m), and net1(19m')-myc9 (19m') cells in msd2-1 or dbf2-1 background were spotted on a YPD plate from right to left, and incubated at the indicated temperature for 2 days before the picture was taken. Two independent isolates for each strain were used.