PP1 Phosphatase assay

1. Pulldown of Glc7-phosphatase from budding yeast cell lysates

Glc7-TAP were enriched from yeast lysates on IgG Sepharose beads (GE Healthcare BioScience) according to manufacturer’s recommendations. In addition, ESM356-1 (a strain without TAP tag) lysates were incubated with IgG Sepharose beads as a no-tag control. Briefly, cell pellets of logarithmically growing cultures were lysed using acid washed glass beads (Sigma) in a lysis buffer containing 50mM Hepes pH:7.5, 150mM NaCl, 1mM EDTA, %1 SDS, 1mM DTT, 2mM PMSF and 1X Complete Protease Inhibitor Coctail (Roche). The lysate was cleared by centrifugation at 10000 rpm at + 4 ^oC and the supernatant was incubated with IgG beads for 2 h at 4 ^oC. Beads were washed with ice cold wash buffer containing 50mM Hepes pH: 7.5, 150mM NaCl, 0.1mM EDTA, %0.1 SDS, %0.025 Tween 20, 1mM DTT and were immediately used for the phosphatase reaction as the phosphatase source.

1. Purification of Bfa1-3HA from budding yeast cell lysates

Bfa1-3HA was purified from BFA1-3HA kin4∆ Gal1 -UPL-TEM1 cells arrested in anaphase. For this, log-phase culture grown in Raffinose/Galactose containing rich medium were transferred into YPAD medium. After >90% of the cell arrest was achieved, cell pellets were lysed using acid washed glass beads in bead beater. Lysis buffer composition was: 50mM Hepes pH:7.5, 150mM NaCl, 5% Glycerol, 1mM EDTA, 1% NP-40, 160mM β-Glycerophosphate, 2mM NaVO₃, 100mM NaF, 2mM PMSF, 4mM Benzamidine and complete Protease Inhibitor Coctail (Roche). After clearing the cell lysate by centrifugation at 10000 rpm at + 4 ^oC, the cell extract was incubated with anti-HA magnetic beads (Thermofisher, Pierce^TM) for 2 h at 4 ^oC. After the incubation, beads were washed in 50mM Hepes pH: 7.5, 150mM NaCl, %1 NP-40, %5 Glycerol, 1mM EDTA and eluted from beads under basic elution conditions according to manufacturer’s protocol. Briefly, beads were incubated with 50mM NaOH for 5 minutes and the eluate was immediately neutralized by 300mM Tris pH:8.5. Next, the eluate was applied on PD MiniTrap G-25 Sephadex (GE Healthcare) columns equilibrated with a buffer containing 50mM Hepes pH: 7.5, 100mM NaCl. Buffer exchange was performed according to manufacturer’s spin protocol. Freshly prepared substrate was used in the phosphatase reaction as hyperphosphorylated Bfa1-3HA diminished upon storage.

1. PP1 Phosphatase reaction

In vitro phosphatase reaction was performed using the IgG-beads that were obtained from protocol 1 (Pulldown of Glc7-phosphatase in association with Bud14 from yeast cell lysates). These were Bud14-TAP containing IgG-beads and control IgG-beads. Bfa1-3HA prepared as explained above (protocol 2) was used as the substrate. Both the substrate and Bud14-TAP was freshly prepared before the assay. Phosphatase reaction buffer composition was 100mM NaCl, 50mM Hepes pH: 7.5, 2mM DTT, %0.025 Tween, 2mM MnCl₂.  MnCl₂ was freshly prepared before the reaction as otherwise caused variability in the reaction efficiency. Three reaction tubes were prepared. All reactions had the Bfa1-3HA. Two of the reactions Bud14-TAP containing IgG-beads and the other reaction had the control IgG-beads. To inhibit phosphatase activity, Okadaic acid (Abcam) was added to 1.5 µM final concentration in one of the reaction tubes that contained Bud14-TAP. Reaction tubes were incubated at 30 ^oC for 1h. When incubation was over, 5x sample buffer was added immediately into the reaction tubes, samples were boiled at 95 ^oC for 5 min and analyzed by western blotting next day.