Lambda phosphatase treatment.

Protein dephosphorylation assays were performed essentially as previously described in reference 34 with slight modifications. Total protein extracts were prepared from 1.5 × 10⁸ cells. Twenty-five-milliliter cultures were mixed with 2.5 ml of 100% (wt/vol) trichloroacetic acid and incubated on ice for 30 min. Cells were centrifuged, and the pellets were washed once with 10 ml of ice-cold acetone and twice with 500 μl of beating buffer (8 M urea, 50 mM ammonium bicarbonate) containing a protease inhibitor cocktail (Sigma-Aldrich). Cells were resuspended in 200 μl of beating buffer plus protease inhibitors and disrupted with 0.5-mm glass beads in a FastPrep cell disruptor for three cycles of 35 s at 5.5 m/s, 4°C. For assays, 150 μg of protein was treated with 400 U of λ-protein phosphatase (New England Biolabs) in the presence/absence of specific phosphatase inhibitor (5 mM sodium orthovanadate) for 50 min at 30°C. The reaction volume was adjusted with water to dilute the urea-containing buffer at a ratio of 1:10. Protein electrophoresis was performed on 10% SDS-PAGE gels, and the Rnc1-HA fusion was detected as indicated.