FIGURE 1.

Acm1 is a phosphoprotein and an in vivo substrate of Cdc28. A, extracted ion chromatograms for the doubly charged unmodified and phosphorylated precursor ions of the indicated peptides obtained from electrospray LC/MS analyses. Phosphorylated residues are underlined. Data are from 3FLAG-Acm1 (from pHLP107) purified from yeast arrested in S phase with HU. The 1st two panels illustrate peptides with high phosphate occupancy, suggesting extensive modification at Thr-161 and Ser-202. The last panel illustrates a peptide with low phosphate occupancy at Ser-126. Similar results were obtained from extracted ion chromatograms generated by MALDI LC/MS analysis. B, recombinant 3FLAG-Acm1 was treated with purified Clb2–3HA·Cdc28 complex or a control purification from yeast lacking the HA₃ tag. Reaction products were processed by SDS-PAGE and detected by anti-FLAG immunoblotting. Panels on the right are control immunoblots for Clb2–3HA and Cdc28 in the IP samples. C, cdc28-4 strain containing pHLP109 or pHLP111 expressing HA-Acm1 or HA-Acm1–5E, respectively, from P_GAL1 was arrested in S phase with HU at 23 °C. Galactose induction was performed at either 23 or 37 °C for 2 h, and proteins were detected by anti-HA immunoblotting. NC, negative control with empty vector. G6PD is a loading control. D, extracted ion chromatograms for unmodified and phosphorylated forms of 3FLAG-Acm1 peptide ¹⁵⁴ISLPSFITPPR¹⁶⁴ from HU-arrested CDC28 or cdc28-4 cells at permissive (25 °C) and nonpermissive (37 °C) temperatures. The CDC28 chromatogram is repeated from A to allow direct comparison to cdc28-4 chromatograms.