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
PGAL1 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.