Phosphorylation of Num1 and Mdm36 in vitro and in vivo. (A) Coomassie-stained gel of 6xHis-T7-Mdm36 purified from E. coli, used in B. (B) In vitro kinase assays containing γ-32P ATP, 1 µg recombinant Mdm36, and 3 pmol Ime2-st purified from yeast or no-kinase control. Reactions were incubated for 15 min at room temperature, then analyzed by SDS-PAGE and autoradiography. Before assembling the kinase assay, half of the input was reserved and analyzed by immunoblot. (C) In vitro kinase assay prepared as in B, but using on-bead substrate immunoprecipitated from vegetative cells using agarose beads conjugated with an anti-V5 antibody. Untagged lysate (no tag) was purified from wild type (UB15) and Num1-3V5 from strain UB12017. Beads never incubated with lysate (none) were also examined. Before assembling the kinase assay, half of the bead volume for each sample was reserved and bound protein was eluted and analyzed by immunoblot. Num1 runs above the highest (190-kD) ladder band. (D) Top: diagram of Num1 domain structure in the SK1 background and in vivo phosphorylation sites detected by MS (red lines and text). Black numbers refer to amino acid positions that define the domain boundaries. The Num1 amino acid sequence in SK1 differs from the S288C reference genome (Yue et al., 2017). Num1 (SK1) contains six copies of a 64-aa repeat, with some repeats separated by a spacer sequence (LEKEVEQ) and an overall length of 2,378 aa (271 kD). CC, coiled coil domain; PH, Pleckstrin homology domain. Bottom: Num1 phosphopeptides detected by LC-MS/MS from Num1-3V5 denaturing IP (see Materials and methods). pS, phosphoserine. The detected number of phosphopeptides and total peptides (phosphorylated and unmodified combined), as well as the overall sequence coverage of Num1 are shown for three biological replicates, is expressed as means ± SD. Num1-3V5 was isolated from ndt80Δ negative control cells (UB17332; prophase I ndt80Δ) and ndt80Δ IME2-st cells (UB16660; prophase I ndt80Δ IME2-st) after 5 h in SPO medium.
