Atg1 kinase activity is upregulated in anaphase I and II. (A) Schematic of the chemical-genetic strategy for monitoring Atg1-as (analog-sensitive Atg1, Atg1-M102G) kinase activity in vitro. Atg1-as thiophosphorylates its substrates with a bulky ATPγS analog (N6-PhEt-ATP-γ-S). Thiophosphorylated substrates of Atg1-as can then be alkylated with para-nitrobenzyl mesylate (PNBM) and detected by IB using anti-thiophosphate ester (α-thioP) antibodies. (B) Atg1-as cells under synchronized meiosis (SPM, t = 1 h after NDT80 induction), mitosis (SD, log phase), and starvation (SD-N, t = 20 min) were collected and treated as in A. Following Atg1 kinase assay, the whole-cell lysates were subjected to IB with indicated antibodies. Hxk1, hexokinase isoenzyme 1 (loading control); Atg1-as-p, Atg1-as autophosphorylation. *, unidentified proteins. NC, no supplemented ATP in starved cell lysates. (C) IB of Atg1-as kinase activity in Atg1-as or Atg1 cells, as in B, during synchronized meiosis. (D) Meiotic kinetics of Atg1-as cells in C determined by IF of Tub1, showing upregulated Atg1-as activity at onset of A-I (2 h), A-II (4 h), and post–A-II (6 h). Source data are available for this figure: SourceData F3.