Bmh1 or Bmh2 overexpression accelerates glucose starvation–induced autophagy by enhancing Atg1 activation. (A) Cells co-expressing an empty vector and GFP-Atg8, or 3✕HA-Bmh1 with the Cup1 promoter and GFP-Atg8 in the wild-type strain were treated with or without CuSO4 for 2 h, and then cultured in glucose starvation medium in the absence or presence of CuSO4 for 0 h, 1 h, 2 h, or 4 h. Autophagic activity was analyzed by Western blot for the cleavage of GFP-Atg8. Pgk1 served as a loading control. (B) Quantification of the ratio of free-GFP/(GFP+GFP-Atg8) from A (n = 3). Data are presented as means ± SD. ***P < 0.001; ns, no significance; two-tailed Student’s t tests were used. (C) Atg1-3✕FLAG Cells expressing an empty vector or 3✕HA-Bmh1 with the Cup1 promoter were treated with or without CuSO4 for 2 h. The cells were then cultured in glucose starvation medium in the absence or presence of CuSO4 for 0 h or 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-p-T226-Atg1 antibody. (D) The phosphorylation level of Atg1 T226 from C was quantified by calculating the ratio of p-Atg1-T226/FLAG-Atg1(n = 3). Data are presented as mean ± SD. ***P < 0.001; ns, no significance; two-tailed Student’s t tests were used. (E) Model depicting Ca2+-triggered assembly of the Atg11–Bmh1/2–Snf1 complex governing the initiation of glucose starvation–induced autophagy. CaMK: Calmodulin (CaM)-dependent protein kinase. Source data are available for this figure: SourceData F9.
