Extended Data Fig. 5: Rpl12 is specifically required in yeast ribophagy under starvation conditions.
a, BY4741, atg1Δ, or rpl12bΔ Rpl12a-AID cells expressing either wild-type (WT) Rpl12 or Rpl12S79D-S101D (2D) were grown to the log phase, harvested, and analyzed using an anti-Rpl12 antibody. The data are representative of two independent experiments. b-e, The indicated yeast cells were treated with IAA for 2 h, followed by incubation in SD-N for 0 or 6 h. Ribophagic activity were analyzed by western blot to detect the cleavage of GFP-fusion proteins. f, Ribophagic activity from (b-e) was quantified. g, The indicated yeast cells were treated with IAA for 2 h, then subjected to SD-N for 6 h, followed by performed the APEX-EM analysis. Scale Bar: 500 nm. h, i, The indicated yeast cells expressing Om45-GFP or Pex14-GFP were treated with IAA for 2 h, then subjected to SD-N for 0 or 8 h. Protein samples were analyzed by western blot using an anti-GFP antibody. j, BY4741 cells, atg1∆ cells, or atg39∆ atg40∆ cells expressing with Sec63-GFP were subjected to SD-N for 0 or 8 h. ER-phagy activity was analyzed by western blot using an anti-GFP antibody. k-m, The indicated cells expressing Rpl3-GFP, Rpl25-GFP, or Rps25a-GFP were subjected to SD-N for 0 or 6 h. Ribophagic activity was analyzed by western blot using an anti-GFP antibody. Pgk1 served as a loading control. The data are representatives of three independent experiments unless specified in the legends. Statistical significance: ***P < 0.001, *P < 0.05. P values were calculated by two-tailed Student’s t tests (f) and the data are presented as mean ± s.d. (n = 3 biological replicates). Exact P values, source numerical data and unprocessed blots are provided in Source data.
