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. 2021 Jun 23;18(2):375–390. doi: 10.1080/15548627.2021.1935522

Figure 7.

Figure 7.

Biological role of autophagy in cells auxotrophic for leucine. (A) The negative effect on lifespan of atg mutants can only be observed 25 days after nutrient deprivation. MM cultures of strains CC10 (WT), CC11 (atg1Δ) and CC20 (atg2Δ) grown in MM until logarithmic phase (day 0) were switched to modified MM without nitrogen (w/o N), sulfur (w/o S) or phosphorus (w/o P) sources during 25 days. Serial dilutions of the cultures were plated onto YE5S plates. (B) The negative effect on lifespan of atg mutants auxotrophic for leucine can be observed 12 h after nitrogen or leucine deprivation. Strains HM123 (WT) and SK1 (atg1Δ), both carrying the leu1-32 allele, were grown in leucine-supplemented MM until logarithmic phase (day 0) and switched to MM without leucine (MM), MM with leucine (MM +Leu), MM without nitrogen (w/o N) or MM without nitrogen with leucine (w/o N +Leu), for 12 or 24 h. Serial dilutions of the cultures were plated onto YE5S plates. (C, D) Microscopy images (C) and percent of survival (D) of HM123 (WT leu1-32) and SK1 (atg1Δ leu1-32) after 48 h in MM and leucine-supplemented MM (+ Leu). For survival measurements, cells were incubated with the permeability-dependent dye PI, and the percentage of non-fluorescent cells, indicative of viability, was determined by flow cytometry. (E) TORC1 kinase activity is unaffected in an autophagy-proficient leu1-32 background. MM cultures of strains CC10 (WT) and CC88 (WT leu1-32) were shifted or not to nitrogen depleted media for 10–30 min and analyzed as in Figure 2A. (F) Leucine deprivation can trigger autophagy in leu1-32 auxotrophic strains. MM cultures of strains CC10 (WT) and CC88 (WT leu1-32), both expressing GFP-Atg8, were shifted or not to leucine (2 h) or nitrogen (3 h) depleted MM and processed and analyzed as in Figure 2 C. Numbers below the panels represent the amount of cleaved GFP relative to the amount after 3 h without nitrogen in WT (with an assigned value of 1). (G) Leucine deprivation can inhibit TORC1 kinase activity in auxotrophic strains. Strains and conditions as in (F) were processed and analyzed as in Figure 2A. (H) The fast negative effect on lifespan of atg mutants auxotrophic for leucine is also displayed by TORC1 mutants with impaired autophagy activation. MM cultures of strains HM123 (WT) and atg1Δ (SK1), CC129 (tsc1Δ), CC134 (tco89Δ) and CC135 (atg1Δ tco89Δ), all carrying the leu1-32 allele, were grown and processed as in (B).