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. Author manuscript; available in PMC: 2016 Mar 2.
Published in final edited form as: Curr Biol. 2015 Feb 5;25(5):546–555. doi: 10.1016/j.cub.2014.12.049

Figure 6. Rim15-dependent phosphorylation of Rph1 upon nitrogen starvation releases its repression on ATG gene expression and autophagy.

Figure 6

(A–B) Rph1 is phosphorylated upon nitrogen starvation. Rph1 was chromosomally tagged with PA tag and Rph1-PA cells (YAB308) were grown in YPD until mid-log phase and then starved for nitrogen for the indicated times.

(A) Protein extracts were analyzed by western blot with or without 50 μM Phos-tag as indicated and with either an antibody that recognizes PA or anti-Pgk1 (loading control) antiserum. Arrowheads indicate shifts in molecular weight of Rph1-PA suggesting phosphorylation.

(B) Rph1-PA band shifts represent phospho-isoforms of the protein. Cells were lysed with or without phosphatase (Ppase) inhibitors. An aliquot of cell lysates was incubated at 30°C for 90 mintes in λ-phosphatase buffer with or without λ-phosphatase (λ Ppase). The reaction was stopped and proteins were precipitated by addition of 10% trichloroacetic acid. Protein extracts were analyzed by western blot as in (A) from gels containing Phos-tag.

(C–D) Rph1 phosphorylation upon nitrogen starvation is blocked in rim15Δ cells. RPH1 was chromosomally tagged with PA in wild-type (WT) and rim15Δ cells. Cells were grown in YPD until mid-log phase and then starved for nitrogen for the indicated times. WT (YAB308) and rim15Δ cells (YAB341) were collected and protein extracts were analyzed by western blot with either an antibody that recognizes PA or anti-Pgk1 (loading control) antiserum. (D) Protein extracts were analyzed by western blot as in (B).

(E) The deletion of RIM15 reduces the induction of ATG gene expression after nitrogen starvation. WT (YAB308) and rim15Δ (YAB341) cells were grown in YPD until mid-log phase and then starved for 1 h (−N). mRNA levels were quantified by RT-qPCR. The mRNA levels of individual ATG genes were normalized to the mRNA level of the corresponding gene in WT cells in nitrogen starvation condition (−N), which was set to 100. Data represent the average of at least 3 independent experiments ± standard deviation.

(F) ATG7 was chromosomally tagged with PA in WT (YAB312) and rim15Δ (YAB342) cells. Cells were grown in YPD until mid-log phase and then starved for nitrogen for the indicated times. Protein extracts were analyzed by western blot with either an antibody that recognizes PA or anti-Pgk1 (loading control) antiserum.

(G) ATG7 was chromosomally tagged with PA in WT (YAB312), rim15Δ (YAB342), rph1Δ (YAB313) and rim15Δ rph1Δ (YAB347) cells. Proteins extracts were analyzed as in (F).

(See also Figure S4.)