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. Author manuscript; available in PMC: 2014 Jul 18.
Published in final edited form as: Cell. 2013 Jul 18;154(2):403–415. doi: 10.1016/j.cell.2013.06.041

Figure 1. Inhibition of NNS-autophagy by Sulfur-Containing Amino Acids.

Figure 1

Cells were grown in YPL rich medium to log phase before switching to SL minimal medium to induce NNS-autophagy. As shown using three different standard assays for autophagy (A-C), NNS-autophagy was significantly inhibited by methionine and cysteine, but not other amino acids. Non-S Mix refers to a non-sulfur amino acid mixture which includes all amino acids except Met, Cys, and Tyr (17 total). See also Figure S1.

(A) Imaging: Note the accumulation of the mitochondria-targeted RFP (mtRFP) reporter in the vacuole following switch to SL. Met or Cys addition was sufficient to inhibit translocation of mtRFP to the vacuole. Vph1-GFP is a vacuolar membrane marker.

(B) GFP-cleavage assay: Note the accumulation of free GFP in strains expressing either mitochondria-targeted OM45-GFP or IDH1-GFP following switch to SL medium. Met or Cys addition was sufficient to significantly reduce the amount of free GFP produced as a result of cleavage in the vacuole.

(C) ALP activity assay: Using a mitochondria or cytosolic targeted alkaline phosphatase reporter, mitophagy and general autophagy were monitored using the alkaline phosphatase assay as described previously (Wu and Tu, 2011). Met or Cys, but not other amino acids, was sufficient to potently inhibit NNS-autophagy. *** p<0.001, ** p<0.01, * p<0.05, ns p >0.05