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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 2. Methionine is the Sole Amino Acid Sufficient for Inhibition of NNS-Autophagy.

Figure 2

(A) In the presence of other amino acids, methionine but not cysteine still inhibited NNS-autophagy. Following switch to SL medium plus the indicated amino acids, general autophagy and mitophagy were measured using the ALP assay (left) and the GFP cleavage assay (right). See also Figure S2.

(B) Diagram outlining sulfur utilization and metabolism in budding yeast.

(C) Homocysteine is unable to inhibit NNS-autophagy in met6Δ cells. Following switch to SL plus the indicated metabolites, general autophagy was measured using the ALP assay. In the met6Δ mutant, metabolic flux from homocysteine to methionine is blocked.

(D) Oxidative stress is not involved in the regulation of NNS-autophagy. General autophagy and mitophagy were assayed following switch to SL plus the indicated concentrations of reductants. Glutathione (GSH) inhibited NNS-autophagy only at much higher concentrations compared to Met or Cys. Two other potent anti-oxidants, dithiothreitol (DTT) and β-mercaptoethanol (βME), did not significantly inhibit NNS-autophagy.

(E) Global oxidative stress is not induced upon switch to SL medium. Intracellular levels of GSSG (oxidized GSH) and GSH were determined by multiple reaction monitoring (MRM) using previously established LC-MS/MS methods (Tu et al., 2007). Relative abundance of two daughter fragments targeting GSH and GSSG are shown.

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