Yeast strains were cultured in synthetic dropout media (2% glucose and 0.67% yeast nitrogen base without amino acids or uracil, supplemented with appropriate nutrients) (Green and Moehle 2001). For yeast strains containing plasmids from the yeast ORF collection, galactose was used as the carbon source in place of glucose because the genes are under the control of the GAL1 promoter. To induce autophagy, nitrogen starvation medium (SD-N or SGal-N; 2% glucose or 2% galactose, 0.17% yeast nitrogen base without amino acids, ammonium sulfate, or vitamins) was used. Yeast cells were first cultured in synthetic dropout media lacking appropriate amino acids or uracil depending on the plasmids they contained, until OD600 = 0.6. They were then incubated at either 30 or 37° in nonstarvation conditions for 30 min before being washing twice in SD-N (or SGal-N) medium and further incubated in SD-N (or SGal-N) at the same temperature for 3 hr. All chemicals for media were from Fisher Scientific except yeast nitrogen base (Sunrise, 1500-500).