Exponential Growth Optimization for Vacuolar and Mitochondrial Studies

Cells were grown exponentially for 15 hours to a maximum density of 5 × 10⁶ cells/ml prior to the initiation of all experiments. Because nutrients and growth phase affected vacuolar and mitochondrial function, extended log-phase growth was necessary to ensure vacuolar and mitochondrial uniformity across the cell population prior to the initiation of all experiments. Cells were cultured in YEPD (1% yeast extract, 2% peptone, 2% glucose) unless otherwise indicated. For CR experiments, cells were cultured in YEP plus the indicated carbon source. Yeast Complete (YC) medium used in the high copy suppressor screen was previously described^{37 (link)}. Concanamycin A (Sigma) was added to cultures at a final concentration of 250 or 500 nM as indicated in figure legends.