Quinacrine Staining for Acidified Vacuoles

Quinacrine (Sigma) staining was performed as previously described^{41 (link)} (Hughes and Gottschling). Briefly, cells were washed once in YEPD + 100 mM HEPES, pH 7.6 and resuspended in 100 μl of the same buffered media containing 200 μM Quinacrine. Cells were incubated for 10 min at 30 °C and then 5 min on ice, followed by pelleting and washing twice with ice cold 100 mM HEPES, pH 7.6 + 2% glucose. Cells were resuspended in 100 mM HEPES, pH 7.6 + 2% glucose for imaging. Quinacrine staining was performed for cells in several conditions: (i) exponentially growing cells from 2% glucose, (ii) cells after 90 min of starvation in 0.2% glucose, (iii) cells after 90 min of starvation in 0.02% glucose, (iv) cells after a 20-min recovery in 2% glucose following starvation in 0.2% glucose, and (v) cells after a 20-min recovery in 2% glucose following starvation in 0.02% glucose. Prior to imaging, cells were kept on ice and all images were obtained within 30 min of staining. Supplementary Fig. 17b presents an example of quicacrine stained (acidified) vacuole, and only such vacuoles were scored as positive for Quinacrine staining, i.e., acidified. Concanamycin A (Sigma) was added to cultures at a final concentration of 500 nM to achieve the inhibition of vacuolar acidification.