Experimental setup to generate the vacuolar biogenesis map. (a) Model of vacuolar protein-targeting routes in yeast. Vacuolar proteins are delivered via the CVT pathway (orange), the CPY pathway (blue), and the AP-3 pathway (green). Deleted genes include APL5 (subunit of the AP-3 complex), the GGA1 and GGA2 adaptor protein coding genes, the CPY receptor VPS10, the S/M protein–encoding gene VPS45, and the CVT receptor ATG19. (b) Vacuolar morphology was analyzed in WT, apl5Δ, vps10Δ, gga1Δgga2Δ, vps45Δ, and atg19Δ cells using the mNeon-tagged transmembrane protein Vph1, the lipophilic dye FM4-64, and the vacuolar luminal dye CMAC. Scale bar, 5 µm. (c) Quality control of purified vacuoles. Vacuoles were purified from the same amounts of WT (Vph1-mCherry) and mutant cells (Vph1-mNeon or ADHpr-GFP-Yck3). Vacuole number and size were determined from 20 pictures (error bars = SD). All mutants, except vps45Δ, show comparable numbers of vacuoles. Scale bar, 5 µm. (d) Experimental setup for QPrevail.