Identification of vacuolar proteins delivered by the Atg19-dependent CVT pathway. (a) Model highlighting the analysis of ATG19 mutants within the CVT trafficking pathway. (b) Volcano plot identifying cargoes that are enriched or depleted at vacuoles of atg19Δ cells. Analysis was performed as in Fig. 3 b. (c) Localization of overproduced GFP-Lap3 and overproduced BFP-Ape1 in WT (upper panel) and atg19∆ (lower panel) under nutrient-rich growth conditions. Overproduction from the CUP1 promoter was induced with 2 mM CuSO₄ for 90 min. Vacuoles were stained with FM4-64. Scale bar, 5 µm. (c and d) Quantification of images in c from three different experiments. The amount of Lap3 co-localizing, in proximity or not colocalizing with Ape1 was calculated. Bars show average of three experiments (red dots, error bars = SD). (e) Analysis of GFP-Lap3 overproduced from the GPD promoter in respect to FM4-64–stained vacuoles is shown in WT cells (top row), atg19Δ cells (top middle row), ape1Δ cells (bottom middle row), and ape1Δatg19Δ cells (bottom row). Scale bar, 5 µm. (f) Proteomic comparison of vacuoles from atg4Δ cells and WT cells. Averaged peptide intensities are plotted against heavy/light SILAC ratios. Significant outliers (P < 1e⁻¹⁴) are color coded in red (P < 0.0001), orange, or blue (P < 0.05); other identified proteins are shown in light blue.