Identification of vacuolar proteins that require Vps45 mediated membrane fusion for their localization. (a) Model highlighting the analysis of VPS45 mutants within the CPY and CVT trafficking pathways. (b) Volcano plot identifying cargoes that are enriched or depleted at vacuoles of vps45Δ cells. Analysis was performed as in Fig. 3 b. Vps10-dependent cargoes are labeled in green, CVT cargo proteins are labeled in red, V-ATPase subunits are labeled in purple, and other proteins are labeled in blue. (c) Localization of C-terminal mNeon-tagged Ncr1 and FM4-64–stained vacuoles was analyzed in WT cells (upper panel) and vps45Δ cells (lower panel). Scale bar, 5 µm. (d) Model of the Ncr1 domain architecture of the full-length protein (left) and the C-terminal truncation (right). (e) Localization of C-terminal mNeon-tagged Ncr1 (upper panel) and Ncr1ΔC (lower panel) with Vph1-mCherry as the vacuole marker was analyzed. Scale bar, 5 µm. (f) Model of the Atg15 full-length protein (left) and the N-terminal truncation (right). (g) Localization of N-terminal GFP-tagged Atg15 (upper panel) and Atg15ΔN (lower panel) with Vph1-mCherry as the vacuole marker was analyzed. Scale bar, 5 µm. (h) N-terminal truncation of Atg15 shows no effect on the vacuolar proteome. Proteomic comparison of vacuoles from NOPpr-Atg15ΔN/NOPpr-Atg15 and atg15Δ/WT cells.