Identification of cargo proteins of the Vps10 sorting receptor. (a) Model highlighting the analysis of the CPY trafficking pathway. (b) Volcano plot identifying cargoes that are enriched or depleted at vacuoles of vps10Δ cells. Fold changes calculated from the independent experiments comparing SILAC ratios from (vps10Δ K8 versus WT K0) with (WT K8 versus WT K0) on the x-axis were plotted against negative logarithmic P values of the t test performed from replicates. The hyperbolic curve separates depleted proteins (left side, blue dots) and enriched proteins (right side, blue dots) from unaffected proteins (black dots). (c) Experimental setup to compare secreted proteins in vps10Δ and WT cells. (d) Identified proteins in the supernatant of vps10∆ compared with WT. Averaged peptide intensities are plotted against average heavy/light SILAC ratios from two different experiments. 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. (e) Validation of MS results via Western blot. Npc2 and CPY were tagged with a 6HA tag and supernatants of WT and mutant cells decorated with anti-HA antibody. (f) Structure of Vps10 variants including full-length Vps10, Vps10-lacking domain 2 (vps10ΔD2), and Vps10 lacking domain 1 (vps10ΔD1). (g and h) Proteomic comparison of vacuoles isolated from vps10ΔD2 cells and WT cells (g) and of vps10ΔD1 cells and WT cells (h). SILAC ratios of two independent experiments are plotted against each other. Previously identified Vps10 cargo proteins are labeled. (i) Proteins which bind to Vps10 domain 2 show leucin or isoleucine as hydrophobic amino acid. Only the domain 1–dependent Ydr415C harbors a phenylalanine. Source data are available for this figure: SourceData F3.