Fig. 5. Conversion of PtdIns(3)P to PtdIns(3.5)P2 changes targeting from early endosomes to late endosomes.

a Diagram showing the probes used for detecting PtdIns(3)P and PtdIns(3.5)P2, and mechanisms proposed for converting PtdIns(3)P to PtdIns(3.5)P2 and for exchanging Rab5 for Rab7. b Binding of PtdInsPx binding proteins to proteoliposomes containing EE-SNAREs or EE-SNAREs plus Rab5(Q79L) in the presence and absence of PtdIns(3)P. All liposomes contained 1% Biotin-PE. To detect PIKfyve recruitment, the proteoliposomes were incubated with cytosolic fractions for 30 min at 37 °C. The proteoliposomes were precipitated with Streptavidin beads and recruited PIKfyve was detected by immunoblotting. PtdIns(3)P or PtdIns(3.5)P2 on proteoliposomes were detected by EGFP-2xFYVE or GFP-ML1N*2 probes that were expressed in HeLa cells (incubation with cytosol from transfected cells), with binding being measured by flotation gradients. Syntaxin 13 indicates loading control. c Targeting specificity of liposomes containing three EE-SNAREs, active Rab5, and 1% PtdIns(3)P in 1 µM YM-201636 treated cells. The inhibition of PtdIns(3.5)P2 generation prevents the targeting of the proteoliposomes to late endosomes/lysosomes. The data show mean values ± SEM of 3–6 independent experiments. At least 100 injected vesicles were analyzed for the colocalization with each organelle marker in each experiment. Stars indicating significance: *P < 0.05, **P < 0.01, all determined by 1-way ANOVA with the Tukey multiple comparison test. d PtdIns(3.5)P2 shifts targeting specificity of EE-SNARE PL to late endosomes/lysosomes. See text for details. The data show mean values ± SEM of 3–7 independent experiments. At least 100 injected vesicles were analyzed for the colocalization with each organelle marker in each experiment. Stars indicating significance: *P < 0.05, **P < 0.01, ***P < 0.001, all determined by 1-way ANOVA with the Tukey multiple comparison test. Source data are provided as a Source Data file.