BLTP2-KO cells show accumulation of intracellular PI(4,5)P 2 -positive vacuoles. (A) Western blot validating knockout (KO) of BLTP2 in HeLaM cells. Endogenous BLTP2 is enriched using IP before detection. Three independent KO clones are verified. (B) BLTP2-KO HeLaM cells showing presence of PI(4,5)P2-positive intracellular vacuoles. Tubular recycling endosomes are still present in these cells. (C and D) Expression of HRas G12V in HeLaM cells (two independent clones: KO-2C3 and KO-2C6) induces formation of macropinosomes/intracellular vacuoles in both WT and BLTP2-KO cells. However, only in the KO cells a large fraction of these vesicles remain PI(4,5)P2 positive. Quantification of PI(4,5)P2-positive macropinosomes is shown in D. One-way ANOVA. Mean ± SEM. n = 3 independent experiments. 123 cells for WT, 137 cells for KO-2C3, 161 cells for KO-2C6, 119 cells for KO-2C3 rescue, and 122 cells for KO-2C6 rescue. (E and F) BLTP2-KO cells expressing HRas G12V together with FAM102A-GFP (E) or FAM102B-GFP (F). FAM102A-GFP, but not FAM102B-GFP, is enriched on PHPLCδ1-labeled PI(4,5)P2-positive macropinosomes. (G and H) PM dye (G), or 10 KD dextran-488 (H) was added to BLTP2-KO cells expressing HRas G12V. A fraction of the preexisting PI(4,5)P2-positive vacuoles were labeled by the dye within minutes after PM dye addition (G), showing internalization of dextran-488 (H). (I) Schematic model of BLTP2 localization at contacts of the ER with PM and PM-connected structures (left). Illustration of the molecular interaction of BLTP2 with phosphoinositides and its binding proteins (FAM102A/B, N-BAR domain proteins) at these contact sites (right). Source data are available for this figure: SourceData F8.