PIP4K2A regulates intracellular cholesterol transport through modulating PI(4,5)P2 homeostasis

Abstract

The transport of LDL-derived cholesterol from lysosomes to peroxisomes is facilitated by membrane contacts formed between the lysosomal protein synaptotagmin VII and the peroxisomal lipid phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P₂]. Here, we used RNA interference to search for regulators of PI(4,5)P₂ and to study the effects of altered PI(4,5)P₂ homeostasis on cholesterol transport. We found that knockdown of phosphatidylinositol 5-phosphate 4-kinase type-2 α (PIP4K2A) reduced peroxisomal PI(4,5)P₂ levels, decreased lysosome-peroxisome membrane contacts, and increased accumulation of lysosomal cholesterol in human SV-589 fibroblasts. Forced expression of peroxisome-localized, kinase-active PIP4K2A in the knockdown cells reduced cholesterol accumulation, and in vitro addition of recombinant PIP4K2A restored membrane contacts. These results suggest that PIP4K2A plays a critical role in intracellular cholesterol transport by upregulating PI(4,5)P₂ levels in the peroxisomal membrane. Further research into PIP4K2A activity may inform future therapeutic interventions for managing lysosomal storage disorders.

Keywords: ATP binding cassette transporter D1; Syt7; lysosome-peroxisome membrane contact; lysosomes; synaptotagmin.

Fig. 1.

Cholesterol accumulated in lysosomes of PIP4K2A-deficient cells. A: Phosphatidylinositol 4,5-bisphosphate related metabolic pathways. B: Knockdown efficiencies of the siRNA duplexes. Scramble siRNAs were used as controls. C: Filipin staining of intracellular cholesterol. Human SV589 fibroblasts were transfected with indicated siRNAs for 48 h. Then the cells grown on coverslips were fixed and stained with filipin (pseudo-colored in red). Representative confocal pictures were selected and shown. Bar = 20 μm. D: Quantification of filipin signal intensity in (C) by Image J. More than 30 cells were randomly selected and counted for each group, statistical analysis, one-way ANOVA. Data are expressed as mean ± SD. E: Filipin staining of cholesterol in CRISPR-Cas9-mediated PIP4K2A-knockout SV589 cells. Genomic regions flanking guide RNA targeting sequences were amplified by PCR and validated by Sanger sequencing. Two individual subclones were selected and stained with filipin (red). F: Costaining of cholesterol and LAMP1. Cholesterol and lysosomes were detected by filipin and the antibody against endogenous LAMP1, respectively. G: Analysis of SREBP-2 cleavage. Wild-type or PIP4K2A-KO cells were treated as described in Materials and Methods. Cell lysates were probed with the monoclonal antibody 1D2 against human SREBP-2, and anti-β-actin. H: Measurement of PM cholesterol. Cholesterol was measured with an Amplex Red Cholesterol Assay Kit. Statistical analysis, one-way ANOVA. Data are expressed as mean ± SD.

Fig. 2.

Knockdown of PIP4K2A reduced lysosome-peroxisome membrane contacts in the cells. A: Immunostaining of endogenous PMP70 (green) and LAMP1 (red). Insets show higher magnification of the area in the white boxes. Bar = 10 μm. B: Quantification of colocalization of LAMP1 and PMP70 signal. Confocal images were analyzed by Image J with the JACoP plugin. Mander’s coefficients were defined by the fraction of LAMP1 signal overlapping PMP70 signal. Forty-six cells were counted for each group; statistical analysis, one-way ANOVA.

Fig. 3.

Silencing PIP4K2A reduced lysosome-peroxisome membrane contacts as measured by proximity-dependent biotinylation assay. A: Schematic illustration of proximity biotinylation assay. B: Colocalization of NPC1-BirA-GFP (green) and lysosome marker LAMP1 (red). C: Validation of proximity-dependent biotinylation assay. HEK-293T cells stably expressing NPC1-BirA-GFP fusion protein were transfected with indicated siRNAs and incubated with biotin-containing culture medium. Then the cells were lysed and biotinylated proteins were pulled down with avidin beads and subjected to Western blotting. D: Proximity-dependent biotinylation assay in control and PIP4K2A-silencing cells. Experiments were performed as described in C. E: Quantification of gray density of PMP70 in D. Triplicate pellet fractions were normalized with input and quantified by Image J.

Fig. 4.

Peroxisomal PIP4K2A was required for lysosome-peroxisome membrane contacts in vitro. A: Schematic illustration of in vitro reconstitution assay. Peroxisomes or lysosomes from HeLa cells stably expressing either scramble or PIP4K2A-targeting shRNA were purified as previously described (8). Peroxisomes were pulled down with Ni-NTA and further incubated with purified lysosomes. Ni-NTA resin was spun down, washed, and the associated lysosomes were detected by Western blotting. B: In vitro reconstitution of lysosome-peroxisome membrane contacts. Lysosomes and peroxisomes were purified from indicated cells and subjected to in vitro reconstitution illustrated in A. C: Purified recombinant Flag-PIP4K2A from HEK-293T cells was resolved on SDS-PAGE and stained with Coomassie Brilliant Blue. D: In vitro lysosome-peroxisome membrane contacts rescued by the recombinant Flag-PIP4K2A protein. Reconstitution assay was performed with or without 1 mg/ml cytosol or 50 μg/ml Flag-PIP4K2A protein. Experiments were performed as described in A.

Fig. 5.

PIP4K2A affected peroxisomal PI(4,5)P₂ level. A: PI(4,5)P₂ dot blot. Lipids were extracted from indicated fractions of cells. Extracts containing equal amounts of protein from either lysosomes or peroxisomes were dotted in 2-fold dilution on a Hybond membrane. PI(4,5)P₂ standards were dotted as indicated. Blots were detected with the anti-PI(4,5)P₂ antibody. B: Rescue experiments using various PIP4K2A constructs. PIP4K2A-knockout cells were transfected with indicated plasmids and stained with filipin. Cells were outlined in white dashed lines. Representative pictures from each experiment were shown.