Intracellular Cholesterol Transport by Sterol Transfer Proteins at Membrane Contact Sites.

Cholesterol is dynamically transported among membrane-bound organelles primarily by nonvesicular mechanisms. Sterol transfer proteins (STPs) bind cholesterol in their hydrophobic pockets and facilitate its transfer across the aqueous cytosol. However, STPs alone may not account for the specific and efficient movement of cholesterol between intracellular membranes. Accumulating evidence has shown that membrane contact sites (MCSs), regions where two distinct organelles are in close apposition to one another, can facilitate STP-mediated cholesterol trafficking in a cell. At some MCSs, cholesterol can move against its concentration by using phosphatidylinositol 4-phosphate (PI4P) metabolism as the driving force. Finally, the emergence of more MCSs and the discovery of a new STP family further highlight the crucial roles of MCSs and STPs in intracellular cholesterol transport.

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

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.

Measurement of Cholesterol Transfer from Lysosome to Peroxisome Using an In Vitro Reconstitution Assay.

Low-density lipoproteins (LDLs) are taken up by the cell mainly through receptor-mediated endocytosis. LDL-derived cholesterol leaves lysosome and further transports to downstream organelles for specific cellular needs. We recently report that cholesterol transfers from lysosome to peroxisome through lysosome-peroxisome membrane contact (LPMC). Here, we use iodixanol density gradient centrifugation to isolate lysosomes and peroxisomes separately for the in vitro reconstitution of LPMC. We also apply 3H-cholesterol-labeled lysosomes and peroxisomes in vitro to measure 3H-cholesterol transfer through LPMC.