Indentification And Studies Of Low-density Lipoprotein Receptor Newly Degradation Pathway

Steroid hormones,includeing sex hormones,mineralocorticoids and glucocorticoids,are crucial signal molecules,which regulate a wide array of physiological functions such as reproduction,initiation and maintenance of sexual characteristics,water-salt balance,and various metabolic processes.Cholesterol is needed as a substrate for steroid hormone biosynthesis in steroidogenic tissues,adrenal gland,gonads and other steroidogenic tissues.LDL-cholesterol is an important source for steroidogenesis.The LDL/LDLR pathway begins with the binding of LDL to LDLR,and the complex is then internalized.In the acidic endosomes,LDL dissociates from LDLR,with LDLR recycled back to the plasma membrane and LDL delivered to lysosome,where cholesteryl esters are hydrolyzed to release cholesterol.However,how LDL-cholesterol is directly conveyed to the mitochondria in steroidogenic organ or cell is poorly defined.Here,through a genome-wide sh RNA screening,we find that the OMM protein phospholipase D6（PLD）acts as a novel regulator of LDLR.PLD6 is anchored to the outer mitochondrial membrane（OMM）via its transmembrane domain（5–27 a.a.）and the156^th histidine residue（H156）is essential for its catalytic activity.Both catalytic activity and mitochondrial localization are crucial for PLD6 to promote LDLR degradation,and PLD6 induces LDLR degradation by modulating PA level in the OMM.By using the Laser-confocal microscopy and transmission electron microscopy,we find LDL/LDLR can be translocated into the mitochondria.Knockdown of PLD6 decreases the co-localization ratio of LDL/LDLR with mitochondria,whereas overexpression of PLD6induces the accumulation of LDL/LDLR in mitochondria,suggesting that PLD6 is involved in LDL/LDLR translocation into mitochondria.Using mitochondrial protease screening and antagonistic experiments,we identidy that LDLR protein is degraded by mitochondrial proteases（HTRA2,PITRM1 and PMPCB）.Through steroid ELISA and cholesteryl ester hydrolase assays,we confirm that LDL can be hydrolyzed into cholesterol for steroidgenesis.Knockdown of PLD6 attenuates the generation of steroid,conversely,overexpression of PLD6 promotes the production of steroid,suggesting that PLD6 is involved in steroidgensis.By immunoprecipitation and cell-free mitochondria recruitment assay,we show that the OMM protein CISD2 binds to the cytosolic tail of LDLR via the C₉₉RCWRS motif and tethers LDLR⁺vesicles to the mitochondria.We further demonstrate the fusogenic lipid PA generated by PLD6 facilitates the membrane fusion of LDLR⁺vesicles with the mitochondria.In summary,we find that a novel LDL/LDLR transport pathwayand unravel a new function of PLD6 in incucing LDL/LDLR⁺vesicles fusion with mitochondria.This work provides insight into the LDL/LDLR pathway and steroidgenesis.