The steroidogenic acute regulatory protein homolog MLN64, a late endosomalcholesterol-binding protein

MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein . The steroidogenic acute regulatory protein is located in the inner membra ne of mitochondria, where it facilitates cholesterol import into the mitoch ondria, Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain, The present work was undertaken to determine which step of the intracellular cholesterol pathway ML64 participates in. U sing immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found spe cifically in late endosomes. Electron microscopy indicates that MLN64 is re stricted to the limiting membrane of late endosomes. Microinjection or endo cytosis of specific antibodies shows that the START domain of MLN64 is cyto plasmic, Deletion and mutagenesis experiments demonstrate that the aminoter minal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signa ls, we show that a dileucine motif (Leu(66)-Leu(67)) and a tyrosine residue (Tyr(89)) are critical for the targeting or the proper folding of the mole cule, Finally, MLN64 colocalizes with cholesterol and Niemann Pick CI prote in in late endosomes. However, complementation assays show that MLN64 is no t involved in the Niemann Pick C2 disease which, results in cholesterol lys osomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s).