The Steroidogenic Acute Regulatory Protein Homolog MLN64, a Late Endosomal Cholesterol-binding Protein

Fabien Alpy (1) , Marie-Elisabeth Stoeckel , Andrée Dierich , Jean-Michel Escola , Corinne Wendling (1) , Marie-Pierre Chenard , Marie T Vanier , Jean Gruenberg , Catherine Tomasetto (1) , Marie-Christine Rio (1)

1 IGBMC - Institut de Génétique et de Biologie Moléculaire et Cellulaire

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 membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. 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 MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the aminoterminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine-or tyrosine-based targeting signals, 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 molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal 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).

Mots clés: PBS; phosphate-buffered saline; NPC; Niemann-Pick C

Domaines: Biologie cellulaire

Fabien Alpy, Marie-Elisabeth Stoeckel, Andrée Dierich, Jean-Michel Escola, Corinne Wendling, et al.. The Steroidogenic Acute Regulatory Protein Homolog MLN64, a Late Endosomal Cholesterol-binding Protein. Journal of Biological Chemistry, 2001, 276 (6), pp.4261-4269. ⟨10.1074/jbc.M006279200⟩. ⟨hal-03415546⟩