MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria.
This study demonstrates that the steroidogenic acute regulatory protein-related lipid transfer (START) domain-containing protein, MLN64, participates in intracellular cholesterol trafficking. Analysis of the intracellular itinerary of MLN64 and MLN64 mutants tagged with green fluorescent protein showed that the N-terminal transmembrane domains mediate endocytosis of MLN64 from the plasma membrane to late endocytic compartments. MLN64 constitutively traffics via dynamic NPC1-containing late endosomal tubules in normal cells; this dynamic movement was inhibited in cholesterol-loaded cells, and MLN64 is trapped at the periphery of cholesterol-laden lysosomes. The MLN64 START domain stimulated free cholesterol transfer from donor to acceptor mitochondrial membranes and enhanced steroidogenesis by placental mitochondria. Expression of a truncated form of MLN64 (DeltaSTART-MLN64), which contains N-terminal transmembrane domains but lacks the START domain, caused free cholesterol accumulation in lysosomes and inhibited late endocytic dynamics. The DeltaSTART-MLN64 dominant negative protein was located at the surface of the cholesterol-laden lysosomes. This dominant negative mutant suppressed steroidogenesis in COS cells expressing the mitochondrial cholesterol side chain cleavage system. We conclude that MLN64 participates in mobilization and utilization of lysosomal cholesterol by virtue of the START domain's role in cholesterol transport.
Zhang
Mei
M
Lipid Cell Biology Section and Cell Biochemistry Section, Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Liu
Pei
P
Dwyer
Nancy K
NK
Christenson
Lane K
LK
Fujimoto
Toshio
T
Martinez
Federico
F
Comly
Marcy
M
Hanover
John A
JA
Blanchette-Mackie
E Joan
EJ
Strauss
Jerome F
JF
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D004705:Endocytosis; D005799:Genes, Dominant; D006801:Humans; D015151:Immunoblotting; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D002352:Carrier Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C105684:STARD3 protein, human; D011374:Progesterone; D002784:Cholesterol