Role of ATAD3 in inter-organelle interactions in hormone-induced Leydig cell steroidogenesis

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 358-380-Steroid Hormone Biosynthesis & Metabolism
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-372
Leeyah Issop*1, Malena Beth Rone2 and Vassilios Papadopoulos3
1Research Institute of the McGill University, QC, Canada, 2McGill Univeristy, Montreal, QC, Canada, 3The Research Institute of the McGill University Health Center, Montreal, QC, Canada
Steroid formation is a multi-step process initiated with the transport of the cholesterol from intracellular stores into the outer mitochondrial membrane (OMM) through a complex termed the transduceosome, in which cytosolic proteins interact with OMM proteins. From there, cholesterol reaches the cytochrome P450 CYP11A1 enzyme in the inner mitochondrial membrane (IMM) through a bioactive 800-kDa protein complex present at the outer/inner membrane contact sites, the steroidogenic metabolon. CYP11A1 converts cholesterol to pregnenolone. Cholesterol transfer from OMM to CYP11A1 is the rate-limiting step of this process. Using BN-PAGE in tandem with mass spectrometry analysis, the AAA+ ATPase ATAD3 was identified to be part in the later complex. ATAD3 is anchored in the IMM and enriched at OMM-IMM contact sites. ATAD3 RNA silencing in MA-10 Leydig cells revealed the critical role of this protein in mitochondrial cristae organization and the hormone-stimulated steroid formation.  Moreover, knocking down ATAD3 resulted in a decrease in OMM-IMM contact site formation and endoplasmic reticulum (ER)-mitochondria physical interactions but had no effect on 22R-hydroxycholesterol-supported steroid formation suggesting a role for this protein in cholesterol transfer to CYP11A1. The C-terminal region of ATAD3 in the matrix contains the ATP-binding domain, named Walker A. Mutation of the Walker A domain resulted in a reduction in Leydig cell steroid formation suggesting a role of ATP in the function of ATAD3 in cholesterol transport. The N-terminus of ATAD3 contains 50 amino proposed to form an alpha-helix that drives the insertion of the protein back into OMM and associated organelles, such as ER. Indeed, ATAD3 has been found in mitochondria associated membranes (MAMs), regions of close apposition between OMM and ER. These structures would allow the transfer of the substrate cholesterol into mitochondria. We hypothesized that ATAD3 functions as a bridge between the mitochondrial membranes and as a contact point between organelles, in the MAMs. Deletion of the ATAD3 N-terminus resulted in the reduction of hormone-stimulated steroidogenesis suggesting a role of ATAD3 in the mitochondria-ER contact site formation. Taken together these results suggest the presence of inter-organelle communications in Leydig cells, mediated by ATAD3, which appear to be crucial for the transfer of cholesterol into mitochondria for steroidogenesis.

Nothing to Disclose: LI, MBR, VP

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