Organelle Plasticity and Interactions in Cholesterol Transport and Steroid Biosynthesis

Author's personal copy

Review

Organelle plasticity and interactions in cholesterol transportand steroid biosynthesis

Leeyah Issop a, Malena B. Rone a, Vassilios Papadopoulos a,b,c,⇑a The Research Institute of the McGill University Health Centre, Department of Medicine, McGill University, Montreal, Quebec, Canada H3G 1A4b Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1A4c Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada H3G 1A4

a r t i c l e i n f o

Article history:Available online 13 December 2012

Keywords:MitochondriaEndoplasmic reticulumLipid dropletsGonadsAdrenalBrain

a b s t r a c t

Steroid biosynthesis is a multi-step process controlled by pituitary hormones, which, via cAMP-dependent signaling pathways, drive tissue-specific steroid formation. Steroidogenesis begins with thetransport of the substrate, cholesterol, from intracellular stores into the inner mitochondrial membrane,where the steroidogenic enzyme CYP11A1 converts cholesterol to pregnenolone. This process is acceler-ated by hormones and involves a number of proteins and protein–protein interactions. Indeed, choles-terol, stored in lipid droplets and membranes, is transferred through a hormone-induced complex ofproteins derived from the cytosol, mitochondria, and other organelles termed the transduceosome tothe outer mitochondrial membrane. From there, cholesterol reaches CYP11A1 through outer/inner mem-brane contact sites. Thus, cholesterol transfer is likely achieved through a hormone-dependent reorgani-zation of organelles and protein distribution and interactions. The findings reviewed herein suggest thepresence of a hormone-dependent organelle communication network mediated by protein–proteininteractions and inter-organelle trafficking, resulting in the efficient and timely delivery of cholesterolinto mitochondria for steroid synthesis.

� 2012 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352. Transduceosome and steroidogenic metabolon: hormone-dependent mitochondrion-targeted cholesterol import and metabolism . . . . . . . . . . 35

2.1. TSPO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362.2. VDAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362.3. ATAD3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372.4. Cyp11a1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372.5. STAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372.6. ACBD3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3. Mitochondria, site of regulation of multiple processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383.1. Mitochondrial bioenergetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383.2. Mitochondrial shaping proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.2.1. MFNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393.2.2. OPA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393.2.3. Drp1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

0303-7207/$ - see front matter � 2012 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.mce.2012.12.003

Abbreviations: cAMP, cyclic AMP; ACAT, acyl-CoA cholesterol acyltransferase; ACSL4, acyl-CoA synthetase 4; ANT, adenine nucleotide transporter; BN-PAGE, blue nativepolyacrylamide gel electrophoresis; CPT, carnitine palmitoyltransferase; ER, endoplasmic reticulum; ETC, electron transfer chain; FDX, ferredoxin; FDXR, ferredoxinreductase; IDH2, isocitrate dehdydrogenase 2; IMM, inner mitochondrial membrane; LAM, lipid droplet-associated membrane; LD, lipid droplet; MAM, mitochondria-associated membrane; MDH2, malate dehydrogenase 2; MFNs, mitofusins; MPTP, mitochondrial permeability transition pore; OMM, outer mitochondrial membrane; PKA,protein kinase A (cAMP-dependent); ROS, reactive oxygen species; SE, sterol ester; TCA, tricarboxylic acid cycle or citric acid cycle; TG, triglyceride; TSPO, translocator protein(18-kDa); VDAC, voltage dependent anion channel.⇑ Corresponding author at: The Research Institute of the McGill University Health Center, Montreal General Hospital, 1650 Cedar Avenue, C10-148, Montreal, Quebec,

Canada H3G 1A4. Tel.: +1 514 934 1934x44580; fax: +1 514 934 8439.E-mail address: [email protected] (V. Papadopoulos).

Molecular and Cellular Endocrinology 371 (2013) 34–46

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4. MAMs: sites of ER–mitochondrial membrane interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395. Inter-organelle interactions between LDs, mitochondria, and the ER. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

5.1. LD–ER interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415.2. LD–mitochondria association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425.3. LD–ER–mitochondria (LERMIT) association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

1. Introduction

Steroids are critical mediators of numerous processes in thebody, from the regulation of development and reproduction tobehavior. Under physiological conditions, steroidogenic cells storea low amount of steroid hormones but in response to circulatingpituitary hormones adrenal and gonadal cells rapidly produce largeamounts of glucocorticoids, mineralocorticoids, progestins, andro-gen, and estrogen to respond to various physiological needs. Theplacenta and brain also have the ability to make steroids that aretargeted to satisfy specific needs of these tissues. At present, thereis no evidence that pituitary hormones control placental and brainsteroidogenesis. Steroid biosynthesis begins at the mitochondrion,where cholesterol, transferred from intracellular stores to the outerand then inner mitochondrial membrane (IMM), is metabolized bythe cytochrome P450 side chain cleavage enzyme CYP11A1, lo-cated in the matrix side of the IMM.

Although steroidogenic cells constitutively produce a certainamount of steroids, this process is accelerated by the pituitary tro-phic hormones, adrenocorticotropic hormone (ACTH), luteinizinghormone (LH), and follicle stimulating hormone (FSH), which in-duce the production of cyclic AMP (cAMP) (Jefcoate, 2002; Simpsonand Waterman, 1988). This rise in intracellular cAMP results in themobilization of free cholesterol (Garren et al., 1971). This choles-terol is transferred to mitochondria to be used for the productionof steroids. The transfer of hydrophobic cholesterol has been pro-posed to occur in three steps: (i) integration at the outer mitochon-drial membrane (OMM), where it remains segregated from thestructural membrane cholesterol; (ii) movement from the OMMto the IMM; and (iii) loading onto CYP11A1 at the matrix side ofthe IMM. Although not much was known about the first step, theidentification of a hormone-induced multiprotein complex com-posed of cytosolic and OMM proteins at the OMM controlling therate of steroid formation named the transduceosome (Liu et al.,2006; Rone et al., 2009a) enhanced our understanding of the mech-anisms underlying cholesterol entry into the OMM and will be dis-cussed later in this review. It has been proposed that the transfer ofcholesterol across the hydrophilic intra-mitochondrial membranespace occurs at specialized contact sites where there is appositionbetween the OMM and the IMM (Stevens et al., 1985; Thomson,2003). This was recently confirmed by the identification of a bioac-tive, multimeric protein complex spanning the OMM–IMM unitthat is responsible for the hormone-induced import, segregation,targeting, and metabolism of cholesterol (Rone et al., 2012) Thus,cholesterol import into the OMM through the transduceosomeand passage into the IMM through the contact sites bring choles-terol to CYP11A1, which is present in this multimeric complex atthe OMM–IMM contact sites, for enzyme loading and conversionto pregnenolone (Hall, 1985; Jefcoate, 2002; Rone et al., 2012).Pregnenolone, the precursor of all steroids, can then be convertedto tissue-specific steroid hormones through steroidogenic enzymeslocated in mitochondria or the endoplasmic reticulum (ER), thoughthe exact mechanism is currently unknown.

The identified multiprotein enzyme complex formed by trans-duceosome proteins and the OMM–IMM contact sites containing

CYP11A1 is able to transfer the substrate cholesterol to its site ofmetabolism without equilibration with and diffusion into the sur-rounding environment (Rone et al., 2012). Such supramolecularcomplexes have been referred to as protein machines or metabo-lons (Srere, 1987).

As mitochondria are cholesterol-poor organelles, the mobiliza-tion of cholesterol from different cytosolic sources, such as lipiddroplets (LDs), the ER, and others, to mitochondria ensures thecontinuation of steroid production. The identification of the trans-duceosome, which allows the association of mitochondria withcytosolic proteins and multiple organelles, provides the missingphysical link between organelles that represent potential sourcesof the steroidogenic cholesterol and mitochondria, the site of cho-lesterol metabolism. The regulation of inter-organelle interactionswith the mitochondria should provide further flexibility of choles-terol sources and regulation of downstream steroid formation.

In this review, we discuss the intracellular mechanisms bywhich steroidogenesis is initiated and tightly regulated, focusingon the transfer of cholesterol via protein–protein and inter-orga-nelle interactions to CYP11A1. We first discuss the initial transferof cholesterol to the IMM through the transduceosome and the for-mation of the steroidogenic metabolon. Then, we discuss the rolemitochondrial morphology plays in steroidogenesis and the rolesthat other intracellular organelles, specifically the ER and LDs, playin making cholesterol available for steroidogenesis via their inter-actions with mitochondria.

2. Transduceosome and steroidogenic metabolon: hormone-dependent mitochondrion-targeted cholesterol import andmetabolism

In 2006, we identified an OMM protein complex termed thetransduceosome that propagates cAMP signaling and initiates cho-lesterol transfer to the mitochondria, thus functioning as a sieve atthe OMM by separating and restricting cholesterol for steroido-genic activity (Liu et al., 2006). This regulated, controlled flow ofcholesterol into mitochondria is accomplished by anchoring ofthe cytosolic components of the transduceosome, acyl-CoA bindingdomain-containing 3 (ACBD3), protein kinase A regulatory subunitI alpha (PKA-RIa), and the hormone-induced mitochondrion-tar-geted steroidogenesis acute regulatory protein (STAR) to theOMM proteins translocator protein (18 kDa, TSPO) and voltage-dependent anion channel (VDAC). While the initial componentsof the proposed metabolon allow cholesterol transfer to theOMM, the transduceosome does not identify the key mechanismof this metabolon of cholesterol transfer to the IMM and CYP11A1.Therefore, to further understand this process and identify theproposed metabolon regulating the rate-limiting step in steroidproduction, we utilized blue native-polyacrylamide gel electropho-resis (BN-PAGE), chemical crosslinking, and mass spectrometry. Inearly 2012, we reported the presence of an 800-kDa proteincomplex composed of OMM and IMM proteins, including TSPO,VDAC, AAA + ATPase ATAD3, and CYP11A1 (Rone et al., 2012). Aschematic representation of the transduceosome and steroidogenicmetabolon is shown in Fig. 1A. While each of these proteins is

L. Issop et al. / Molecular and Cellular Endocrinology 371 (2013) 34–46 35


discussed below, more detailed information is available elsewhere(Papadopoulos and Miller, 2012; Rone et al., 2009a). In this section,we will focus on the formation of the transduceosome and the roleof the identified metabolon in steroidogenesis, presenting itsmitochondrial and cytosolic components.

2.1. TSPO

TSPO, previously known as peripheral benzodiazepine receptor,was identified when radiolabeled benzodiazepine was found tohave binding sites in the kidney and further confirmed to be pres-ent in most tissues of the body (Anholt et al., 1985; Papadopoulos,1993). A specific role in steroid production was suggested whenTSPO was further recognized to be present at higher concentra-tions in steroidogenic tissues, where it was found to be located pri-marily at the OMM (Anholt et al., 1985; Woods et al., 1996).Confirmation of its role in steroidogenesis was obtained when li-gands specific to TSPO were shown to increase pregnenolone andsteroid formation in multiple steroidogenic cell lines, primary cellcultures, and isolated mitochondria as well as by the absence ofsteroid formation in genetically engineered steroidogenic cells de-void of TSPO (Lacapere and Papadopoulos, 2003). In vivo studiesfurther demonstrated a correlation between TSPO levels and ste-roidogenesis and the effect of TSPO drug ligands on steroid forma-tion (Lacapere and Papadopoulos, 2003; Rupprecht et al., 2010).

Through 3D modeling and docking simulations, it was proposedthat TSPO spanned the OMM in five alpha helixes and was able toaccommodate and transfer a cholesterol molecule (Culty et al.,1999). TSPO was found to bind cholesterol with high affinity andthe cholesterol recognition amino acid consensus (CRAC) domainof TSPO was identified in the C-terminal region of the protein (Liand Papadopoulos, 1998; Li et al., 2001b). More recent structuralstudies supported by cryoelectron microscopy and nuclear mag-netic resonance further confirm both the five alpha helix morphol-ogy and the role of TSPO in cholesterol binding and transport(Korkhov et al., 2010; Murail et al., 2008; Teboul et al., 2012).

Upon hormonal stimulation, TSPO was shown to undergo poly-merization (Boujrad et al., 1996; Papadopoulos et al., 1994), due tothe formation of dityrosine bonds (Delavoie et al., 2003). TSPOdimerization and polymerization resulted in increased ligand

binding affinity and cholesterol translocation to the IMM (Delavoieet al., 2003). TSPO was found to be concentrated at OMM–IMMcontact sites (Culty et al., 1999) and subsequently identified as partof a transduceosome protein complex in MA-10 Leydig cell mito-chondria that binds photoactivatable cholesterol (Liu et al.,2006). Using BN-PAGE and photoactivatable cholesterol under na-tive conditions, TSPO was further identified to be part of two dis-tinct complexes migrating at 66- and 800-kDa (Rone et al., 2012).The 66-kDa complex contained TSPO monomers, whereas TSPOdimers/polymers were found in the 800-kDa complex shown tocontain OMM and IMM proteins, forming the steroidogenicmetabolon.

2.2. VDAC

VDAC is a 32-kDa OMM beta-barrel protein where it functionsin regulating the transfer of ions and small molecules across themitochondria membrane. Through this process, it can influencemultiple cellular events related to mitochondrial bioenergeticsand regulate apoptosis. It is also proposed to be an importantmember of the transduceosome complex, where it interacts withTSPO to aid in the transfer of cholesterol to the IMM.

VDAC was first identified to interact with TSPO in digitonin-sol-ubilized mitochondria separated through gel filtration columnchromatography (McEnery et al., 1992). The IMM adenine nucleo-tide translocase (ANT) was also identified as part of the same com-plex. Interestingly, VDAC and ANT are part of the mitochondrialpermeability transition pore (MPTP) which plays an important rolein the regulation of apoptosis. TSPO was also shown to be part ofand play a regulatory role in the function of this pore (Ricchelliet al., 2011). Interestingly, it has also been proposed that this poreis part of the OMM–IMM contact sites (Brdiczka et al., 2006).Although we were able to demonstrate a role for VDAC in the func-tion of TSPO, we were unable to provide clear evidence for a rolefor ANT (Garnier et al., 1994). VDAC, unlike ANT, was identifiedto be part of the transduceosome (Liu et al., 2006) and present inthe 800-kDa steroidogenic metabolon (Rone et al., 2012). These re-sults were confirmed by knocking down or inhibiting the functionof these proteins in MA-10 Leydig cells (Rone et al., 2012). It shouldbe noted that in recent studies knocking down VDAC and ANT had

Fig. 1. Mitochondrial proteins involved in the regulation of steroidogenesis. (A) The transduceosome complex and the steroidogenic metabolon. In response to hormonetreatment, the OMM TSPO and VDAC1 complex recruits ACBD3, which brings PKA-RIa to mitochondria. The hormone-induced STAR protein contains a mitochondrial signalsequence and is targeted to the OMM, where it interacts with VDAC and is locally phosphorylated by PKA for maximal activity. This complex is termed the transduceosomebecause it transduces the cAMP signal directly at the OMM. The OMM proteins TSPO and VDAC together with the IMM proteins ATAD3 and CYP11A1 are part of the larger800-kDa metabolon composed of proteins that bring cholesterol directly to CYP11A1 for metabolism. (B) Mitochondrial fusion and fission proteins. The principal actors inmitochondrial fusion are the mitofusins (MFN1/2) in the OMM and OPA1 in the IMM. Different isoforms of OPA1 are displayed: short (s) or long (l), soluble or anchored. Theseisoforms are able to form oligomers involved in the maintenance of cristae. Drp1 is a cytosolic protein involved in the mitochondrial fission mechanism.

36 L. Issop et al. / Molecular and Cellular Endocrinology 371 (2013) 34–46


no impact on the regulation of the permeability transition pore andapoptosis, challenging their role as essential components of thepore (Baines et al., 2007; Halestrap, 2009). Taken together, theseresults confirm the importance of VDAC-TSPO interactions in cho-lesterol import into mitochondria and transfer to CYP11A1. Therole of VDAC in steroidogenesis was further supported by the find-ing that it interacts with bioactive phosphorylated STAR at theOMM and facilitates the processing of this protein to a matureinactive form (Bose et al., 2008).

Due to its role in the transfer of small molecules, such ascalcium, ADP/ATP, glutamate, and NADH, (Shoshan-Barmatz andGincel, 2003; Shoshan-Barmatz et al., 2010) at the OMM–IMM con-tact sites, VDAC could further alter the energetics of mitochondria,thus regulating steroid production (Allen et al., 2006). It has alsobeen demonstrated that VDAC interacts with proteins present inthe ER, an observation that will be addressed in more detail below.Taken together, our data on TSPO and VDAC1 (Rone et al., 2012),the results reporting an interaction between STAR and VDAC1(Bose et al., 2008) and the data showing that TSPO drug ligandsregulate MPTP (Ricchelli et al., 2011; Sileikyte et al., 2011; Zulianet al., 2011) suggest that TSPO mediates a signal across the OMMto the permeability channel in the IMM and that this plays animportant role in cholesterol transfer.

2.3. ATAD3

ATAD3 belongs to the large family of ATPases associated withdiverse activities (AAA). ATAD3 is a highly conserved protein and,while genomic studies revealed that the Atad3 gene has no ortho-log in prokaryotic organisms, it does appear in higher eukaryoticorganisms from plants to humans (Li and Rousseau, 2012). ATAD3was identified in 2003 and, although the exact structure of the pro-tein remains unknown, it is enriched at mitochondrial contact sitesand strongly anchored in the IMM (Da et al., 2003; Gilquin et al.,2010; He et al., 2007). Among the various identified isoforms ofthe protein, these studies revealed the original structure of a longisoform of the protein containing at the N terminus an additional50 amino acids, which are suggested to form an alpha helix loop.This specific domain of the protein contains a flexible region richin proline and coiled coil domains, which through oligomerizationis proposed to interact with the inner surface of the OMM. The C-terminal region contains an ATP-binding domain named Walker Aand an ATPase domain named Walker B. The latter domain hasbeen shown to be involved in the oligomerization responsible forthe interaction of the ATPase with the OMM or eventually organ-elles such as the ER, although the mechanisms involved remainunclear.

Among multicellular organisms, this abundant and ubiquitousprotein is associated with many activities, including embryogene-sis in C. elegans and Drosophila (Gilquin et al., 2010; Hoffmannet al., 2009), cellular growth in Drosophila, association with frataxin(Correia et al., 2008), and coordination of the fusion–fission eventsto the channeling of diverse metabolites (Gilquin et al., 2010). Re-cent studies have implicated ATAD3 in steroidogenesis (Gilquinet al., 2010; Rone et al., 2012).

First, ATAD3 was shown to affect mitochondrial organizationand function and to be involved in H295R adrenal cortical steroidformation (Gilquin et al., 2010). Using BN-PAGE in tandem withmass spectrometry analysis, ATAD3 was identified in the 800-kDa bioactive molecular complex together with TSPO and VDAC(Rone et al., 2012). RNA silencing studies demonstrated the criticalrole of the protein in MA-10 Leydig cell steroid formation. RNAsilencing of ATAD3 also caused a profound modification of mito-chondrial ultrastructure, observed by electron microscopy. Thelocalization of ATAD3 as a bridge between the mitochondrial mem-branes indicates that this protein may not be just a component of

the mitochondrial contact sites, but also might serve as a contactpoint between organelles, such as the ER and mitochondria, to al-low the transfer of cholesterol. We also hypothesized that ATAD3may function in transporting out of the mitochondria the pregnen-olone and progesterone formed. However, the fact that ATAD3-de-pleted cells make steroids in the presence of the hydrosolublecholesterol 22R-hydroxy-cholesterol indicates that its role ismainly in cholesterol import. Indeed, the presence of this proteinhas recently been shown at ER–mitochondrion specialized regions,sites of trafficking of many molecules, such as calcium and lipids.Reduced expression of ATAD3 results in loss of interaction betweenmitochondrial and ER membranes and reduced cholesterol flowbetween the two organelles (Li and Rousseau, 2012).

2.4. Cyp11a1

CYP11A1 is constitutively active on the matrix side of the IMM,its activity depending on substrate availability (Papadopoulos andMiller, 2012). Like all enzyme members of the P450 family,CYP11A1 activates molecular oxygen using its heme iron coreand electrons provided by nicotinamide adenine dinucleotidephosphate (NADPH). Electrons are provided by an electron transferchain containing a ferredoxin reductase (FDXR), a monoflavin pro-tein, and ferredoxin (FDX), a small iron-sulfur protein, both presentin the IMM. CYP11A1 was identified in the 800-kDa complex, andits levels were found to increase in response to hormone treat-ment, indicating hormone-dependent mitochondrial plasticity(Rone et al., 2012). To demonstrate that CYP11A1 in the 800-kDacomplex was active, we incubated the complex with a choles-terol-resorufin probe that fluoresces upon cleavage by CYP11A1(Rone et al., 2012). The results showed that the 800-kDa complex,which also contains FDX and FDXR, is functional in steroid produc-tion upon addition of isocitrate and NADP and its activity increasesfollowing hormone treatment, thus fulfilling the characterizationof the steroidogenic metabolon.

2.5. STAR

STAR was first identified due to the rapid increase in its proteinexpression and phosphorylation after the addition of hormones orcAMP in steroidogenic cells (Miller, 2007; Pon et al., 1986; Pon andOrme-Johnson, 1986; Stocco and Clark, 1996). It was further iden-tified that inactivating mutations of STAR result in a very severedisorder, namely congenital lipoid adrenal hyperplasia, in whichsteroid biosynthesis is inhibited (Miller and Bose, 2011). Newlysynthesized, STAR is a 37-kDa protein that contains an N-terminalmitochondrial targeting sequence, which, upon import into themitochondria, is cleaved to form the 30-kDa mature form of theprotein (Jefcoate, 2002). The targeting of STAR to mitochondriaand its rapid upregulation that parallels the rise in steroid produc-tion has led to the hypothesis that STAR assists with the targetingand transfer of cholesterol to the OMM. There is now evidence thatonly newly synthesized STAR is functional (Artemenko et al.,2001), and that STAR does not need to enter mitochondria to stim-ulate steroidogenesis (Bose et al., 2002). Rather, it functions at theOMM by activating a mitochondrial receptor or transport mecha-nism (Miller and Bose, 2011; Strauss et al., 2003). Although thisseems to be true in vitro, in non-steroidogenic and steroidogeniccell models, discrepancies in the efficacy of STAR lacking its aminoterminal mitochondrial targeting sequence to stimulate steroidformation in steroidogenic cells to the same extent as in non-ste-roidogenic cells, and in vivo studies indicating that cleavage ofSTAR at the mitochondria is linked to the steroidogenic activityof STAR, suggest that there is a tight control of STAR targetingand processing in steroidogenic cells (Granot et al., 2007b; Mannaet al., 2009). Indeed, recent data indicated the presence of



hormone-induced adaptor proteins that bind STAR and control therate of its activity (Aghazadeh et al., 2012).

Molecular modeling of STAR identified the STAR-related lipidtransfer (START) domain, a conserved amino acid motif that func-tions in cholesterol and lipid binding (Clark, 2012). START bindscholesterol with 30 nM affinity (Miller, 2007; Tuckey et al., 2002)and transfers free cholesterol to mitochondria (Alpy and Tomaset-to, 2005; Petrescu et al., 2001; Strauss et al., 2003). Although thismotif has not been conclusively demonstrated to be the mecha-nism through which cholesterol is transferred to the mitochondria,this motif has been shown to function in the stabilization of thetertiary structure of STAR, which is required for proper steroido-genic activity (Barbar et al., 2009). The stable tertiary structureupon cholesterol binding could play an import role in protein–pro-tein interactions at the mitochondria, specifically between TSPOand STAR.

The presence of STAR in the transduceosome was confirmedthrough the incubation of photo-crosslinkable amino acids in hor-mone-treated, but not control, MA-10 cells, which demonstratedthat the presence of STAR increased in the transduceosome in atime-dependent manner (Liu et al., 2006). The further interactionof STAR with the transduceosome was also seen in TSPO-depletedcells. Besides a dramatic decrease in steroid production, it was alsodemonstrated in these cells that STAR is not processed into the ma-ture 30-kDa form, suggesting that TSPO also functions in STAR im-port into mitochondria (Hauet et al., 2005). Therefore, the presenceof STAR in the transduceosome complex functions in the hormone-induced increase in cholesterol movement to mitochondria. STARwas not present in the 800-kDa complex, most likely due to its ra-pid processing by LONP1 protease (Granot et al., 2007a), which wasidentified in the 800-kDa complex by mass spectrometry (Roneet al., 2012). It is also likely that STAR is not tightly bound to the800-kDa mitochondrial complex and it is released during the isola-tion of the mitochondria. However, addition of recombinant STARto isolated mitochondria accelerated cholesterol transport and ste-roid synthesis by the 800-kDa metabolon, demonstrating that thisprotein machinery is its site of action in mitochondria.

2.6. ACBD3

ACBD3, previously named PAP7, is a cytosolic and Golgi-associ-ated protein that binds to both TSPO and PKA-RIa (Li et al., 2001a).Upon hormone stimulation, ACBD3 dissociates from the Golgi andmoves to mitochondria, thus bringing PKA to its site of action.Indeed, PKA-RIa is proposed to be brought in close proximity toSTAR through this scaffolding network to allow for the efficientphosphorylation required for maximal steroid production. Theimportance of ACBD3 has been demonstrated by transient overex-pression, which stimulates progesterone production in MA-10cells; ACBD3 knockdown and transient transfection of the TSPO-and PKA-RIa-binding domain of ACBD3 inhibited hormone-stimulated steroid formation (Li et al., 2001a). It should be notedthat ABCD3 also binds PKARIIa although with less affinity (Liet al., 2001a). Crosslinking and immunoblotting studies demon-strated that ACBD3 was present in the transduceosome (Liuet al., 2006). However, it was not found in the 800-kDa complex,suggesting that, like STAR, it acts outside mitochondria but withOMM proteins. In this case, ACBD3 binds to TSPO.

3. Mitochondria, site of regulation of multiple processes

Mitochondria are dynamic organelles for which there is a closelink between structure and function. Besides its role as the site ofsteroidogenesis initiation, this organelle also functions in the regu-lation of numerous parameters crucial for cell viability, such as ATP

production, apoptosis, free radical production, and calcium levels.A review of how various mitochondrial functions could play a roleon steroidogenesis is presented below.

3.1. Mitochondrial bioenergetics

Mitochondrial bioenergetics results from the tightly controlledcommunication between different processes occurring in mito-chondria, such as the electron transfer chain (ETC) and the citricacid cycle (TCA). This regulation is essential for mitochondrialhomeostasis and may act as another level of regulation ofsteroidogenesis.

Indeed, studies using different inhibitors of the ETC demon-strated that steroid formation is dependent on the energy stateof mitochondria (Allen et al., 2006). In addition, modulation ofthe various proteins involved in steroidogenesis can modulateETC. For example, specific TSPO drug ligands were shown to mod-ulate mitochondrial respiration (Hirsch et al., 1989; Zisterer et al.,1992), and mitochondrial respiration could be implicated in theregulation of steroid formation. Previous studies have also shownthat ATP synthesis is also critical for steroidogenesis in both MA-10 mouse tumor and primary Leydig cells (Allen et al., 2006). Itis important to note that, although the role of ATP in steroid pro-duction is not questioned, its source is cell-dependent. MA-10 isa Leydig cell line that uses ATP originating from cytosolic glycoly-sis, like all tumor cell lines, whereas ATP production is dependenton the ETC in primary Leydig cells (Midzak et al., 2011).

ATP is not only present to provide energy for the cell but couldalso be associated with other functions, such as the targeting ofproteins like TSPO to mitochondria, an ATP-dependent process(Rone et al., 2009b), or used as a substrate for mitochondrial ATP-ases, such as ATAD3. Indeed, it has been shown that the ATP-bind-ing domain of ATAD3A (Walker A) is involved in OMM-IMMinteractions (Gilquin et al., 2010).

The electron donors of the ETC, such as NADH or FADH2, areprovided by the TCA. The regulation of the TCA also plays a rolein the control of the energetic state of mitochondria. In our recentstudy, mass spectrometry analysis revealed the presence of twoenzymes in the 800-kDa metabolon, isoform 2 of isocitrate dehy-drogenase (IDH2) and isoform 2 of malate dehydrogenase(MDH2) (Rone et al., 2012). These two enzymes catalyze the reac-tions providing NADPH and NADH, both used in the ETC. IDH2functions in the production of NADPH for the FED system, the elec-tron source for CYP11A1 activity. The formation of NADPH is alsoassociated with the production of reactive oxygen species (ROSs),an aspect of the mitochondrial bioenergetic process that has tobe taken into account in the regulation of steroid formation. Actu-ally, all reactions happening in the ETC, meaning the flow of elec-trons to a molecular oxygen acceptor lead to the production of ROS.These species are highly active and could play both physiologicaland pathological roles. Since IDH2 and MDH2 are involved in theETC, NADPH production, and ROS production, these enzymesshould be considered novel targets in the study of the regulationof steroidogenesis. In addition, the activity of both enzymes istightly regulated by calcium (de Brito and Scorrano, 2010), whichis a crucial regulator of steroidogenesis (Kimura, 1986).

3.2. Mitochondrial shaping proteins

Mitochondria continuously undergo fusion and fission depend-ing on their cellular and physiological state. Specific proteins calledmitochondrial shaping proteins (Sauvanet et al., 2010; Zorzanoet al., 2010) are responsible for the balance between fusion and fis-sion of the mitochondrial network. This developing field of re-search provides a potential novel mechanism in the regulation ofmitochondrial steroid biosynthesis. In the last decade, several



genes that modulate mitochondrial fusion and fission have beenidentified (Yaffe, 1999). The principal proteins involved in this pro-cess are the mitofusins (MFN 1 and 2), which drive the tetheringbetween OMMs; optical atrophy 1 (OPA-1), which drives not onlythe fusion of IMMs but also has an important role in the mainte-nance of mitochondrial cristae remodeling; and dynamin-relatedprotein 1 (Drp1), which regulates mitochondrial fission. Fig. 1Bshows the distribution of the mitochondrial shaping proteins.

3.2.1. MFNSMFN1 and 2 are the homologs of the yeast protein Fzo1 (Rojo

et al., 2002) initially discovered in Drosophila male mutants inwhich spermatozoa had the appearance of ‘‘fuzzy onions’’ (Halesand Fuller, 1997). MFNs belong to the GTPase family of proteinsand are associated with the OMM (Bourne et al., 1990, 1991).Knockout studies in mice indicated a role for Mfn2 in placentalfunction (Chen et al., 2007). Mitochondrial fragmentation observedafter the loss of each of these isoforms is different, suggesting adifference in membrane tethering likely due to GTPase activity(Palmer et al., 2011). Further studies indicated that MFN2 not onlylocalizes to the OMM but also to the ER membranes and is found atspecific contact sites between the two organelles (de Brito andScorrano, 2008). This supports the idea that MFN2 could play a rolein the regulation of steroidogenesis by transporting cholesterol orions to support steroid production. In support of this hypothesis,recent studies demonstrated that MFN2 expression is induced inresponse to cAMP treatment of MA-10 Leydig cells, whereas silenc-ing Mfn2 results in reduced cAMP-dependent steroid formation bythese cells (Duarte et al., 2012). Although MFN1 was found in the800-kDa mitochondrial complex (Rone et al., 2012), silencingMfn1 in MA-10 Leydig cells did not affect hormone-dependent ste-roid formation (unpublished data).

3.2.2. OPA1OPA1 was discovered as the gene carrying the mutations leading

to autosomal dominant optic atrophy (Alexander et al., 2000;Delettre et al., 2000). It was further identified as a homolog ofthe yeast proteins Mgm1 (Saccaharomyces cerevisiae) and Msp1(Schizosaccharomyces pombe). OPA1 belongs to the dynamin-re-lated protein family, in which a GTPase domain is required foractivity (Alexander et al., 2000).

OPA1 is found in the mitochondrial intermembrane space orstrongly anchored to the IMM (Delettre et al., 2001). Several stud-ies analyzing the consequences of OPA1 loss on the mitochondrialnetwork revealed the existence of diverse proteases that targetOPA1 at specific cleavage sites. This proteolytic process is respon-sible for the generation of two isoforms, a long isoform critical forthe fusion process and a short isoform that cannot drive fusion byitself but is required by the long isoform for fusion and the main-tenance of cristae morphology (Duvezin-Caubet et al., 2006;Estaquier et al., 2012; Palmer et al., 2011). Recently, the two isoformswere shown to undergo oligomerization with the balance betweenthese two isoforms altering mitochondrial homeostasis. For in-stance, the induction of apoptosis, dissipation of mitochondrialmembrane potential, and alteration of mitochondrial cristae asso-ciated with mitochondrial fragmentation has been documentedupon degradation of the long isoform and increase of the solubleshort isoform (Arnoult et al., 2005; Frezza et al., 2006; Lee et al.,2004). OPA-1 activity in apoptosis and mitochondrial fusion seemto be independent of each other (Zorzano et al., 2010).

OPA-1 was identified to be part of the 800-kDa metabolon inMA-10 Leydig cells. Silencing of Opa1 induced fragmentation ofthe mitochondrial network in these cells but has no effect on hor-mone-induced steroid production (Rone et al., 2012). At the sametime, OPA-1 was shown to play a role in placental steroid produc-tion (Wasilewski et al., 2012). The differences in the role of OPA-1

in testis Leydig cell versus placental steroidogenesis may be due tothe fact that a placenta forms steroid in a constitutive, hormone-independent manner, whereas Leydig cell steroid formation is in-duced by LH and cAMP. Thus, although OPA-1, which regulatesmitochondrial fusion and cristae morphology, may play a role inbasal steroid formation in placenta, its role may be attenuated bythe hormone-induced changes in Leydig cell mitochondrialstructure.

These findings on the role of MFN and OPA-1 in steroidogenesisimplicate for the first time the role of mitochondrial fusion, as wellas structure, in the regulation of cholesterol import into mitochon-dria and subsequent steroid formation. Considering that mitochon-drial morphology and function are closely linked to thebioenergetic state of mitochondria, the aforementioned studiessuggest that cholesterol transport into mitochondria is a tightlyregulated process involving multiple partners, the main functionof which may not be related to steroid formation but which di-rectly or indirectly control this process.

3.2.3. Drp1Drp1 was discovered as a homolog of Dnm1 in yeast, in which it

functions as a cytosolic GTPase that mediates mitochondrial fission(Smirnova et al., 1998). Upon stimulation, cytosolic Drp1 is re-cruited to the OMM, where it initiates mitochondrial fission; how-ever, how this process occurs remains unclear (Palmer et al., 2011).There is no information about the role of Drp1 in steroidogenesis.Nevertheless, it has been reported that Drp1 may play a role inthe formation of contact sites between the ER and mitochondriaand thus could affect mitochondrial and ER morphology (Fanget al., 2010). This finding is further supported by the observationthat the ER plays a role in mitochondrial division and Drp1 couldparticipate at the site of division (Friedman et al., 2011; Palmeret al. 2011). These observations suggest that Drp1 and mitochon-dria fission could indirectly participate in the regulation of steroidformation by modulating the formation of mitochondria-associ-ated membranes (MAMs) (see below).

4. MAMs: sites of ER–mitochondrial membrane interaction

The ER is a highly interconnected and dynamic tubular networkthat has many cellular functions, including protein folding, transferof metabolites, and serving as a reservoir for calcium. The ER hasalso been proposed to play important roles in the regulation ofsteroidogenesis. This may occur through multiple processes, ascalcium plays a crucial role in the regulation of steroidogenesis(Cooke, 1999; Kimura, 1986; Spat and Hunyady, 2004), and cal-cium homeostasis is controlled by the ER (Prins and Michalak,2011). Electron microscopy coupled to biochemical studies re-vealed the existence of specific regions of close apposition betweenER membranes and the OMM (Mannella et al., 1998; Rizzuto et al.,1993, 1998). More recently electron tomography studies confirmedthe presence of MAMs (Csordas et al., 2010). These regions repre-sent 5–20% of the mitochondrial surface (Rizzuto et al., 1998).MAMs are crucial for highly efficient transmission of calcium fromthe ER to mitochondria, thus controlling mitochondrial bioenerget-ics, morphology, and certain functions like apoptosis (Bononi et al.,2012). In the last couple of years, evidence has been presentedshowing accumulation of free cholesterol in specific domains ofthe MAMs, especially on a specific site called lipid raft-like micro-domains formed by cholesterol and lipids such as ceramides(Hayashi and Su, 2010). These findings suggest that it is likely thata source of cholesterol for steroidogenesis may be coming from theER via the MAMs.

Most steroidogenic enzymes are located in the ER. It is believedthat pregnenolone—and some progesterone—formed in mitochondria



freely diffuses to the ER to be converted into final tissue-specificsteroid products, such as glucocorticoids, mineralocorticoids,androgen, estrogen, and neurosteroids. It is likely that pregneno-lone and progesterone transfer to the ER through MAMs, thusallowing for the targeted delivery of mitochondrial steroids tothe steroidogenic enzymes in the ER.

Imaging studies have supported the idea that the distances be-tween the ER and mitochondria are important for their properfunction (Rizzuto et al., 1993, 1998). This distance can be modu-lated by proteins, such as MFN2, which is able to bind both mem-branes. Mfn2 RNA silencing induced both complete fragmentationof the mitochondrial network and swollen and aggregated ERmembranes. At the same time, calcium transfer was altered (deBrito and Scorrano, 2008). While MFN2 has been shown to play arole in MAM formation, no such role has been shown for MFN1,suggesting a different role for the MFNs in relationship to theirlocalization. These data are in agreement with the findings thatMFN2 is important for cAMP-induced Leydig cell steroid formation(Duarte et al., 2012), whereas MFN1 does not seem to play a role(unpublished data). In addition to MFN2, PACS2 has been shownto control ER–mitochondria communication (Simmen et al.,2010), although no role of this protein in steroidogenesis has beenshown.

Acyl-CoA synthetase 4 (ACSL4), an enzyme originally shown tobe involved in fatty acids biosynthesis has also been shown to playa crucial role in the regulation of steroids synthesis (Malobertiet al., 2005, 2007). ACSL4 is present in the ER and it is part of theMAMs (Lewin et al., 2002; Simmen et al., 2005). The associationof ACSL4 with the Leydig cell mitochondria is regulated by LHand cAMP and a recent study demonstrated that this associationalso depends on mitochondrial fusion (Duarte et al., 2012).

ATAD3 has also been found in MAMs, and knocking down thisprotein resulted in a decrease in mitochondrial contact site formation

and ER–mitochondria physical interactions (Fang et al., 2010; Liand Rousseau, 2012). The role of ATAD3 in this case remainsunclear; however, since the function of this protein is ATP-depen-dent, it is likely to be involved in MAM formation as an ATP sensor.

Calcium homeostasis is very important for the proper function-ing of both the ER and mitochondria, where it could be an impor-tant regulator of steroidogenesis, especially in the transfer ofcholesterol and steroid products between the ER and mitochon-dria. Furthermore, calcium homeostasis in the two organelles isalso linked to the energy state of the organelles and could be mod-ulated in these MAM regions.

Cytosolic calcium levels are very low; however, upon hormonalstimulation, an intra-mitochondrial rise of calcium rapidly occurs.Studies of this rapid increase in calcium revealed the existence oflipid microdomains at the MAMs, which contain high concentra-tions of calcium, thus providing an explanation for the rapid up-take of calcium by mitochondria (Fujimoto et al., 2012; Hayashiand Su, 2010; Rizzuto et al., 1998). There are a number of proteinspresent at MAMs, including certain ER proteins involved in calciumsignaling, including the inositol-1,4,5-triphosphate receptor (IP3R),molecular chaperones, such as the sigma-1 receptor and BiP, andmitochondrial proteins, such as MFN2 and VDAC1. VDAC1 has beensuggested to interact with GRP75, a chaperone involved in theregulation of calcium levels by modulating the activity of IP3R(Szabadkai and Duchen, 2008). Interestingly, this interaction hasno impact on ER–mitochondrial distance but acts specifically incalcium homeostasis and thus could play a role in steroidogeniccell mitochondrial function.

The integrity of each organelle involved in MAM formation de-pends on energy state, and the regulation of the physical interac-tions between the ER and mitochondria could play an importantrole in various pathologies (Ozcan and Tabas, 2012). Indeed, uponER stress, the reduced distance between the ER and mitochondria

Fig. 2. Formation of mitochondria-associated membranes (MAMs) and regulation of steroid production. (1) The mitochondrial VDAC interacts with GRP75, leading to themodulation of the IP3 receptor and alteration of calcium uptake by the mitochondrion. The activity of enzymes involved in the TCA cycle (IDH2, MDH2, or alpha-ketoglutarate[AKG]) can be regulated by calcium, leading to increased ATP and steroid production. (2) PACS2 is a scaffolding protein involved in the regulation of distance between the ERand mitochondria, which can regulate calcium transport. (3) The isoform 4 of ACSL (ACSL), an enzyme involved in fatty acid synthesis, is also involved in the regulation ofsteroid formation likely by regulating the formation of MAMs. (4) Mitochondrial shape proteins that colocalize in MAMs, such as MFN2, ATAD3, and Drp-1, could participatein the regulation of this process.



is a way for mitochondria to supply the ER with ATP for oxidativeprotein folding (Simmen et al., 2010). It is also likely that the dis-tance between the ER and mitochondria and MAM formation couldbe influenced by the state of the cytoskeleton. Indeed, numerousstudies have shown the role of actin, microtubules, and microfila-ments in the regulation of cholesterol transport into mitochondriaand hormone-induced steroid formation (Hall and Almahbobi,1992; Sewer and Li, 2008; Shen et al., 2012).

In conclusion, the distance between the ER and mitochondriaand the formation of MAMs, in addition to tethering domains be-tween mitochondria and the ER, may play multiple and criticalroles in steroidogenesis at the juncture of calcium signaling and li-pid transfer. A schematic representation of the processes of andproteins in MAMs is shown in Fig. 2.

5. Inter-organelle interactions between LDs, mitochondria, andthe ER

LDs are cytoplasmic organelles present in many cells and crucialin lipid storage and metabolism (Walther and Farese, 2012). In ste-roidogenic cells, LDs store cholesterol in the form of cholesterol es-ters. The sizes of LDs in adrenal and gonadal cells are hormonallycontrolled. Cholesterol storage is essential for making available en-ough cholesterol substrate for steroidogenesis at times of need,such as during a stress response. Any dysfunction in cholesteroltransport and/or metabolism to steroids would lead to lipid accu-mulation with detrimental effects, as is the case in lipid congenitaladrenal hyperplasia (Hu et al., 2010; Miller and Bose, 2011). LDscontain a hydrophobic core of neutral lipids surrounded by amonolayer of surface phospholipids and specific proteins. The ste-rol esters (SEs) and triglycerides (TGs) are the storage forms of cho-lesterol and fatty acids, respectively, and represent the majority ofthe neutral lipids.

Synthesis of these neutral lipids requires specific enzymes, suchas diacylglycerol acyltransferases 1 and 2 for the synthesis of TGsand acyl-CoA cholesterol acyl transferases (ACAT) 1 and 2 for theSEs. On the other hand, as previously mentioned the synthesis offatty acids and SEs, as in the process of steroidogenesis, is per-formed by ACSLs defined as ATP-dependent enzymes. The regula-tion of the synthesis and hydrolysis of SEs must be tightlycontrolled to avoid lipotoxicity. In addition to hormones, inter-organellar interactions seem to play a role in the regulation of lipidmetabolism.

LDs were previously thought to be inert organelles, but ad-vances in imaging technology combined with proteomic ap-proaches revealed that LDs are dynamic, functionally activestructures. Physical contacts sites between LDs and other organ-elles, such as the ER, peroxisomes, endosomes, and mitochondria,have been identified (Digel et al., 2010; Zehmer et al., 2009) anddesignated as lipid droplet-associated membranes (LAMs) (Ozekiet al., 2005). Considering the role of LDs in steroid production,we will focus on the relationship between LDs, the ER, andmitochondria.

5.1. LD–ER interaction

It has been known since the 1970s that the ER is closely associ-ated with LDs. Different models exist for the origin of LDs, thoughin yeast and mammalian cells it is proposed that LDs bud from ERmembranes (Beller et al., 2010; Kalantari et al., 2010; Walther andFarese, 2012; Zehmer et al., 2009). In various steroid-producingcells, apposition of the ER and LDs has frequently been observedand suggested to be involved in the synthesis and catabolism ofstored cholesterol esters for steroid synthesis (Christensen andFawcett, 1966; Ozeki et al., 2005; Rhodin, 1971). Proteomic ap-proaches revealed the presence of different members of the RAB

Fig. 3. Lipid droplet (LD)-associated membranes (LAMs). LDs are composed of a monolayer of phospholipids and surface proteins, such as perilipins (PLIN3, 5) and Rab18. Theenzymes DGAT and ACAT, present in the ER, are involved in the synthesis of triglycerides and sterol esters, respectively, which form the neutral lipids stored in lipid droplets.DGAT2 is also present in LDs.



protein family, especially Rab 18, overexpression of which in-creases the LD–ER interaction in HepG2 cells, indicating a role forthis protein in LD–ER contact site formation (Ozeki et al., 2005).The precise mechanism and structure of the LD–ER associationhave not been defined yet. In yeast, LDs and the ER may be contin-uously in contact, and exchanges between the two organelles seemto be bidirectional (Jacquier et al., 2011); suggesting cholesteroltrafficking at this site. These observations clearly indicate thatthere are dynamic LD–ER contact sites that could be of importancein cholesterol trafficking from LDs to mitochondria in steroido-genic cells. A schematic representation of an LAM is shown inFig. 3.

5.2. LD–mitochondria association

Lipid droplets are important sources of fatty acids (FAs). FAs arean important source of energy in the cell; FA hydrolysis in mito-chondria during b-oxidation provides a source of ATP, an importantfactor in steroid formation (Walther and Farese, 2012). In the liver,this process starts with the transport of free FAs across the OMMby carnitine palmitoyltransferase Ia (CPT-Ia) and the subsequenttransfer across the IMM by carnitine. Once inside the mitochon-drial matrix, the FA-carnitine reacts with coenzyme A to releasethe FA and produce acetyl-CoA. CPT-Ia is believed to be the rate-limiting step in FA oxidation. In rat liver, VDAC was found to beassociated with CPT-Ia and acyl-CoA synthetase isoform 1 at the

OMM (Lee et al., 2011). In the mitochondrial matrix during b-oxi-dation, two-carbon molecules of acetyl-CoA are repeatedly cleavedfrom the FA. Acetyl-CoA can then enter the TCA cycle, which pro-duces two essential cofactors, NADH and FADH2, subsequentlyused in the electron transport chain to produce ATP. This repre-sents one more link between the energy state in mitochondriaand steroidogenesis, as discussed earlier.

The association between mitochondria and LDs was first ob-served with electron microscopy and further confirmed with pro-teomic approaches. However, the mechanisms underlying suchinteractions have not been identified yet. A recent study indicatedthat vimentin could increase the physical ER–LD contact sites andplay a role in this organelle interaction (Walther and Farese, 2012).This latter observation supports the link between cytoskeleton andorganelle plasticity, especially in the case of steroidogenic LDs andmitochondria, previously shown to be attached to intermediate fil-aments in adrenal cells (Almahbobi et al., 1992a, b).

Imaging studies reveal the presence of members of a family ofscaffolding proteins called perilipins in LDs. These proteins couldplay a significant role in the interaction between LDs and otherorganelles, including mitochondria (Fong et al., 2002). Perilipinsare present in steroidogenic cell LDs (Servetnick et al., 1995) andcould be candidates in the regulation of cholesterol transport tomitochondria and steroidogenesis (Kimmel et al., 2010). For in-stance, it has been demonstrated that in tissues with high levelsof oxidative stress, such as cardiomyocytes, perilipin 5, through

Fig. 4. Proposed model of LD–ER–mitochondrial interactions (LERMIT). In this model, interactions between the three organelles are proposed to participate in the regulationand promote the efficiency of steroid production. This is accomplished through the formation of LERMIT, which will allow cholesterol transfer from both the ER and LDs tomitochondria to be metabolized into pregnenolone. The conversion of pregnenolone into the final steroid product can then be controlled through the energetics of themitochondria and contact site regulation with the ER. Cytoskeletal proteins, such as vimentin, can regulate the distance between LDs and mitochondria. Lipids, such ascholesterol esters from LDs are transferred to the mitochondria and hydrolyzed by the ACSL1, which forms a complex for the import of the lipids with VDAC and CPT. Theresultant free FA enters the OMM and is transferred across to the IMM by carnitine. Once inside the mitochondrial matrix, the FA-carnitine reacts with coenzyme A to releasethe FA and produce acetyl-CoA. During b-oxidation, two-carbon molecules of acetyl-CoA are repeatedly cleaved from the fatty acid. Acetyl-CoA can then enter the TCA cycle,which produces NADH and FADH2, which are subsequently used in the ETC to produce ATP, which can then affect the regulation of steroidogenesis.



Fig. 5. Mitochondria–ER–LD interactions in steroidogenic cells. Transmission electron microscopy images of MA-10 Leydig cells showing the various inter-organelleinteractions reviewed. Blue circle, schematic representations of the various interactions seen upon hormonal stimulation of the cells: (A) ER–mitochondria MAM, (B) LD–mitochondria LAM, (C) LD–ER–mitochondria LERMIT. (D) Wild-type mitochondrion with visible cristae. N indicates the nucleus. Scale bars represent 500 nm. Cell culture andelectron microscopy were performed as described (Rone et al., 2012).



its C-terminal region, recruits mitochondria (Wang et al., 2011).These interactions prevent excessive hydrolysis of FA in mitochon-dria under stress conditions. This study clearly showed a physicaland metabolic link between LDs and mitochondria. Perilipin 3 alsoassociates under stress conditions with mitochondria through itsN-terminal domain (Hocsak et al., 2010). This kind of regulationthrough LD-mitochondria tethering could happen in steroidogeniccells, in which there is increased oxidative stress during steroidformation due to the fact that steroidogenic P450s are monoxygen-ases. Overproduction of reactive oxygen species could impair theactivity of steroidogenic enzymes (Georgiou et al., 1987).

5.3. LD–ER–mitochondria (LERMIT) association

Upon hormonal stimulation, modifications of steroidogenic cellshape occur through cytoskeletal rearrangements (Betz and Hall,1987; Hall and Almahbobi, 1992), providing a mechanism throughwhich organelle plasticity can occur. The study of this plasticitybrings to light the possible existence of physical and tightly regu-lated interactions between LDs, the principal site of cholesterolstorage; the ER, the site of regulation of lipid trafficking; and mito-chondria, the crucial site for steroidogenesis. This inter-organellecommunication indicates the presence of a coordination of func-tion and shared multifunctional protein components between thethree organelles. For example, VDAC, which functions in mitochon-dria as a porin in ion transfer regulation and is part of the trans-duceosome and the steroidogenic metabolon, in MAMs as acalcium regulator, and also part of the complex involved in FAcatabolism, seems to be the prototypical candidate to study thebridges between the three organelles. This may also be the casefor diglyceride acyltransferase, an ER protein present in LDs andable to translocate from MAMs to LDs (McFie et al., 2011; Stoneet al., 2009). ACAT isoform I could be a regulator of steroidogenesisin LERMIT. ACAT 1 is present in the 800-kDa bioactive metabolonin Leydig cells (Rone et al., 2012). As previously mentioned, thisprotein, only found in the ER, induces the synthesis of SE in LDs.Thus, we hypothesize the flow of cholesterol passing through spe-cialized sites where this protein provides the link between LDs, theER, and mitochondria. We believe that knowledge of the specificinteractions between these three organelles could provide insightinto the regulation of hormone-stimulated steroidogenesis drivenby the localization of different components. This type of interac-tions is designated LERMIT (Fig. 4). Fig. 5 shows transmission elec-tron microscopy images of MA-10 Leydig cells as examples of thevarious inter-organelle interactions reviewed herein.

6. Conclusion

The odyssey of steroid formation from cholesterol to the finalproduct represents an ensemble of tightly regulated multi-stepprocesses involving various organelles and proteins working in ahormone-dependent, timely, and well-orchestrated manner to de-liver cholesterol to CYP11A1 in the IMM. The regulatory complexesidentified in LDs, the ER, and mitochondria, from those associatedwith calcium regulation to mitochondrial bioenergetic state reflectvery important processes in steroidogenesis. While the study ofprotein–protein interactions and the formation of the transduceo-some and the steroidogenic metabolon are crucial to understandthe mechanisms and efficiency of the steroidogenic process, therearrangement of the cytoskeleton together with the morphologi-cal and functional changes occurring in mitochondria and its LDand ER partners should be considered important parameters inthe regulation of steroid biosynthesis. The interest in these inter-organelle contact sites also raises new questions about how thishormone-induced plasticity takes place, the role of these different

specialized regions during steroid formation, and how miscommu-nication can lead to human pathologies.

Acknowledgments

The work of the authors presented in this review was supportedby a grant from the Canadian Institutes of Health Research(MOP102647) and a Canada Research Chair in BiochemicalPharmacology to V.P. M.R. was supported in part by a postdoctoralfellowship from The Research Institute of MUHC. The ResearchInstitute of MUHC was supported by a Center grant from Le Fondsde la recherche du Québec-santé.

References

Aghazadeh, Y., Rone, M.B., Blonder, J., Ye, X., Veenstra, T.D., Hales, D.B., Culty, M.,Papadopoulos, V., 2012. Hormone-induced 14-3-3gamma adaptor proteinregulates steroidogenic acute regulatory protein activity and steroidbiosynthesis in MA-10 Leydig cells. J. Biol. Chem. 287, 15380–15394.

Alexander, C., Votruba, M., Pesch, U.E., Thiselton, D.L., Mayer, S., Moore, A.,Rodriguez, M., Kellner, U., Leo-Kottler, B., Auburger, G., Bhattacharya, S.S.,Wissinger, B., 2000. OPA1, encoding a dynamin-related GTPase, is mutated inautosomal dominant optic atrophy linked to chromosome 3q28. Nat. Genet. 26,211–215.

Allen, J.A., Shankara, T., Janus, P., Buck, S., Diemer, T., Hales, K.H., Hales, D.B., 2006.Energized, polarized, and actively respiring mitochondria are required for acuteLeydig cell steroidogenesis. Endocrinology 147, 3924–3935.

Almahbobi, G., Williams, L.J., Hall, P.F., 1992a. Attachment of mitochondria tointermediate filaments in adrenal cells: relevance to the regulation of steroidsynthesis. Exp. Cell Res. 200, 361–369.

Almahbobi, G., Williams, L.J., Hall, P.F., 1992b. Attachment of steroidogenic lipiddroplets to intermediate filaments in adrenal cells. J. Cell Sci. 101 (Pt 2), 383–393.

Alpy, F., Tomasetto, C., 2005. Give lipids a START: the StAR-related lipid transfer(START) domain in mammals. J. Cell Sci. 118, 2791–2801.

Anholt, R.R., De Souza, E.B., Oster-Granite, M.L., Snyder, S.H., 1985. Peripheral-typebenzodiazepine receptors: autoradiographic localization in whole-bodysections of neonatal rats. J. Pharmacol. Exp. Ther. 233, 517–526.

Arnoult, D., Grodet, A., Lee, Y.J., Estaquier, J., Blackstone, C., 2005. Release of OPA1during apoptosis participates in the rapid and complete release of cytochrome cand subsequent mitochondrial fragmentation. J. Biol. Chem. 280, 35742–35750.

Artemenko, I.P., Zhao, D., Hales, D.B., Hales, K.H., Jefcoate, C.R., 2001. Mitochondrialprocessing of newly synthesized steroidogenic acute regulatory protein (StAR),but not total StAR, mediates cholesterol transfer to cytochrome P450 side chaincleavage enzyme in adrenal cells. J. Biol. Chem. 276, 46583–46596.

Baines, C.P., Kaiser, R.A., Sheiko, T., Craigen, W.J., Molkentin, J.D., 2007. Voltage-dependent anion channels are dispensable for mitochondrial-dependent celldeath. Nat. Cell Biol. 9, 550–555.

Barbar, E., Lehoux, J.G., Lavigne, P., 2009. Toward the NMR structure of StAR. Mol.Cell Endocrinol. 300, 89–93.

Beller, M., Thiel, K., Thul, P.J., Jackle, H., 2010. Lipid droplets: a dynamic organellemoves into focus. FEBS Lett. 584, 2176–2182.

Betz, G., Hall, P.F., 1987. Steroidogenesis in adrenal tumor cells: influence of cellshape. Endocrinology 120, 2547–2554.

Bononi, A., Missiroli, S., Poletti, F., Suski, J.M., Agnoletto, C., Bonora, M., De, M.E.,Giorgi, C., Marchi, S., Patergnani, S., Rimessi, A., Wieckowski, M.R., Pinton, P.,2012. Mitochondria-associated membranes (MAMs) as hotspot Ca(2+) signalingunits. Adv. Exp. Med. Biol. 740, 411–437.

Bose, H.S., Lingappa, V.R., Miller, W.L., 2002. Rapid regulation of steroidogenesis bymitochondrial protein import. Nature 417, 87–91.

Bose, M., Whittal, R.M., Miller, W.L., Bose, H.S., 2008. Steroidogenic activity of StARrequires contact with mitochondrial VDAC1 and phosphate carrier protein. J.Biol. Chem. 283, 8837–8845.

Boujrad, N., Vidic, B., Papadopoulos, V., 1996. Acute action of choriogonadotropin onLeydig tumor cells: changes in the topography of the mitochondrial peripheral-type benzodiazepine receptor. Endocrinology 137, 5727–5730.

Bourne, H.R., Sanders, D.A., McCormick, F., 1990. The GTPase superfamily: aconserved switch for diverse cell functions. Nature 348, 125–132.

Bourne, H.R., Sanders, D.A., McCormick, F., 1991. The GTPase superfamily:conserved structure and molecular mechanism. Nature 349, 117–127.

Brdiczka, D.G., Zorov, D.B., Sheu, S.S., 2006. Mitochondrial contact sites: their role inenergy metabolism and apoptosis. Biochim. Biophys. Acta 1762, 148–163.

Chen, H., McCaffery, J.M., Chan, D.C., 2007. Mitochondrial fusion protects againstneurodegeneration in the cerebellum. Cell 130, 548–562.

Christensen, A.K., Fawcett, D.W., 1966. The fine structure of testicular interstitialcells in mice. Am. J. Anat. 118, 551–571.

Clark, B.J., 2012. The mammalian START domain protein family in lipid transport inhealth and disease. J. Endocrinol. 212, 257–275.

Cooke, B.A., 1999. Signal transduction involving cyclic AMP-dependent and cyclicAMP-independent mechanisms in the control of steroidogenesis. Mol. CellEndocrinol. 151, 25–35.



Correia, A.R., Pastore, C., Adinolfi, S., Pastore, A., Gomes, C.M., 2008. Dynamics,stability and iron-binding activity of frataxin clinical mutants. FEBS J. 275,3680–3690.

Csordas, G., Varnai, P., Golenar, T., Roy, S., Purkins, G., Schneider, T.G., Balla, T.,Hajnoczky, G., 2010. Imaging interorganelle contacts and local calciumdynamics at the ER–mitochondrial interface. Mol. Cell. 39, 121–132.

Culty, M., Li, H., Boujrad, N., Amri, H., Vidic, B., Bernassau, J.M., Reversat, J.L.,Papadopoulos, V., 1999. In vitro studies on the role of the peripheral-typebenzodiazepine receptor in steroidogenesis. J. Steroid Biochem. Mol. Biol. 69,123–130.

Da, C.S., Xenarios, I., Langridge, J., Vilbois, F., Parone, P.A., Martinou, J.C., 2003.Proteomic analysis of the mouse liver mitochondrial inner membrane. J. Biol.Chem. 278, 41566–41571.

de Brito, O.M., Scorrano, L., 2008. Mitofusin 2 tethers endoplasmic reticulum tomitochondria. Nature 456, 605–610.

de Brito, O.M., Scorrano, L., 2010. An intimate liaison: spatial organization of theendoplasmic reticulum–mitochondria relationship. EMBO J. 29, 2715–2723.

Delavoie, F., Li, H., Hardwick, M., Robert, J.C., Giatzakis, C., Peranzi, G., Yao, Z.X.,Maccario, J., Lacapere, J.J., Papadopoulos, V., 2003. In vivo and in vitroperipheral-type benzodiazepine receptor polymerization: functional signifi-cance in drug ligand and cholesterol binding. Biochemistry 42, 4506–4519.

Delettre, C., Lenaers, G., Griffoin, J.M., Gigarel, N., Lorenzo, C., Belenguer, P.,Pelloquin, L., Grosgeorge, J., Turc-Carel, C., Perret, E., Astarie-Dequeker, C.,Lasquellec, L., Arnaud, B., Ducommun, B., Kaplan, J., Hamel, C.P., 2000. Nucleargene OPA1, encoding a mitochondrial dynamin-related protein, is mutated indominant optic atrophy. Nat. Genet. 26, 207–210.

Delettre, C., Griffoin, J.M., Kaplan, J., Dollfus, H., Lorenz, B., Faivre, L., Lenaers, G.,Belenguer, P., Hamel, C.P., 2001. Mutation spectrum and splicing variants in theOPA1 gene. Hum. Genet. 109, 584–591.

Digel, M., Ehehalt, R., Fullekrug, J., 2010. Lipid droplets lighting up: insights fromlive microscopy. FEBS Lett. 584, 2168–2175.

Duarte, A., Poderoso, C., Cooke, M., Soria, G., Cornejo, M.F., Gottifredi, V., Podesta, E.J.,2012. Mitochondrial fusion is essential for steroid biosynthesis. PLoS One 7,e45829.

Duvezin-Caubet, S., Jagasia, R., Wagener, J., Hofmann, S., Trifunovic, A., Hansson, A.,Chomyn, A., Bauer, M.F., Attardi, G., Larsson, N.G., Neupert, W., Reichert, A.S.,2006. Proteolytic processing of OPA1 links mitochondrial dysfunction toalterations in mitochondrial morphology. J. Biol. Chem. 281, 37972–37979.

Estaquier, J., Vallette, F., Vayssiere, J.L., Mignotte, B., 2012. The mitochondrialpathways of apoptosis. Adv. Exp. Med. Biol. 942, 157–183.

Fang, H.Y., Chang, C.L., Hsu, S.H., Huang, C.Y., Chiang, S.F., Chiou, S.H., Huang, C.H.,Hsiao, Y.T., Lin, T.Y., Chiang, I.P., Hsu, W.H., Sugano, S., Chen, C.Y., Lin, C.Y., Ko,W.J., Chow, K.C., 2010. ATPase family AAA domain-containing 3A is a novel anti-apoptotic factor in lung adenocarcinoma cells. J. Cell Sci. 123, 1171–1180.

Fong, T.H., Yang, C.C., Greenberg, A.S., Wang, S.M., 2002. Immunocytochemicalstudies on lipid droplet–surface proteins in adrenal cells. J. Cell Biochem. 86,432–439.

Frezza, C., Cipolat, S., Martins de, B.O., Micaroni, M., Beznoussenko, G.V., Rudka, T.,Bartoli, D., Polishuck, R.S., Danial, N.N., De, S.B., Scorrano, L., 2006. OPA1 controlsapoptotic cristae remodeling independently from mitochondrial fusion. Cell126, 177–189.

Friedman, J.R., Lackner, L.L., West, M., DiBenedetto, J.R., Nunnari, J., Voeltz, G.K.,2011. ER tubules mark sites of mitochondrial division. Science 334, 358–362.

Fujimoto, M., Hayashi, T., Su, T.P., 2012. The role of cholesterol in the association ofendoplasmic reticulum membranes with mitochondria. Biochem. Biophys. Res.Commun. 417, 635–639.

Garnier, M., Dimchev, A.B., Boujrad, N., Price, J.M., Musto, N.A., Papadopoulos, V.,1994. In vitro reconstitution of a functional peripheral-type benzodiazepinereceptor from mouse Leydig tumor cells. Mol. Pharmacol. 45, 201–211.

Garren, L.D., Gill, G.N., Masui, H., Walton, G.M., 1971. On the mechanism of action ofACTH. Recent Prog. Horm. Res. 27, 433–478.

Georgiou, M., Perkins, L.M., Payne, A.H., 1987. Steroid synthesis-dependent, oxygen-mediated damage of mitochondrial and microsomal cytochrome P-450enzymes in rat Leydig cell cultures. Endocrinology 121, 1390–1399.

Gilquin, B., Taillebourg, E., Cherradi, N., Hubstenberger, A., Gay, O., Merle, N., Assard,N., Fauvarque, M.O., Tomohiro, S., Kuge, O., Baudier, J., 2010. The AAA+ ATPaseATAD3A controls mitochondrial dynamics at the interface of the inner and outermembranes. Mol. Cell Biol. 30, 1984–1996.

Granot, Z., Kobiler, O., Melamed-Book, N., Eimerl, S., Bahat, A., Lu, B., Braun, S.,Maurizi, M.R., Suzuki, C.K., Oppenheim, A.B., Orly, J., 2007a. Turnover ofmitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease:the unexpected effect of proteasome inhibitors. Mol. Endocrinol. 21, 2164–2177.

Granot, Z., Melamed-Book, N., Bahat, A., Orly, J., 2007b. Turnover of StAR protein:roles for the proteasome and mitochondrial proteases. Mol. Cell Endocrinol.265–266, 51–58.

Hales, K.G., Fuller, M.T., 1997. Developmentally regulated mitochondrial fusionmediated by a conserved, novel, predicted GTPase. Cell 90, 121–129.

Halestrap, A.P., 2009. What is the mitochondrial permeability transition pore? J.Mol. Cell Cardiol. 46, 821–831.

Hall, P.F., 1985. Role of cytochromes P-450 in the biosynthesis of steroid hormones.Vitam. Horm. 42, 315–368.

Hall, P.F., Almahbobi, G., 1992. The role of the cytoskeleton in the regulation ofsteroidogenesis. J. Steroid Biochem. Mol. Biol. 43, 769–777.

Hauet, T., Yao, Z.X., Bose, H.S., Wall, C.T., Han, Z., Li, W., Hales, D.B., Miller, W.L.,Culty, M., Papadopoulos, V., 2005. Peripheral-type benzodiazepine receptor-mediated action of steroidogenic acute regulatory protein on cholesterol entryinto leydig cell mitochondria. Mol. Endocrinol. 19, 540–554.

Hayashi, T., Su, T.P., 2010. Cholesterol at the endoplasmic reticulum: roles of thesigma-1 receptor chaperone and implications thereof in human diseases.Subcell. Biochem. 51, 381–398.

He, J., Mao, C.C., Reyes, A., Sembongi, H., Di, R.M., Granycome, C., Clippingdale, A.B.,Fearnley, I.M., Harbour, M., Robinson, A.J., Reichelt, S., Spelbrink, J.N., Walker,J.E., Holt, I.J., 2007. The AAA+ protein ATAD3 has displacement loop bindingproperties and is involved in mitochondrial nucleoid organization. J. Cell Biol.176, 141–146.

Hirsch, J.D., Beyer, C.F., Malkowitz, L., Beer, B., Blume, A.J., 1989. Mitochondrialbenzodiazepine receptors mediate inhibition of mitochondrial respiratorycontrol. Mol. Pharmacol. 35, 157–163.

Hocsak, E., Racz, B., Szabo, A., Mester, L., Rapolti, E., Pozsgai, E., Javor, S., Bellyei, S.,Gallyas Jr., F., Sumegi, B., Szigeti, A., 2010. TIP47 protects mitochondrialmembrane integrity and inhibits oxidative-stress-induced cell death. FEBS Lett.584, 2953–2960.

Hoffmann, M., Bellance, N., Rossignol, R., Koopman, W.J., Willems, P.H., Mayatepek,E., Bossinger, O., Distelmaier, F., 2009. C. elegans ATAD-3 is essential formitochondrial activity and development. PLoS One 4, e7644.

Hu, J., Zhang, Z., Shen, W.J., Azhar, S., 2010. Cellular cholesterol delivery,intracellular processing and utilization for biosynthesis of steroid hormones.Nutr. Metab. (Lond.) 7, 47.

Jacquier, N., Choudhary, V., Mari, M., Toulmay, A., Reggiori, F., Schneiter, R., 2011.Lipid droplets are functionally connected to the endoplasmic reticulum inSaccharomyces cerevisiae. J. Cell Sci. 124, 2424–2437.

Jefcoate, C., 2002. High-flux mitochondrial cholesterol trafficking, a specializedfunction of the adrenal cortex. J. Clin. Invest. 110, 881–890.

Kalantari, F., Bergeron, J.J., Nilsson, T., 2010. Biogenesis of lipid droplets – how cellsget fatter. Mol. Membr. Biol. 27, 462–468.

Kimmel, A.R., Brasaemle, D.L., McAndrews-Hill, M., Sztalryd, C., Londos, C., 2010.Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular lipid storage droplet proteins. J. Lipid Res. 51, 468–471.

Kimura, T., 1986. Transduction of ACTH signal from plasma membrane tomitochondria in adrenocortical steroidogenesis. Effects of peptide,phospholipid, and calcium. J. Steroid Biochem. 25, 711–716.

Korkhov, V.M., Sachse, C., Short, J.M., Tate, C.G., 2010. Three-dimensional structureof TspO by electron cryomicroscopy of helical crystals. Structure 18, 677–687.

Lacapere, J.J., Papadopoulos, V., 2003. Peripheral-type benzodiazepine receptor:structure and function of a cholesterol-binding protein in steroid and bile acidbiosynthesis. Steroids 68, 569–585.

Lee, Y.J., Jeong, S.Y., Karbowski, M., Smith, C.L., Youle, R.J., 2004. Roles of themammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 inapoptosis. Mol. Biol. Cell. 15, 5001–5011.

Lee, K., Kerner, J., Hoppel, C.L., 2011. Mitochondrial carnitine palmitoyltransferase1a (CPT1a) is part of an outer membrane fatty acid transfer complex. J. Biol.Chem. 286, 25655–25662.

Lewin, T.M., Van Horn, C.G., Krisans, S.K., Coleman, R.A., 2002. Rat liver acyl-CoAsynthetase 4 is a peripheral-membrane protein located in two distinctsubcellular organelles, peroxisomes, and mitochondrial-associated membrane.Arch. Biochem. Biophys. 404, 263–270.

Li, H., Papadopoulos, V., 1998. Peripheral-type benzodiazepine receptor function incholesterol transport. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern. Endocrinology 139,4991–4997.

Li, S., Rousseau, D., 2012. ATAD3, a vital membrane bound mitochondrial ATPaseinvolved in tumor progression. J. Bioenergy Biomembr. 44, 189–197.

Li, H., Degenhardt, B., Tobin, D., Yao, Z.X., Tasken, K., Papadopoulos, V., 2001a.Identification, localization, and function in steroidogenesis of PAP7: aperipheral-type benzodiazepine receptor- and PKA (RIalpha)-associatedprotein. Mol. Endocrinol. 15, 2211–2228.

Li, H., Yao, Z., Degenhardt, B., Teper, G., Papadopoulos, V., 2001b. Cholesterol bindingat the cholesterol recognition/ interaction amino acid consensus (CRAC) of theperipheral-type benzodiazepine receptor and inhibition of steroidogenesis byan HIV TAT-CRAC peptide. Proc. Natl. Acad. Sci. USA 98, 1267–1272.

Liu, J., Rone, M.B., Papadopoulos, V., 2006. Protein–protein interactions mediatemitochondrial cholesterol transport and steroid biosynthesis. J. Biol. Chem. 281,38879–38893.

Maloberti, P., Castilla, R., Castillo, F., Cornejo, M.F., Mendez, C.F., Paz, C., Podesta, E.J.,2005. Silencing the expression of mitochondrial acyl-CoA thioesterase I andacyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis. FEBS J. 272,1804–1814.

Maloberti, P., Cornejo, M.F., Castillo, A.F., Castilla, R., Duarte, A., Toledo, M.F., Meuli,F., Mele, P., Paz, C., Podesta, E.J., 2007. Enzymes involved in arachidonic acidrelease in adrenal and Leydig cells. Mol. Cell Endocrinol. 265–266, 113–120.

Manna, P.R., Dyson, M.T., Stocco, D.M., 2009. Regulation of the steroidogenic acuteregulatory protein gene expression: present and future perspectives. Mol. Hum.Reprod. 15, 321–333.

Mannella, C.A., Buttle, K., Rath, B.K., Marko, M., 1998. Electron microscopictomography of rat-liver mitochondria and their interaction with theendoplasmic reticulum. Biofactors 8, 225–228.

McEnery, M.W., Snowman, A.M., Trifiletti, R.R., Snyder, S.H., 1992. Isolation of themitochondrial benzodiazepine receptor: association with the voltage-



dependent anion channel and the adenine nucleotide carrier. Proc. Natl. Acad.Sci. USA 89, 3170–3174.

McFie, P.J., Banman, S.L., Kary, S., Stone, S.J., 2011. Murine diacylglycerolacyltransferase-2 (DGAT2) can catalyze triacylglycerol synthesis and promotelipid droplet formation independent of its localization to the endoplasmicreticulum. J. Biol. Chem. 286, 28235–28246.

Midzak, A.S., Chen, H., Aon, M.A., Papadopoulos, V., Zirkin, B.R., 2011. ATP synthesis,mitochondrial function, and steroid biosynthesis in rodent primary and tumorLeydig cells. Biol. Reprod. 84, 976–985.

Miller, W.L., 2007. Steroidogenic acute regulatory protein (StAR), a novelmitochondrial cholesterol transporter. Biochim. Biophys. Acta 1771, 663–676.

Miller, W.L., Bose, H.S., 2011. Early steps in steroidogenesis: intracellular cholesteroltrafficking. J. Lipid Res. 52, 2111–2135.

Murail, S., Robert, J.C., Coic, Y.M., Neumann, J.M., Ostuni, M.A., Yao, Z.X.,Papadopoulos, V., Jamin, N., Lacapere, J.J., 2008. Secondary and tertiarystructures of the transmembrane domains of the translocator protein TSPOdetermined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding.Biochim. Biophys. Acta 1778, 1375–1381.

Ozcan, L., Tabas, I., 2012. Role of endoplasmic reticulum stress in metabolic diseaseand other disorders. Annu. Rev. Med. 63, 317–328.

Ozeki, S., Cheng, J., Tauchi-Sato, K., Hatano, N., Taniguchi, H., Fujimoto, T., 2005.Rab18 localizes to lipid droplets and induces their close apposition to theendoplasmic reticulum-derived membrane. J. Cell Sci. 118, 2601–2611.

Palmer, C.S., Osellame, L.D., Stojanovski, D., Ryan, M.T., 2011. The regulation ofmitochondrial morphology: intricate mechanisms and dynamic machinery. CellSignal. 23, 1534–1545.

Papadopoulos, V., 1993. Peripheral-type benzodiazepine/diazepam bindinginhibitor receptor: biological role in steroidogenic cell function. Endocr. Rev.14, 222–240.

Papadopoulos, V., Miller, W.L., 2012. Role of mitochondria in steroidogenesis. BestPract. Res. Clin. Endocrinol. Metab. 26, 771–790.

Papadopoulos, V., Boujrad, N., Ikonomovic, M.D., Ferrara, P., Vidic, B., 1994.Topography of the Leydig cell mitochondrial peripheral-type benzodiazepinereceptor. Mol. Cell Endocrinol. 104, R5–R9.

Petrescu, A.D., Gallegos, A.M., Okamura, Y., Strauss III, J.F., Schroeder, F., 2001.Steroidogenic acute regulatory protein binds cholesterol and modulatesmitochondrial membrane sterol domain dynamics. J. Biol. Chem. 276, 36970–36982.

Pon, L.A., Orme-Johnson, N.R., 1986. Acute stimulation of steroidogenesis in corpusluteum and adrenal cortex by peptide hormones. Rapid induction of a similarprotein in both tissues. J. Biol. Chem. 261, 6594–6599.

Pon, L.A., Hartigan, J.A., Orme-Johnson, N.R., 1986. Acute ACTH regulation of adrenalcorticosteroid biosynthesis. Rapid accumulation of a phosphoprotein. J. Biol.Chem. 261, 13309–13316.

Prins, D., Michalak, M., 2011. Organellar calcium buffers. Cold Spring Harb. Perspect.Biol. 3.

Rhodin, J.A., 1971. The ultrastructure of the adrenal cortex of the rat under normaland experimental conditions. J. Ultrastruct. Res. 34, 23–71.

Ricchelli, F., Sileikyte, J., Bernardi, P., 2011. Shedding light on the mitochondrialpermeability transition. Biochim. Biophys. Acta 1807, 482–490.

Rizzuto, R., Brini, M., Murgia, M., Pozzan, T., 1993. Microdomains with high Ca2+

close to IP3-sensitive channels that are sensed by neighboring mitochondria.Science 262, 744–747.

Rizzuto, R., Pinton, P., Carrington, W., Fay, F.S., Fogarty, K.E., Lifshitz, L.M., Tuft, R.A.,Pozzan, T., 1998. Close contacts with the endoplasmic reticulum asdeterminants of mitochondrial Ca2+ responses. Science 280, 1763–1766.

Rojo, M., Legros, F., Chateau, D., Lombes, A., 2002. Membrane topology andmitochondrial targeting of mitofusins, ubiquitous mammalian homologs of thetransmembrane GTPase Fzo. J. Cell Sci. 115, 1663–1674.

Rone, M.B., Fan, J., Papadopoulos, V., 2009a. Cholesterol transport in steroidbiosynthesis: role of protein–protein interactions and implications in diseasestates. Biochim. Biophys. Acta 1791, 646–658.

Rone, M.B., Liu, J., Blonder, J., Ye, X., Veenstra, T.D., Young, J.C., Papadopoulos, V.,2009b. Targeting and insertion of the cholesterol-binding translocator proteininto the outer mitochondrial membrane. Biochemistry 48, 6909–6920.

Rone, M.B., Midzak, A.S., Issop, L., Rammouz, G., Jagannathan, S., Fan, J., Ye, X.,Blonder, J., Veenstra, T., Papadopoulos, V., 2012. Identification of a dynamicmitochondrial protein complex driving cholesterol import, trafficking, andmetabolism to steroid hormones. Mol. Endocrinol.

Rupprecht, R., Papadopoulos, V., Rammes, G., Baghai, T.C., Fan, J., Akula, N., Groyer,G., Adams, D., Schumacher, M., 2010. Translocator protein (18 kDa) (TSPO) as atherapeutic target for neurological and psychiatric disorders. Nat. Rev. DrugDiscov. 9, 971–988.

Sauvanet, C., Arnaune-Pelloquin, L., David, C., Belenguer, P., Rojo, M., 2010.Mitochondrial morphology and dynamics: actors, mechanisms and functions.Med. Sci. (Paris) 26, 823–829.

Servetnick, D.A., Brasaemle, D.L., Gruia-Gray, J., Kimmel, A.R., Wolff, J., Londos, C.,1995. Perilipins are associated with cholesteryl ester droplets in steroidogenicadrenal cortical and Leydig cells. J. Biol. Chem. 270, 16970–16973.

Sewer, M.B., Li, D., 2008. Regulation of steroid hormone biosynthesis by thecytoskeleton. Lipids 43, 1109–1115.

Shen, W.J., Zaidi, S.K., Patel, S., Cortez, Y., Ueno, M., Azhar, R., Azhar, S., Kraemer, F.B.,2012. Ablation of vimentin results in defective steroidogenesis. Endocrinology153, 3249–3257.

Shoshan-Barmatz, V., Gincel, D., 2003. The voltage-dependent anion channel:characterization, modulation, and role in mitochondrial function in cell life anddeath. Cell Biochem. Biophys. 39, 279–292.

Shoshan-Barmatz, V., De Pinto, V., Zweckstetter, M., Raviv, Z., Keinan, N., Arbel, N.,2010. VDAC, a multi-functional mitochondrial protein regulating cell life anddeath. Mol. Asp. Med. 31, 227–285.

Sileikyte, J., Petronilli, V., Zulian, A., Dabbeni-Sala, F., Tognon, G., Nikolov, P.,Bernardi, P., Ricchelli, F., 2011. Regulation of the inner membrane mitochondrialpermeability transition by the outer membrane translocator protein (peripheralbenzodiazepine receptor). J. Biol. Chem. 286, 1046–1053.

Simmen, T., Aslan, J.E., Blagoveshchenskaya, A.D., Thomas, L., Wan, L., Xiang, Y.,Feliciangeli, S.F., Hung, C.H., Crump, C.M., Thomas, G., 2005. PACS-2 controlsendoplasmic reticulum–mitochondria communication and Bid-mediatedapoptosis. EMBO J. 24, 717–729.

Simmen, T., Lynes, E.M., Gesson, K., Thomas, G., 2010. Oxidative protein folding inthe endoplasmic reticulum: tight links to the mitochondria-associatedmembrane (MAM). Biochim. Biophys. Acta 1798, 1465–1473.

Simpson, E.R., Waterman, M.R., 1988. Regulation of the synthesis of steroidogenicenzymes in adrenal cortical cells by ACTH. Annu. Rev. Physiol. 50, 427–440.

Smirnova, E., Shurland, D.L., Ryazantsev, S.N., van der Bliek, A.M., 1998. A humandynamin-related protein controls the distribution of mitochondria. J. Cell Biol.143, 351–358.

Spat, A., Hunyady, L., 2004. Control of aldosterone secretion: a model forconvergence in cellular signaling pathways. Physiol. Rev. 84, 489–539.

Srere, P.A., 1987. Complexes of sequential metabolic enzymes. Annu. Rev. Biochem.56, 89–124.

Stevens, V.L., Tribble, D.L., Lambeth, J.D., 1985. Regulation of mitochondrialcompartment volumes in rat adrenal cortex by ether stress. Arch. Biochem.Biophys. 242, 324–327.

Stocco, D.M., Clark, B.J., 1996. Regulation of the acute production of steroids insteroidogenic cells. Endocr. Rev. 17, 221–244.

Stone, S.J., Levin, M.C., Zhou, P., Han, J., Walther, T.C., Farese Jr., R.V., 2009. Theendoplasmic reticulum enzyme DGAT2 is found in mitochondria-associatedmembranes and has a mitochondrial targeting signal that promotes itsassociation with mitochondria. J. Biol. Chem. 284, 5352–5361.

Strauss III, J.F., Kishida, T., Christenson, L.K., Fujimoto, T., Hiroi, H., 2003. STARTdomain proteins and the intracellular trafficking of cholesterol in steroidogeniccells. Mol. Cell Endocrinol. 202, 59–65.

Szabadkai, G., Duchen, M.R., 2008. Mitochondria: the hub of cellular Ca2+ signaling.Physiology (Bethesda.) 23, 84–94.

Teboul, D., Beaufils, S., Taveau, J.C., Iatmanen-Harbi, S., Renault, A., Venien-Bryan, C.,Vie, V., Lacapere, J.J., 2012. Mouse TSPO in a lipid environment interacting witha functionalized monolayer. Biochim. Biophys. Acta 1818, 2791–2800.

Thomson, M., 2003. Does cholesterol use the mitochondrial contact site as a conduitto the steroidogenic pathway? Bioessays 25, 252–258.

Tuckey, R.C., Headlam, M.J., Bose, H.S., Miller, W.L., 2002. Transfer of cholesterolbetween phospholipid vesicles mediated by the steroidogenic acute regulatoryprotein (StAR). J. Biol. Chem. 277, 47123–47128.

Walther, T.C., Farese Jr., R.V., 2012. Lipid droplets and cellular lipid metabolism.Annu. Rev. Biochem. 81, 687–714.

Wang, H., Sreenevasan, U., Hu, H., Saladino, A., Polster, B.M., Lund, L.M., Gong, D.W.,Stanley, W.C., Sztalryd, C., 2011. Perilipin 5, a lipid droplet-associated protein,provides physical and metabolic linkage to mitochondria. J. Lipid Res. 52, 2159–2168.

Wasilewski, M., Semenzato, M., Rafelski, S.M., Robbins, J., Bakardjiev, A.I., Scorrano,L., 2012. Optic atrophy 1-dependent mitochondrial remodeling controlssteroidogenesis in trophoblasts. Curr. Biol. 22, 1228–1234.

Woods, M.J., Zisterer, D.M., Williams, D.C., 1996. Two cellular and subcellularlocations for the peripheral-type benzodiazepine receptor in rat liver. Biochem.Pharmacol. 51, 1283–1292.

Yaffe, M.P., 1999. The machinery of mitochondrial inheritance and behavior. Science283, 1493–1497.

Zehmer, J.K., Huang, Y., Peng, G., Pu, J., Anderson, R.G., Liu, P., 2009. A role for lipiddroplets in inter-membrane lipid traffic. Proteomics 9, 914–921.

Zisterer, D.M., Gorman, A.M., Williams, D.C., Murphy, M.P., 1992. The effects of theperipheral-type benzodiazepine acceptor ligands, Ro 5-4864 and PK 11195, onmitochondrial respiration. Methods Find. Exp. Clin. Pharmacol. 14, 85–90.

Zorzano, A., Liesa, M., Sebastian, D., Segales, J., Palacin, M., 2010. Mitochondrialfusion proteins: dual regulators of morphology and metabolism. Semin. CellDev. Biol. 21, 566–574.

Zulian, A., Sileikyte, J., Petronilli, V., Bova, S., Dabbeni-Sala, F., Cargnelli, G., Rennison,D., Brimble, M.A., Hopkins, B., Bernardi, P., Ricchelli, F., 2011. The translocatorprotein (peripheral benzodiazepine receptor) mediates rat-selective activationof the mitochondrial permeability transition by norbormide. Biochim. Biophys.Acta 1807, 1600–1605.


Organelle Plasticity and Interactions in Cholesterol Transport and Steroid Biosynthesis

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