Tracking the Role of a StAR in the Sky of the New Millennium

Douglas M. Stocco

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Address all correspondence and requests for reprints to: Dr. Douglas Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, 3601 4th Street, 5B108 HSC Building, Lubbock, Texas 79430. E-mail: doug.stocco{at}ttmc.ttuhsc.edu


    ABSTRACT
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 ABSTRACT
 INTRODUCTION
 THE CASE FOR StAR...
 TRANCRIPTIONAL REGULATION OF THE...
 HOW DOES StAR MEDIATE...
 REFERENCES
 
The steroidogenic acute regulatory protein is indispensable for the biosynthesis of steroid hormones. Steroidogenic acute regulatory protein mediates the rate-limiting step in steroidogenesis, the transfer of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane where it is cleaved to pregnenolone. Its essential role in steroidogenesis was shown when it was discovered that mutations in the steroidogenic acute regulatory protein gene in humans cause the lipoid form of congenital adrenal hyperplasia, a potentially lethal disease resulting from an inability to synthesize steroids. Also, the steroidogenic acute regulatory protein null mouse has a phenotype that is essentially the same as that observed with human mutations. Studies on the regulation of the expression of the steroidogenic acute regulatory protein gene has enjoyed considerable progress, yet the complexity of this regulation indicates that much work remains. The mechanism whereby steroidogenic acute regulatory protein mediates the transfer of cholesterol to the inner mitochondrial membrane remains a mystery, but the recent solving of the structure of the cholesterol transferring domain of a steroidogenic acute regulatory protein homolog coupled with structure-function studies of steroidogenic acute regulatory protein in natural and synthetic membranes has allowed for at least two models to be proposed. This review will briefly attempt to summarize what is currently known about the regulation of the steroidogenic acute regulatory protein gene and its mechanism of action, fully understanding that in both areas considerable gaps in our knowledge remain.


    INTRODUCTION
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 ABSTRACT
 INTRODUCTION
 THE CASE FOR StAR...
 TRANCRIPTIONAL REGULATION OF THE...
 HOW DOES StAR MEDIATE...
 REFERENCES
 
STEROIDS ARE SYNTHESIZED in specialized steroidogenic cells in the adrenal, ovary, testis, placenta, and brain and are essential for maintaining normal body homeostasis and reproductive capacity. While the steroid hormones have diverse physiological actions, the biosynthesis of all steroids begins with the conversion of cholesterol to the first steroid synthesized, pregnenolone. This reaction is catalyzed by the cytochrome P450 side-chain cleavage enzyme (P450scc), which is located on the matrix side of the inner mitochondrial membrane (1). For many years the activity of the P450scc enzyme was considered as the rate-limiting step in steroidogenesis, and in the presence of adequate supplies of substrate cholesterol, it can indeed be rate limiting. However, it later became clear that the activity of this enzyme was not the true regulated step, and it was determined that the delivery of the substrate cholesterol to the inner mitochondrial membrane and to the P450scc was the real rate-limiting step in steroid hormone synthesis (2, 3, 4, 5). A fundamental observation in this process was that this regulation had an absolute requirement for the synthesis of new proteins (6, 7). Further studies demonstrated that inhibition of protein synthesis had no effect on the delivery of cellular cholesterol to the outer mitochondrial membrane, but that the delivery of this substrate from the outer membrane to the inner mitochondrial membrane was completely inhibited (5, 8). With these observations, the action of the putative regulator protein had been precisely characterized. The role of this protein was to mediate the transfer of cholesterol from the outer mitochondrial membrane, through the aqueous intermembrane space, to the inner membrane. It was clear that this process could not occur without a mediator since the hydrophobic nature of cholesterol would not allow for its delivery to the P450scc in amounts required to support the observed level of steroidogenesis after hormonal stimulation. Several candidate proteins have been proposed for this role, and a listing of these proteins and the data supporting their candidacies have been discussed in a number of previous reviews (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). This review will focus mainly on one of the candidate proteins, the steroidogenic acute regulatory (StAR) protein, which was cloned in our laboratory in 1994. Now, some 7 yr later, it is an ideal time to reflect very briefly on the reasons why I feel that StAR is the regulatory protein and, more importantly, on areas that represent perhaps the greatest challenge in the study of this protein into the new millennium. My intent will be to first briefly provide enough background to indicate why StAR is the best candidate for the putative regulator of steroidogenesis. I will then focus on what I perceive to be two of the most interesting areas in future StAR research, namely, the means by which this interesting gene is regulated and the mechanism of its action. Other aspects of research on StAR including its possible role in neurosteroid biosynthesis, its relationship to proteins showing high degrees of homology to segments of the StAR protein, and the role of StAR as recently cloned from lower vertebrates are of great interest and will continue to progress. However, the manner in which the StAR gene is so specifically expressed in both a temporal and spatial manner and the mechanism by which it mediates the transfer of cholesterol to the inner mitochondrial membrane are, in my opinion, the most interesting and important areas to explore as we embark on the new millennium.


    THE CASE FOR StAR AS THE ACUTE REGULATOR
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StAR was initially described by Orme-Johnson and colleagues as a rapidly induced 30-kDa phosphoprotein in ACTH-treated rat and mouse adrenocortical cells, and in LH-treated rat corpus luteum cells and mouse Leydig cells, and was later characterized in hormone-stimulated MA-10 mouse Leydig tumor cells by Stocco and colleagues (reviewed in Ref. 9). In both laboratories, these proteins were found to be localized to the mitochondria and consisted of several forms of a newly synthesized 30- kDa protein. In addition to the 30-kDa proteins, 37-kDa precursor forms of these proteins containing N-terminal mitochondrial signal sequences were also detected (21, 22). The cDNA for the 37-kDa mitochondrial protein was cloned from MA-10 cells in our laboratory in 1994 and when compared with other sequences in the data base, both the nucleic acid and protein sequences indicated that it represented a novel protein (23). Transient transfection experiments demonstrated that expression of the cDNA-derived protein in MA-10 cells in the absence of hormone stimulation, or in COS-1 cells that were rendered steroidogenic by transfection, resulted in a several fold increase in the conversion of cholesterol to pregnenolone (9, 23, 24, 25). These results indicated a direct cause-and-effect role for the 37- and 30-kDa proteins in hormone-regulated steroid production.

However, the observation that provided the most compelling evidence for the essential requirement for StAR in steroidogenesis came with the finding that mutations in the StAR gene were the cause of the potentially lethal condition known as congenital lipoid adrenal hyperplasia (lipoid CAH) (25). Patients with this disease are unable to synthesize adequate amounts of steroids, are characterized by excessive levels of cholesterol and cholesterol esters in adrenal and testicular steroidogenic cells, and will not survive unless appropriate steroid replacement therapy is quickly administered. The events involved in the pathogenesis of lipoid CAH were further elucidated with the description of the two-hit model. This model hypothesized an initial loss in steroidogenesis due to mutations in StAR followed by a subsequent and complete loss of steroidogenesis due to the damage caused by the accumulation of cholesterol esters in the cell (26). The case for StAR playing an essential role in regulated steroid synthesis was greatly bolstered when StAR-specific knockout mice were generated and found to have a phenotype that was essentially identical to the human condition (27). Thus, biochemical and genetic studies converged to demonstrate an indispensable role for StAR in steroid hormone biosynthesis in adrenal and gonadal tissues.


    TRANCRIPTIONAL REGULATION OF THE StAR GENE
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Positive Regulation of the StAR Gene
After the cloning of StAR, experiments designed to learn more about both the StAR protein and its gene were initiated. Predictably, many studies focused on the factors involved in the regulation of the StAR gene. In general it has been shown that the regulation of the StAR gene is most complex and is subject to both positive and negative regulation (28). While the regulation of the StAR gene has been the subject of several recent reviews (28, 29, 30, 31), given its importance it deserves some consideration here.

StAR expression can be regulated both positively and negatively by agents that presumably act on its promoter. Studies to determine which regulatory elements were present within the StAR promoter and the manner in which transcription factors and other proteins interact with these elements were conducted. In early studies it was demonstrated that hormone-stimulated steroid synthesis was accompanied by a rapid increase in StAR mRNA levels (32). Since trophic hormone stimulation usually results in a rapid increase in intracellular cAMP, the role of cAMP in the regulation of StAR was investigated and found to have a positive and rapid effect (33, 34, 35, 36, 37, 38). Most studies demonstrated that the cAMP-responsive site was found within the first 254 nucleotides relative to the transcription start site (33, 35, 36, 37, 38), and this region has been the focus of studies to identify promoter elements and their cognate-binding proteins that mediate the cAMP response. However, the StAR promoter lacks a consensus full cAMP response element, raising the possibility that the cAMP response element binding protein might not act directly on sequences found in the StAR promoter.

Steroidogenic factor 1 (SF-1), was the first transcription factor to be studied as a potential regulator of the StAR gene. Several SF-1 consensus binding sites have been identified in the StAR promoter (35, 37, 38). Two of these sites, located at positions -97 and -42, are highly conserved in several species whose promoter regions have been sequenced, whereas the -132 site may only be present in mouse and rat. Utilizing transient transfection protocols, SF-1 has been demonstrated to transactivate the StAR promoter in several cell types (35, 36, 37, 38). It is also possible that SF-1 may play some role in the developmental regulation of the StAR gene since StAR mRNA is not detected in the urogenital ridge of the SF-1 knockout mouse during embryonic development (32).

While SF-1 plays a role in the regulation of the StAR gene, it became apparent that other elements were also involved in StAR’s tissue and temporal specific expression. The CCAAT/enhancer binding proteins (C/EBPs) are a family of basic region/leucine zipper transcription factors implicated as regulators of differentiation and function in multiple cell types (39). Previous studies have demonstrated that two family members, C/EBP{alpha} and C/EBPß, are expressed in steroidogenic cells, including Leydig cells and ovarian granulosa cells (40, 41). Two putative C/EBP binding sites in the StAR promoter have been identified (42, 43, 44). These studies have determined that the StAR promoter is transactivated by C/EBPß during transient transfection assays and that SF-1 transactivation of the StAR promoter is dependent on the presence of functional C/EBP binding sites, suggesting that SF-1 and C/EBPß form a complex on this promoter (42, 43). In other studies, functional assays of the StAR promoter were performed using FSH-induced primary granulosa cell cultures from prepubertal rat ovaries (43). This led to the identification of two trans-acting proteins, C/EBPß and GATA-4, which were required for the transcriptional activation of the StAR promoter. This study revealed a non-consensus binding sequence for C/EBPß (-81/-72), located 10 nucleotides upstream from a consensus motif for GATA-4 binding (-61/-66). Site-directed mutagenesis reinforced the observation that these two binding elements are required for transactivation of the StAR promoter in these cells. Western analyses demonstrated that while GATA-4 was constitutively expressed in granulosa cells, the C/EBPß isoforms were induced by FSH. This suggests that GATA-4 may play a permissive role and C/EBPß may play a regulated role in the acute rate of StAR transcription.

Findings from other laboratories have demonstrated that the sterol regulatory element binding protein-1a (SREBP-1a) may also be involved in the regulation of the StAR gene (45, 46). These studies have demonstrated the presence of potential sterol regulatory element binding sites in both the human and the rat StAR promoters and have demonstrated that SREBP-1a is capable of transactivating the StAR promoter. They further indicated that other transcription factors such as SF-1, nuclear factor-Y, yin yang 1, and Sp1 may also be involved in the action of SREBP1a on the StAR promoter. Since the SREBP family of transcription factors are involved in the up-regulation of proteins involved in steroid biosynthesis and the uptake of cholesterol, it is intriguing to speculate that StAR may be coordinately regulated by SREBP-1a along with these other steroidogenesis-supporting proteins.

In summary, it is has been determined that several currently identified transcription regulatory elements and their binding sequences are involved in the up-regulation of the StAR gene. However, it is also possible that additional regulatory elements will continue to be uncovered as it is clear that the regulation of the StAR gene is complex. In this regard, studies in our laboratory have indicated that CREB can also bind to and transactivate the StAR promoter and can do so in a rapid manner (in review). The major challenge in this area will be to determine how the different promoter sequences and their cognate binding factors interact with each other to bring about the rapid expression of the StAR gene.

Negative Regulation of the StAR gene
Interestingly, the StAR promoter may also harbor elements involved in repression of its transcription. DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X, gene 1) is an unusual member of the nuclear hormone receptor family, retaining homology with only the ligand-binding domain of the nuclear hormone receptors (47). The DNA-binding domain lacks zinc finger motifs and consists of a repeat of a 65- to 67-amino acid sequence in its N terminus (47). DAX-1 overexpression was demonstrated to inhibit the synthesis of steroids in Y-1 mouse adrenal tumor cells (48). Indeed, the DAX-1 protein has been demonstrated to contain a powerful transcriptional silencing domain in its C-terminal region (49). As a possible mechanism of action for this observation, DAX-1 has been shown to interact directly with a hairpin structure in the StAR promoter to inhibit its expression in one study (48) and to bind directly to SF-1, resulting in an inhibition of SF-1-mediated transactivation in another study (50). Combined, these data implicate DAX-1 as a key factor in the regulation of StAR gene expression.


    HOW DOES StAR MEDIATE CHOLESTEROL TRANSFER?
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Early Models and Ideas
Since its discovery, arguably the most intriguing question that has been asked concerning StAR is, "How does it work?" There has been ample proof presented that StAR is required to mediate cholesterol transfer to the inner mitochondrial membrane, and it is understandable that the question of how it does this would be of great interest and importance. Taking into consideration the observation that StAR is rapidly imported and processed by the mitochondria, we originally proposed a model hypothesizing that during the course of import, contact sites were formed between the outer and inner mitochondrial membranes and, in some unknown manner, cholesterol could be transferred to the inner membrane during this process (9, 23). This model proposed that the formation of contact sites collapsed the intermitochondrial membrane aqueous space that served as a barrier to keep the hydrophobic cholesterol from crossing to the inner membrane and in this way allowed for its transfer via this newly formed lipid bridge. This seemed a reasonable model in that other investigators had shown the existence of contact sites at loci where proteins were imported into the mitochondria (51, 52). This model was severely questioned when it was found that cells transfected to express StAR proteins lacking the N-terminal 62 amino acids (and thus all of the signal sequence) supported steroid synthesis to the same extent as wild-type StAR, yet never entered the mitochondria (53). This indicated that StAR could act on the outside of the mitochondria to mediate cholesterol transfer to the inner membrane. Similarly, recombinant StAR lacking the N-terminal 62 amino acids could fully support steroid synthesis in isolated mitochondria, once again, without entering the organelle (54). Experiments employing constructs truncated in their C- terminal end indicated that the cholesterol transferring capability of the StAR protein resided in the C-terminal portion of the molecule (25, 26, 53, 55). In hindsight this finding seems most reasonable since we now know that mutations causing lipoid CAH are all found in the C-terminal region of the StAR protein (15), thus indicating a critical role for this segment of the protein in cholesterol transfer. The role of the C-terminal region of StAR became even more extensively investigated when it was found that a protein known as MLN64 contained an amino acid sequence that was highly homologous to the C terminus of StAR (56) and could stimulate steroid synthesis when transfected into COS-1 cells (57). Interestingly, while MNL64 is found in many tissues, it is also found in the human placenta where it can be proteolytically cleaved to produce a fragment that has significant steroidogenic activity (58). Conversely, StAR is not found in the human placenta, and it has been speculated that MLN64 may play a role in steroidogenesis in this tissue (31).

At the time the above experiments were being performed, information on the mechanism by which StAR mediated cholesterol transfer to the inner mitochondrial membrane was indeed scarce. In one effort to explain StAR action, Kallen et al. (59) demonstrated that StAR can act as a sterol transfer protein and that the function of the StAR protein may be to enhance desorption of cholesterol from one sterol-containing membrane to another. In this model, StAR is specifically directed to the mitochondria via its N terminus and, upon its arrival at the outer mitochondrial membrane, the C terminus produces alterations in this membrane that in some manner result in the transfer of cholesterol from the outer to the inner membrane. Interestingly, the transfer of cholesterol to both trypsin-treated and heat-treated mitochondria or to heat-treated microsomes by purified StAR protein was specific in that identical experiments employing phosphatidylcholine failed to show transfer of this phospholipid. This is particularly pertinent to the situation found in steroidogenic mitochondria in which the desorption of cholesterol from the sterol-rich outer membrane to the sterol-poor inner membrane (60) would serve to enhance pregnenolone synthesis by the P450scc enzyme. While indicating that StAR had this capability, this study, however, failed to provide a molecular mechanism of how StAR could act as a sterol carrier protein.

The Molten Globule Hypothesis
Another approach was taken by Miller and colleagues (61) in which they attempted to determine the physical characteristics of the StAR protein under different physico-chemical conditions and then utilize these characteristics to provide insights into the mechanism of StAR action. In one of their studies they subjected StAR to limited proteolysis at different pH values and found that the molecule behaves differently as the pH decreases. They demonstrated that at pH values in the 3.5–4.0 range StAR undergoes conformational changes that result in a partial unfolding of the protein and a transition to a molten globule state. Molten globules are structures within proteins that have lost at least some of their tertiary structure but which have retained virtually all of their secondary structure. They speculated that if the pH microenvironment surrounding the mitochondria is acidic, which might be caused by the expulsion of protons from the mitochondrial matrix by the proton pump and/or by the presence of the negatively charged head groups of the phospholipids in the outer mitochondrial membrane, the StAR molecule may undergo a conformational shift. They further hypothesized that as the transition to a molten globule occurs, this structural change could result in an opening of the StAR protein, possibly exposing a hydrophobic region, or it may prolong the interval with which StAR can reside on the outer membrane, thus allowing increased transfer of cholesterol during this period.

With the demonstration 1) that StAR was fully active even without its signal sequence, 2) that it could act as a sterol carrier protein, and 3) that it could form a molten globule while interacting with the mitochondrial outer membrane, a picture began to emerge in which StAR might be causing perturbations of the outer membrane that resulted in cholesterol movement from the outer to the inner mitochondrial membrane. An obvious possibility was that StAR was interacting with other mitochondrial outer membrane proteins and/or phospholipids to produce this effect. However, attempts to identify such binding partners using the yeast two-hybrid system, coimmunoprecipitation, and binding assays utilizing radioactive StAR and isolated mitochondria have thus far failed to produce any components that specifically interact with StAR (62). The methods used to identify StAR binding partners can be technically difficult and subject to artifacts. However, to date they have produced no positive results. In support of these findings it was demonstrated that StAR could promote cholesterol transfer to mitochondria in which the outer membrane proteins have been removed by partial proteolysis with trypsin (59). This would suggest that StAR does not have protein binding partners on the outer mitochondrial membrane and can, instead, interact directly with membrane phospholipids. A recent study using fluorescence energy transfer has demonstrated that StAR and the peripheral benzodiazepine receptor (PBR) are closely associated on the outer mitochondrial membrane, being less than 100 A from each other (63). PBR is a membrane protein found in high abundance in the outer mitochondrial membrane of steroidogenic cells and has been shown to be involved in cholesterol delivery to the inner mitochondrial membrane (64). Based on this association, the authors proposed a model in which StAR targets cholesterol to the PBR, which then facilitates its transfer to the inner mitochondrial membrane. While PBR appears to be involved in cholesterol transfer to the inner mitochondrial membrane, little is known concerning the mechanism of its action in this process, and, in any event, it appears that StAR can transfer cholesterol into the mitochondria in the absence of outer mitochondrial membrane proteins (59).

StAR-Related Lipid Transfer (START) Domains
As indicated earlier, the cholesterol-transferring region of the StAR protein appears to be located in the C-terminal region of the protein as demonstrated in studies with both N-terminally truncated StAR proteins and MLN64. A hint of how the C terminus of these proteins might transfer cholesterol has recently been put forth by Ponting and Aravind (65), who demonstrated that sequences in the C-terminus of StAR are homologous to sequences in several other proteins, including MNL64, which display diverse functions. They named these sequences START domains, for StAR-related lipid transfer domains. START domains consist of approximately 200–210 amino acid stretches, and the significance of these domains is that they are capable of binding lipids. Thus, with this information, the possibility that StAR was a lipid-binding carrier protein would have to be considered. This possibility received an exciting boost recently when Tsujishita and Hurley (66) succeeded in obtaining crystals and solving the structure for the START domain of the MNL64 protein. Because of problems in purifying and crystallizing the START domain from StAR, they focused on the START domain of MLN64, which shows the highest degree of homology to the StAR-START domain and found that it readily crystallized. They demonstrated that both StAR-START and MLN64-START could bind cholesterol in an essentially identical manner and that binding occurred in a ratio of 1:1. These studies were important in that they demonstrated that the START domains of both MLN64 and StAR behaved similarly and, thus, were a confirmation of earlier studies that had been performed in vitro using transfected cells (26, 57). The crystal structure of the MLN64-START at 2.2 Å indicated that it consisted of an {alpha} + ß fold built around a U-shaped incomplete ß-barrel. MLN64-START contains a nine-stranded antiparallel ß-sheet, four {alpha}-helices, and two {Omega}-loops. Most importantly, the tertiary structure of MLN64-START revealed a hydrophobic tunnel that was 26 x 12 x 11 Å in size and was large enough to bind a single molecule of cholesterol. Interestingly, when three of the most common mutations resulting in lipoid CAH are projected onto the MLN64-START domain model, they are all found to reside quite close to each other, and two of these mutations reside within the cholesterol-binding hydrophobic tunnel. These mutations would be expected to disrupt the structure of the tunnel and quite likely result in a decrease in cholesterol binding. Based on these findings, the authors propose that StAR functions in transferring cholesterol to the inner mitochondrial membrane via its ability to bind and function as an intermitochondrial membrane cholesterol-shuttling protein. However, several aspects of this model are in conflict with observations that have been made previously and with some hypotheses of other models that have been proposed. These conflicts will be discussed later.

Probing the Physical Characteristics of StAR
While the controversy continues of whether StAR can act as a cholesterol-shuttling protein in the intermembrane space or whether it can act on the outer mitochondrial membrane to effect cholesterol transfer, the Miller laboratory has continued to add new structure-function studies to the field. In one study they examined the structural properties of a bacterially produced segment of the StAR protein corresponding to amino acids 63–193, the protease-resistant region (67). They found that expression of the 63–193 domain in the absence of the molten globule 194–285 domain altered its structure, rendering it more susceptible to protease digestion and devoid of tertiary structure. Treatment with detergents increased the secondary structure of this domain, indicating that, like the 194–285 domain, the 63–193 domain could also form a molten globule. Most importantly, addition of 63–193 StAR to liposomes consisting of phosphatidylcholine or phosphatidylserine induced the formation of stable protein-liposome complexes. These data indicate the N-terminal region of the StAR protein can form a molten globule and that this structure can interact directly with membranes. This finding is important since, when in the molten globule state, proteins lose tertiary structure and can open, thus exposing a hydrophobic interior (if one exists), allowing them to interact with phospholipid membranes. The strong interaction of water-soluble proteins with phospholipid membranes after their transition to molten globule states has been well documented previously (68, 69, 70, 71, 72), and thus a case for StAR interaction with a phospholipid environment can be made. This observation has important implications in that StAR has been shown to closely interact with the outer mitochondrial membrane during the course of cholesterol transfer, and this interaction apparently does not require that it bind to other proteins (59).

These observations were followed by more extensive studies on StAR and its interactions with artificial membranes. Utilizing unilamellar artificial membranes composed of phosphatidylcholine or phosphatidylcholine:cholesterol (73), Miller’s group demonstrated that recombinant StAR can readily bind to these membranes in the complete absence of other proteins, supporting the hypothesis that StAR can interact directly with the outer mitochondrial membrane, and does not require a receptor protein. Also, this binding occurred maximally at low pH, conditions favoring the formation of molten globule structures. While the degree of binding of StAR to these membranes varied with the heterogeneity of the membrane composition, a most interesting observation was that StAR was able to bind preferentially to the cholesterol-rich domains in cholesterol-containing membranes. Cholesterol-rich domains have been previously demonstrated in biological membranes (74), and it is intriguing to speculate that StAR binds to such regions in the relatively cholesterol-rich mitochondrial outer membrane to more easily facilitate transfer of this substrate to the cholesterol-poor inner membrane. Importantly, StAR proteins harboring mutations that cause lipoid CAH, and thus impaired steroidogenesis, did not bind to the artificial membranes as efficiently as did wild-type StAR (74). They also found that when StAR bound to artificial membranes containing cardiolipin in concentrations approximating that found in authentic mitochondrial outer membranes, it underwent a conformational change to a molten globule more readily than when cardiolipin-free membranes were used.

THE ARGUMENTS
It is eminently clear at this point in time that we do not know precisely how StAR works to mediate cholesterol transfer to the inner mitochondrial membrane. Many of the observations that have been made to date cannot be disputed, but they also do not tell us how StAR works. For example, the data that StAR can act as a sterol carrier protein and promote desorption from one membrane to another is most convincing, but is not placed within the context of what is occurring in a steroidogenic cell. Also, the observation that StAR and PBR are in close proximity to each other on the outer mitochondrial membrane is very convincing but does not tell us if they act together to promote cholesterol transfer. In fact, it is hard to imagine that StAR would target cholesterol to the outer mitochondrial membrane where PBR would transfer it to the inner membrane. First, if it performs this function via its cholesterol recognition/interaction amino acid sequence and consensus pattern (CRAC) sequence, that is known to bind cholesterol, then N-62 StAR should be completely inactive since the CRAC domain in StAR is found at positions 5–18 in the N terminus (75). This is not the case as many studies have shown N-62 to be fully active in steroidogenesis when transfected into various cell lines, and one study even demonstrated that N-62 StAR could transfer cholesterol to membranes of isolated mitochondria (59). Second, if it delivers cholesterol to the PBR for subsequent transfer via its hydrophobic cholesterol binding START domain, it is most difficult to imagine that it could perform this task up to 400 times per molecule of StAR, as calculated by Jefcoate et al. (76), before being taken up by the mitochondria. From yet another point of view, it seems unlikely that StAR functions as a cytosolic cholesterol carrier protein in that virtually every study on StAR has found it to be tightly associated with, and rapidly taken up by, the mitochondria after its synthesis.

The two models that would appear to have the most credence at this time are the intermembrane shuttle model and the molten globule model. In the former, StAR acts as a carrier of cholesterol from the outer to the inner mitochondrial membrane, and in the latter StAR acts to promote cholesterol transfer via changes in its conformation that might produce a hydrophobic tunnel or region through which cholesterol might pass. Each model has strong points and each model has facets that appear to be incompatible with the other. For example, the cholesterol shuttle model is inconsistent with the observation that StAR can act on the outer mitochondrial membrane and promote cholesterol transfer without ever entering the intermembrane space or matrix. There may also be some problems with stoichiometry with this model in that StAR appears to become inactivated very quickly, and it does not appear that transferring one cholesterol molecule at a time would account for the large number of steroid molecules that are formed. Also, the openings of the hydrophobic core of the START domain do not appear to be large enough to allow cholesterol molecules to enter or exit the pocket without some sort of conformational change in the protein. It is highly speculative, but interesting, that perhaps the transformation of the START domain to something approximating a molten globule would allow for the opening of the hydrophobic core and thus cholesterol could enter and exit the tunnel more readily. As for the molten globule hypothesis, the data clearly indicate that StAR can form this structure at low pH. The questions that arise from this model are several. Does the local pH in the vicinity of the outer mitochondrial membrane reach the pH required (3.0–4.0) to form a molten globule? In support of this model it is argued that between the charged head groups of the membrane phospholipids and the extrusion of protons by the proton pump, an acidic environment is produced, but no evidence is available to what this pH actually is in vivo. This model however, is in agreement with observations that an active electrochemical force, in which protons would be actively extruded from the matrix, is required for the support of steroid hormone synthesis (77). In addition, the shuttle model argues that it is not reasonable to hypothesize that a molecule that has evolved such a highly ordered three-dimensional cholesterol-binding pocket would function by eliminating the structure of this pocket through the formation of a molten globule. This argument makes sense, but it is clearly teleological in nature.

Perhaps there is some common ground in these two theories. Miller’s group (73) has recently stated that their fluorescence energy transfer data are compatible with StAR acting either on the outer mitochondrial membrane or in the intermembrane space. If N-62 StAR can indeed act in the intermembrane space, perhaps a fusion of the two models is possible. One can envision StAR coming into contact with the mitochondria and, as supported by observations with artificial membranes, the StAR-START segment of the molecule becomes buried in the outer mitochondria membrane. Simultaneously, this buried segment of the StAR protein becomes transformed to a molten globule by the low pH of the membrane environment. Then, if StAR can traverse the outer membrane and appear at the intermembrane space, it is possible that it does so with the hydrophobic cholesterol-binding interior of the StAR-START domain partially open. This would allow for the rapid binding and release of cholesterol located in the outer membrane, perhaps by desorption, to the inner membrane. Thus, the START domain of StAR may act like a tunnel through which cholesterol can enter at one end and exit at the other. While this is occurring, the N-terminal domain of StAR is interacting with a mitochondrial import complex, and eventually the entire StAR molecule enters the matrix where it is no longer active in further cholesterol transfer (see Fig. 1Go.) Thus, the hydrophobic core, consisting of a partially opened START domain, is the conduit for cholesterol transfer for the length of time that is required for StAR to be imported into the matrix. It is possible that this transfer could occur in locations in the membranes where contact sites have formed. For example, if the N terminus of the StAR protein is being imported simultaneously, the START domain could utilize the contact sites formed by the import of the N terminus for cholesterol transfer. Such contact sites have been characterized in steroidogenic cells and have been shown to contain higher levels of cholesterol (55, 78), StAR (78), and the first two enzymes in the steroidogenic pathway, cytochrome P450scc and 3ß-hydroxysteroid dehydrogenase (78).



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Figure 1. Proposed Model of StAR Action

In this hypothetical model StAR comes into contact with the outer mitochondrial membrane probably via its N-terminal signal sequence (not shown). At the same time the START domain (MLN64-START domain shown here) becomes buried in the phospholipid bilayer while simultaneously being transformed to a molten globule, thus losing some of its tertiary structure. The START domain then traverses the outer membrane and appears at the surface of the intermembrane space. Transformation to the molten globule state could result in a conformational change in the START domain resulting in the partial opening of the cholesterol entrance to this hydrophobic pocket. This would allow for cholesterol, which is rich in the outer membrane, to enter the START domain pocket and move along its hydrophobic face. If the inner membrane is very close to or in contact with the outer membrane, the cholesterol exiting by way of the lower entrance could be transferred to the inner mitochondrial membrane and the cytochrome P450scc system, perhaps by desorption as hypothesized by others. Thus, the START domain may act like a hydrophobic tunnel through which cholesterol can enter at one end and exit at the other, an action highly dependent upon conformational changes in the START domain. It might be envisioned that this hydrophic pocket can shuttle cholesterol molecules one at a time or that the cholesterol molecules might simply slide along the hydrophobic face of the pocket once it is exposed by conformational changes in the START domain. Not shown in this model, but inherent in it, the N-terminal domain of StAR would be simultaneously interacting with a mitochondrial import complex, and eventually the entire StAR molecule enters the matrix where it is no longer active in further cholesterol transfer. Thus, the hydrophobic core, consisting of a partially opened START domain, is the conduit for cholesterol transfer for the length of time that is required for StAR to be imported into the matrix. This model is, of course, highly speculative at this time, and until more specific information on the interactions between StAR, the mitochondrial membranes, and cholesterol are available, the mechanism of action of StAR in mediating cholesterol transfer in steroidogenic mitochondria remains a mystery.

 
This model, of course, is entirely speculative, and until more specific information regarding the interactions between StAR, the mitochondrial membranes, and cholesterol are available, the mechanism of action of StAR in mediating cholesterol transfer in steroidogenic mitochondria remains one of the most intriguing questions to be answered in the field of steroidogenesis.


    ACKNOWLEDGMENTS
 
The author gratefully acknowledges Dr. Walter Miller of the University of California at San Francisco for sharing preprints of his work and for a critical and helpful reading of the text. He also thanks Ms. Deborah Alberts for help in preparing this manuscript.


    FOOTNOTES
 
This work was supported by NIH Grant HD-17481.

Abbreviations: CAH, congenital adrenal hyperplasia; C/EBP, CCAAT/enhancer binding protein; CRAC, cholesterol recognition/interaction amino acid sequence and consensus; DAX-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X, gene 1; P450scc, cytochrome P450 side-chain cleavage enzyme; PBR, peripheral benzodiazepine receptor; SF-1, steroidogenic factor-1; SREBP, sterol regulatory element binding protein-1a; START, StAR-related lipid transfer.

Received for publication April 5, 2001. Accepted for publication June 4, 2001.


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