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
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ABSTRACT
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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.
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INTRODUCTION
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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.
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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.
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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 StARs
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
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.
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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 3.54.0 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 200210 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
+ ß fold built around a
U-shaped incomplete ß-barrel. MLN64-START contains a nine-stranded
antiparallel ß-sheet, four
-helices, and two
-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 63193, the protease-resistant
region (67). They found that expression of the 63193
domain in the absence of the molten globule 194285 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 194285
domain, the 63193 domain could also form a molten globule. Most
importantly, addition of 63193 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), Millers 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 518 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 (3.04.0) 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. Millers
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. 1
.) 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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