From the Department of Zoology, Brigham Young
University, Provo, Utah 84602 and the § Department of
Pediatrics and ¶ Metabolic Research Unit, University of
California, San Francisco, California 94143-0978
Received for publication, January 31, 2001, and in revised form, March 5, 2001
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ABSTRACT |
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Steroidogenic acute regulatory protein (StAR)
mediates cholesterol transport from the outer to the inner
mitochondrial membrane during steroid biosynthesis. The mechanism of
StAR's action is not established. To address mechanistic issues, we
assessed the binding of StAR to artificial membranes by fluorescence
resonance energy transfer using endogenous StAR tryptophan residues as
the donor and dansyl-phosphatidylethanolamine in the bilayer as the acceptor. Mixing StAR with dansyl-labeled vesicles composed of phosphatidylcholine increased the fluorescence intensity of
dansyl emission excited at 280 nm by 10-40%. This interaction was
dependent on pH, with a maximum at pH 3.0-3.5 and essentially no
change above pH 5. Binding experiments at different temperatures and various combinations of phosphatidylcholine, phosphatidylglycerol, cardiolipin, and cholesterol showed that binding involves an
electrostatic step and one or more other steps. Although binding
prefers a thermodynamically ordered bilayer, the rate-limiting step
occurs either when the bilayer is in a fluid state or when there is
cholesterol-induced membrane heterogeneity. Experiments with
fluorescence and light scattering indicate that StAR binding promotes
ordering and aggregation of anionic membranes. The inactive StAR mutant
R182L had lower affinity for the membrane, and the partially active
mutant L275P had intermediate affinity. Far-UV CD spectroscopy of StAR
in PC membranes show more The steroidogenic acute regulatory protein
(StAR)1 rapidly increases the
movement of cholesterol into adrenal and gonadal mitochondria (1, 2)
for conversion to pregnenolone by cytochrome P450scc to initiate
steroidogenesis (3). Although low levels of steroid biosynthesis can
occur in the absence of StAR (4), the StAR protein is required for the
rapid gonadotropin-induced and ACTH-induced steroidogenic responses
necessary for reproduction and responses to stress (2). The crucial
role of StAR in human physiology is best illustrated by the findings in
congenital lipoid adrenal hyperplasia, which is caused by mutations in
the StAR gene (4, 5). Affected individuals have female external
genitalia irrespective of chromosomal sex and die in infancy from
mineralocorticoid and glucocorticoid deficiency if not diagnosed and
treated with hormonal replacement therapy (6).
The mechanism of StAR's action is unknown. StAR is synthesized as a
37-kDa phosphoprotein that is cleaved to a 30-kDa form upon
mitochondrial entry (1, 2). However, StAR's action requires new
protein synthesis, and the 15-30-min time course of StAR's action
correlates more closely with the presence of the cytoplasmic 37-kDa
precursor than with the 2-3-h half-life of the intramitochondrial
30-kDa form (7-9). Furthermore, deletion of 62 amino-terminal residues
(N-62 StAR), which includes the mitochondrial leader peptide, results
in an extramitochondrial protein that is fully active both when
expressed in living cells and when expressed in bacteria and added to
isolated mitochondria in vitro (10-13). By contrast, StAR
is inactivated by deleting just 28 C-terminal amino acids (5), and all
StAR mutations that cause congenital lipoid adrenal hyperplasia are
found in C-terminal domains, suggesting that this region is responsible for StAR's activity (4, 5). In addition, biophysical studies indicate
that StAR acts on the outer mitochondrial membrane (OMM) while
undergoing a pH-dependent transition to a molten globule (14). Thus, it appears that the 37-kDa "preprotein" acts on the OMM
as a molten globule and is then inactivated by mitochondrial import and
cleavage to generate the inactive 30-kDa intramitochondrial protein
(13).
Recently, Tsujishita and Hurley (15) suggested a different model based
on the 2.2-Å resolution structure of the StAR-like domain of MLN64
(MLN64 residues 216-445), a protein that has 35% amino acid sequence
identity with StAR (16) and has StAR-like activity in vivo
and in vitro (12, 17). This structure showed that StAR-like
proteins have a large hydrophobic tunnel that binds one molecule of
cholesterol. The data were interpreted to mean that StAR acts in the
mitochondrial intramembranous space (IMS) to shuttle cholesterol one
molecule at a time from the OMM to the inner mitochondrial membrane.
This "IMS/shuttle" model of StAR's action was viewed as being
inconsistent with the interpretation that N-62 StAR acts on the OMM and
forms a molten globule (15).
StAR is active with isolated mitochondria in which proteins associated
with the OMM have been inactivated by heat denaturation or partial
proteolysis with trypsin, and StAR can transfer cholesterol to
intracellular membranes other than OMM. These results suggest that StAR
interacts directly with membrane phospholipids and does not require a
specific receptor protein (18). Therefore, we sought to study the
interactions of biosynthetic N-62 StAR with lipids in synthetic
unilamellar vesicles of known composition. By measuring resonance
energy transfer to synthetic dansylated membranes, we now show that
N-62 StAR interacts with membrane lipids in a pH-dependent
fashion that supports the molten globule hypothesis. CD spectroscopy of
N-62 StAR in lipid environments also indicates a
pH-dependent molten globule. Features of both the
OMM/molten globule hypothesis and the IMS/shuttle hypothesis appear to
be necessary to account for StAR's behavior.
Reagents--
Wild type and mutant 6-His-N-62 StAR proteins were
expressed in Escherichia coli and purified as described
(19). Dansyl-phosphatidylethanolamine (dansyl-PE) and laurdan were
obtained from Molecular Probes, Inc. (Eugene, OR). Egg
phosphatidylcholine (PC), dimyristoylphosphatidylcholine (DMPC),
tetraoleoyl cardiolipin, and
1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG)
were purchased from Avanti Polar Lipids (Birmingham, AL). All other
reagents were obtained from standard sources.
Vesicle Preparation--
Vesicles were prepared by mixing
phospholipids with or without 2 mol % (of the final lipid
concentration) dansyl-PE and with or without 50 mol % cholesterol
(dissolved in chloroform). The lipids were dried under N2
to remove chloroform and then hydrated with 20 mM acetate
buffer (pH 4) containing 150 mM KCl to give a 10 mM final bulk lipid concentration. The hydrated lipids were heated to 37 °C to ensure that the mixture was above the lipid phase
transition temperature and incubated for 1 h with intermittent mixing by rapid agitation. The resulting multilamellar vesicles were
sonicated three times for 3 min each using a probe sonicator to obtain
small (100-200-Å diameter) unilamellar vesicles (SUV). Ambient
temperature was maintained constant in the sample during sonication
with a water bath. DMPC vesicles were sonicated at 35 °C to ensure
that SUV formation occurred above the phase transition temperature.
Fluorescence Measurements--
The binding of wild type and
variant StAR proteins to phospholipid vesicles was assessed by
fluorescence resonance energy transfer using tryptophan residues in the
protein as the donor and dansyl-PE in the membrane as the acceptor.
Energy transfer was assayed using a photon-counting spectrofluorometer
(Instruments SA) (20). Excitation was set at the maximum for Trp (280 nm), and the emission of dansyl-PE was measured at 510 nm (4.25-nm band
pass). Simultaneous assessment of fluorescence intensity at multiple
excitation and emission wavelengths was obtained by rapid sluing of
monochromator mirrors using control software provided with the
instrument. Alternatively, energy transfer was also assessed from
excitation spectra with emission at 510 nm. Measurements of the
physical state of membranes containing the bilayer probe, laurdan, were
obtained using a PC1 fluorometer from ISS (Urbana, IL). Diluents ranged
in pH from 2.0 to 7.0 and contained 150 mM KCl with 20 mM phosphate, citrate, acetate, or HEPES buffers as appropriate for the target pH. The temperature was maintained at
37 °C or as indicated in the relevant figure using circulating water
baths. Continuous gentle magnetic stirring preserved sample homogeneity
in both instruments.
For binding experiments by energy transfer, SUV (50 µM
lipid) were equilibrated to the temperature in the sample compartment of the fluorometer, and base-line fluorescence intensity was recorded. The procedure was then repeated following incremental additions of StAR
protein. Energy transfer was observed as an increase in fluorescence
intensity (excited at 280 nm) upon the addition of protein. Some direct
excitation of dansyl-PE also occurs at 280 nm, and this excitation may
likewise be affected by the binding of protein. To distinguish these
effects from true energy transfer, the direct fluorescence of dansyl-PE
was measured simultaneously (excitation = 340 nm, emission = 510 nm). The energy transfer was then corrected for changes in dansyl
intrinsic fluorescence by calculating the ratio of fluorescence excited
at 280 nm to that excited at 340 nm. When experiments involved the pH
dependence of energy transfer with charged lipids, the results were
normalized to the intensity at 300 nm. This change in analysis
procedure was required by the presence of an optical artifact at 340 nm with anionic membranes at low pH.
Laurdan (1.2 µM) dissolved in Me2SO
was incorporated into liposomes prior to sonication, and SUV were
prepared as described above without dansyl-PE. SUV were equilibrated in
the fluorometer sample chamber to the indicated temperatures, and
emission spectra were acquired (excitation = 350 nm). The
procedure was then repeated at each temperature in the presence of N-62
StAR. Effects of temperature and the binding of the protein on the
emission spectrum were quantified by calculating the value of
generalized polarization (GP) from the spectra as described (21).
CD Spectroscopy--
Far UV (200-250 nm) CD measurements were
carried out in a 1.0-mm path length flat cuvette in a Jasco-715
spectropolarimeter equipped with a Peltier-controlled temperature
system. All lipids and protein samples were mixed with the same buffer
system that was used for the fluorescence resonance energy transfer
measurements and were incubated at 37 °C for 10 min prior to
recording the spectrum. Appropriate base lines were obtained under the
same experimental conditions and were subtracted from the sample
spectra. In some spectra, the noise was reduced by application of a
Savitsky-Golay filter.
Binding of StAR Proteins--
The apparent binding of N-62 StAR to
vesicle surfaces was readily detected by resonance energy transfer. The
fluorescence excitation spectrum of SUV containing 2% dansyl-PE has a
minimum at 280 nm and a peak at 340 nm, representing intrinsic dansyl fluorescence. The addition of StAR elicits a small decrease in dansyl
intrinsic fluorescence and a substantial increase in fluorescence intensity at 280-300 nm (Fig.
1A). The ratio of the two
spectra from Fig. 1A shows a peak at 280 nm consistent with
a tryptophan absorption spectrum and indicating resonance energy
transfer between one or more StAR tryptophans and dansyl-PE in the
membrane (Fig. 1B).
Increasing concentrations of N-62 StAR enhanced dansyl fluorescence of
SUV made of either PC or 1:1 PC/cholesterol (Fig.
2A). The effect was saturable
and fit a simple binding model with an apparent KD
of ~0.2 µM in each case. The binding was very sensitive
to pH; no binding was detected at pH 7.0 (not shown), and binding was
maximal at pH 3.0-3.5 (Fig. 2B), which corresponds to the
pH range at which StAR forms a molten globule structure (14). This
ability of StAR to bind to unilamellar lipid bilayers lacking intrinsic
membrane proteins supports the view that StAR acts directly on
phospholipid membranes and does not require a receptor.
The binding of StAR was also critically dependent on small variations
in the sequence of the StAR protein. The StAR missense mutant R182L
causes severe congenital lipoid adrenal hyperplasia and is wholly
inactive in transfected cells, whereas the mutant L275P causes less
severe disease and retains about 10% of normal activity in transfected
cells (4). Spectroscopic analyses have shown that the R182L mutant is
grossly misfolded, while the L275P mutant has a nearly normal fold
(19). The R182L and L275P mutants of N-62 StAR bound to SUV composed of
either PC or 1:1 PC/cholesterol but differed from wild-type N-62 StAR
and from each other in their affinities for the bilayer (Fig.
3). The average values for
KD measured between pH 4.0 and 2.0 were 0.16 ± 0.02 µM for wild type, 1.03 ± 0.26 µM
for L275P, and 3.1 ± 1.2 µM for R182L (mean ± S.E., n = 28, 8, and 10, respectively). These
differences were statistically significant by two-way analyses of
variance (p < 0.0001) at all pH values tested with or
without the presence of cholesterol.
pH Effect--
The statistical analysis of KD
for the binding of StAR to PC SUV detected a major effect of pH and
differences in the effect of pH on the wild-type and mutant StAR
sequences (Fig. 4). Below pH 4, wild-type
N-62 StAR bound more efficiently to SUV composed of PC alone, whereas
the two mutants showed little difference in binding between PC and
PC/cholesterol SUV as a function of pH (Fig. 4A). These
results were confirmed statistically by two-way analysis of variance
for both the effects of pH (p < 0.0001) and
cholesterol content (p = 0.002) as well as the
interaction between the two variables apparent for wild type protein in
Fig. 4A (p = 0.011). A similar analysis
revealed no statistical significance for either mutant (Fig. 4,
B and C). Cholesterol tends to aggregate into
cholesterol-rich domains when mixed 1:1 with PC (22), therefore creating domains relatively deficient in dansyl groups. The data in
Fig. 4 suggest that wild type, but not the mutant forms of N-62 StAR,
binds with increasing avidity to cholesterol-rich domains as pH
decreases so that N-62 StAR is sequestered away from regions of the
membrane that contain dansyl groups. This interpretation is consistent
with the effect of N-62 StAR to make the environment of dansyl-PE more
rigid in the absence of cholesterol but more fluid in the presence
(Fig. 5). Thus, although the presence of cholesterol is not required for StAR to interact with PC membranes, it
substantially alters StAR's binding characteristics.
Effects on Dansyl Intrinsic Fluorescence--
The StAR-induced
reduction in dansyl intrinsic fluorescence (340 nm) (Fig.
1A) suggested that the binding of StAR alters the physical
state of the membrane. Because CD data show that changes in pH affect
StAR's conformation (14), we considered whether changes in pH would
also affect the physical state of SUV and their interaction with StAR.
In the presence or absence of N-62 StAR, the fluorescence was stable at
pH Effects of Membrane Order--
To pursue the suggestion that StAR
binding might affect membrane order, we varied the fluidity of the
bilayer by altering the temperature. Because the phase transition of PC
from gel to liquid crystalline phases occurs at temperatures below
0 °C, we instead prepared vesicles from DMPC, whose phase transition
occurs at ambient temperature. This choice of lipid allowed us to test the binding of StAR to both the liquid crystalline and the gel phases
of SUV without freezing the sample. It also permitted us to reach the
liquid phase at temperatures low enough to avoid denaturing the protein.
As with PC vesicles, increasing concentrations of N-62 StAR enhanced
dansyl fluorescence in DMPC and 1:1 DMPC/cholesterol SUV in a saturable
manner (Fig. 6A). The binding
of N-62 StAR to DMPC SUV was similar to its binding to PC or
PC/cholesterol SUV (compare with Fig. 2A; maximum
I280/I340 = 0.16-0.19
for PC, PC/cholesterol, and DMPC). However, the binding to
DMPC/cholesterol SUV was more than doubled. Increasing the temperature
from 15 to 25 °C increased energy transfer, but there was little if
any effect of increasing the temperature further to 35 °C (Fig.
6B). Thus the binding of N-62 StAR appeared favored by
increased membrane fluidity.
The binding of N-62 StAR to SUV of various compositions suggests that
StAR should prefer a more ordered membrane over one that is fluid
(Figs. 5A and 6A); however, binding apparently
preferred the more fluid liquid crystalline state of DMPC above the
phase transition temperature of 22 °C (Fig. 6B). To
explore this apparent inconsistency, we assessed bilayer fluidity in
DMPC and DMPC/cholesterol SUV as a function of temperature, with or
without StAR, using the fluorescent membrane probe laurdan. The
emission spectrum of laurdan is very sensitive to the polarity of the
membrane environment; the spectrum displays large red shifts when the
number and/or mobility of water molecules partitioned at the level of
the phospholipid glycerol backbones has increased (21). Thus, enhancing
bilayer fluidity allows water to have greater access to laurdan and
thus shifts the emission spectrum toward longer wavelengths. These spectral shifts are quantified by calculation of GP, which varies from
Thus, the temperature dependence results (Fig. 6B) required
further analysis. One possibility is that the binding results obtained
with DMPC membranes represent kinetic effects rather than the
thermodynamics of the binding interaction. To test this possibility, we
assayed the binding of StAR to DMPC SUV by increasing the temperature
incrementally from 15 to 35 °C and then decreasing the temperature
back to 15 °C (Fig. 6D). Consistent with the data in Fig.
6B, binding increased as the temperature was raised, but instead of returning to the original level when the sample was cooled,
the binding increased. Thus, the system was not at equilibrium at each
temperature because the binding of StAR to lipid in the gel state
depended on sample history; heating the vesicles to a temperature above
the phase transition apparently removed a kinetic barrier to binding.
The increased order of DMPC SUV when they contain cholesterol helps to
explain the effect of cholesterol to enhance binding above 22 °C.
However, differences in bilayer fluidity cannot account for the
differences in StAR binding between DMPC SUV with and without
cholesterol at lower temperatures, because the fluidity of the two
types of vesicles detected by laurdan was similar at 15-20 °C (Fig.
6C). These observations may be explained by the tendency of
cholesterol to form domains in membranes (22). The kinetic barrier
evidenced in Fig. 6D is probably created by a homogeneous
and well ordered membrane, such as that formed by DMPC SUV in the gel
phase. The domain structure of DMPC/cholesterol vesicles may behave
like the fluid phase of DMPC membranes by disrupting the regular
surface structure and thus removing the kinetic barrier. Similar
phenomena have been observed with other proteins where kinetic barriers
to binding and/or function are removed either by
temperature-dependent changes in the physical state of the
membrane or by promoting structural heterogeneity through compositional
diversity (20, 25-27). In addition to explaining the apparent
inconsistencies in the data of Figs. 5 and 6, A-C, the data
of Fig. 6D show that the binding of StAR occurs in multiple steps. If binding were a simple adsorption reaction, it would be
limited kinetically only by diffusion of the protein and vesicles. However, the observed kinetic limitation dependent on physical properties of the bilayer indicates that there must be at least one
additional step following the initial collision of the protein with the membrane.
Effects of Membrane Charge--
StAR's apparent preference for
binding to ordered membranes suggests that electrostatic interactions
might be involved in the binding reaction (24). Such an electrostatic
mechanism would be consistent with the binding of N-62 StAR to the
mitochondrial outer membrane, which contains about 18% cardiolipin
(28), a polyanionic phospholipid that is essentially a dimer of two
phosphatidylglycerol molecules.
To determine whether the binding of StAR might be assisted by the
presence of charged lipids in the membrane, we prepared PC SUV with
18% tetraoleoyl cardiolipin. As with SUV composed of PC (Fig.
2A) or DMPC (Fig. 6A), N-62 StAR bound well to
vesicles containing cardiolipin, but the energy transfer at saturating protein concentrations (0.5-0.75 µM) was about 5-fold
greater (Fig. 7A). Consistent
with the data in Figs. 3 and 4, StAR mutant R182L bound with lower
affinity than the wild type, and L275P bound with intermediate
affinity. However, unlike the results with either PC or DMPC vesicles,
both wild type and L275P N-62 StAR appeared to bind cooperatively to
PC/cardiolipin SUV. The very low affinity of binding with R182L made it
difficult to assess whether cooperativity was present. Measurements of
laurdan GP showed that N-62 StAR increased the order of membranes
containing cardiolipin (Fig. 7B).
To determine whether the effect of cardiolipin was due to electrostatic
charge rather than a specific effect of cardiolipin, we assayed the
binding of N-62 StAR to PC SUV containing 18 or 30 mol % DPPG.
Vesicles with a ratio of 82:18 PC/DPPG contain a molar concentration of
DPPG equal to the molar concentration of cardiolipin in the
PC/cardiolipin vesicles; those containing 30 mol % DPPG contain the
same number of ionizable species as the PC/cardiolipin vesicles.
Both the magnitude of N-62 StAR binding and the apparent cooperativity
to DPPG vesicles (Fig. 7C) was very similar to that observed with
PC/cardiolipin SUV (Fig. 7A), suggesting that membrane
surface charge is the important element in the effect of cardiolipin to
enhance binding of N-62 StAR.
The pH dependence of the interaction of N-62 StAR with membranes
containing cardiolipin showed a maximum at pH 3.0-3.5 (Fig. 8A). This is similar to the pH
dependence of interaction with PC/cholesterol vesicles (Fig.
4A) and argues that the pH effect is a function of the
protein rather than the properties of the bilayer and may be associated
with StAR's transition to a molten globule at this pH (14). Consistent
with this, analysis of the effects of pH on membrane fluidity as
assessed by laurdan GP shows essentially no pH-dependent
changes in the fluidity of vesicles containing cholesterol and no
changes above pH 3 in those without cholesterol (Fig. 8B).
Thus, all of the data assessing the interaction of StAR with lipid
membranes favors a pH-dependent interaction with membranes
containing cardiolipin at a composition approximating that of the
OMM.
Increasing concentrations of StAR caused aggregation of vesicles
containing cardiolipin or DPPG. To quantify the amount of aggregation,
we fit the dansyl region of the excitation spectra (310-400 nm) to a
Gaussian curve and calculated the S.D. of the data from the curve.
Comparing the standard deviation (i.e. the noise) as a
function of StAR concentration demonstrated a systematic trend in
vesicles containing cardiolipin or DPPG with increasing concentrations
of protein (Fig. 8C). This effect was absent in vesicles
composed of DMPC or PC vesicles devoid of either cardiolipin or DPPG.
The tendency of the anionic vesicles to aggregate in the presence of
StAR may be responsible for the apparent cooperativity of binding. If
the binding of a single protein molecule to the surface of one vesicle
increases the likelihood of a second vesicle to adsorb to the complex,
then StAR would bind with higher affinity when the two vesicles are
aggregated. Hence, the binding of a second protein would be enhanced,
and repetition of the process would lead to both the high order
aggregates and the cooperative binding. This phenomenon of protein
binding causing aggregation of anionic vesicles has been reported with
other proteins such as the plant toxin thionin (25) and cytochrome
c (29).
Conformational Changes of StAR in Lipid Environments--
In
aqueous Tris or phosphate buffers, the CD spectra of N-62 StAR show
minima at the 208 nm (
In contrast to the relatively modest effect elicited by adding
cholesterol to PC membranes, adding 18% cardiolipin has a profound effect (Fig. 9C). At pH 3.5-4.0, the CD signal is reduced
dramatically, suggesting that the StAR protein is buried more deeply in
the lipid, thus attenuating its signal, and the broad spectral minima at 220-230 nm indicate a predominance of stable Our data lead to five conclusions concerning the binding of StAR
to artificial membranes. First, StAR binds to membranes without assistance from other proteins. Second, binding involves at least two
steps; the first step is the initial collision of the protein with the
bilayer surface, and the second step appears to be limited by the
structure of the bilayer. The second step is facilitated by
heterogeneity of membrane surface structure induced by compositional diversity or by increased membrane fluidity; thermodynamically, the
binding prefers an ordered membrane. Third, in addition to promoting
bilayer heterogeneity, cholesterol also appears to exert a specific
effect to promote the binding of StAR to cholesterol-rich domains. This
interaction may involve StAR's cholesterol binding site, since the
mutant StAR proteins did not display this effect. Fourth, StAR's
preference for an ordered membrane reflects, at least in part, an
electrostatic component to the binding. Apparently, the preference for
negative charge in the bilayer is sufficient to compensate for the
kinetic subtleties and cholesterol dependence of binding observed in
the absence of anionic lipids. Fifth, StAR undergoes a conformational
change to a molten globule while interacting with membranes, especially
when cardiolipin is present.
Two apparently conflicting models of StAR's action have been proposed,
the OMM/molten globule model (14) and the IMS/shuttle model (15). Our
fluorescence energy transfer experiments are compatible with StAR
acting either on the OMM or in the IMS, but they also strongly support
the concept that StAR undergoes a pH-dependent transition
to a molten globule state while interacting with cholesterol-rich membranes, such as the OMM. The CD spectroscopy in membrane
environments strongly supports the pH-dependent molten
globule model. There can be little doubt that the x-ray crystal
structure of 216-444 MLN64 (15) corresponds closely with the as yet
undetermined structure of N-62 StAR, since these two proteins share
about 35% amino acid sequence identity and >50% amino acid
similarity (16), and the two proteins behave similarly in
vivo and in vitro (12, 17). However, the IMS/shuttle
model of StAR's action (15) is based on parsimony and analogy with the
action of other lipid transfer proteins. The molten globule data were
considered inconsistent with the crystal structure, because StAR
appeared to have evolved a fold ideally suited to transporting lipids,
and hence it seemed unlikely that it would then function in
vivo only in its molten globule form (15). Nevertheless, the
crystal structure also shows that the two potential openings of the
cholesterol-binding tunnel are too small to admit a cholesterol
molecule. Hence, StAR must undergo a conformational change while
binding cholesterol. This required conformational change may correspond
to the pH-dependent structural transition to a molten
globule that is detected spectroscopically. Similar
pH-dependent transitions to a molten globule structure have
been observed with other proteins that interact with membranes (32-34). Our fluorescence energy transfer experiments corroborate the
suggestion that StAR undergoes a pH-dependent transition to a molten globule while active. Furthermore, our data suggest that this
molten globule transition favors binding to cholesterol-rich membranes
at pH <4 and that single amino acid replacement mutants that decrease
or ablate StAR's activity also inhibit membrane binding and transition
to the molten globule. Thus, while our studies do not establish the
specific mitochondrial site of StAR's action, both the spectroscopic
evidence and our fluorescence energy transfer data provide strong
evidence for the changes in the shape of the StAR protein that appear
to be mandated by the crystallographic data.
-structure than in aqueous buffers, and
the presence of cardiolipin or cholesterol in the membrane fosters a
molten globule state. Our data suggest that StAR binds to membranes in
a partially unfolded molten globule state that is relevant to the
activity of the protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Resonance energy transfer. A,
relative fluorescence intensity of PC SUV (50 µM lipid)
containing 2% dansyl-PE before (solid curve) and
after (dashed curve) the addition of 0.14 µM N-62 StAR at pH 3.5. B, the ratio of the
spectra in A. The ratio was calculated by dividing the
spectrum obtained after the addition of protein by the spectrum
obtained before.
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Fig. 2.
Binding of wild type N-62 StAR to SUV
measured by fluorescence energy transfer. Energy transfer is shown
as the ratio of the dansyl fluorescence (measured at 510 nm) elicited
by excitation at 280 nm (tryptophan absorption maximum) to the dansyl
fluorescence elicited by excitation at 340 nm (intrinsic dansyl
fluorescence) A, binding at pH 4.0 to SUV composed of PC
alone (open circles) or to SUV composed of 1:1
PC/cholesterol (closed triangles). B, binding to SUV
composed of 1:1 PC/cholesterol at pH 2.0 (squares), pH 2.5 (triangles), pH 3.0 (inverted
triangles), pH 3.5 (diamonds), and pH 5.0 (circles).
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Fig. 3.
Binding of StAR mutants to SUV.
Fluorescence energy transfer from wild-type (solid
squares), L275P (open triangles), and
R182L (closed inverted triangles) N-62
StAR was measured with SUV composed of 1:1 PC/cholesterol at pH 4.0 (A) and pH 3.0 (B) and to SUV composed of PC
alone at pH 4.0 (C) and pH 3.0 (D).
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Fig. 4.
Binding of wild-type and mutant N-62 StAR
proteins to SUV composed of PC alone (open
circles) or 1:1 PC/cholesterol (closed
triangles) as a function of pH. Protein
concentrations were 0.16 µM wild-type (A), 0.6 µM L275P (B), and 1.1 µM R182L
(C). The data represent the mean ± S.E.,
n = 1-6.
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Fig. 5.
Effect of pH on SUV membranes.
A, the intrinsic fluorescence of the dansyl-PE in vesicles
composed of PC alone (circles) or 1:1 PC/cholesterol
(triangles) was measured before (closed
symbols) and after (open symbols) the
addition of 0.14 µM N-62 StAR at the pH values shown. The
data represent one example of four independent experiments.
B, the difference in intrinsic dansyl fluorescence induced
by the addition of StAR was calculated from A for SUV
composed of PC alone (open circles) and 1:1
PC/cholesterol (closed triangles). The data
represent the mean ± S.E., n = 4. The effect of
cholesterol (p < 0.0001) and the interaction between
the pH and cholesterol content (p = 0.0027) were
significant by two-way analysis of variance. C, the
similarity in the effects of StAR on both PC and PC/cholesterol SUV at
pH 4 and the gross differences in the effects of StAR on PC and
PC/cholesterol SUV at pH 2.5 were consistent at all StAR concentrations
above 0.14 µM.
4.0 and decreased proportionately as pH was reduced (Fig.
5A). The fractional decrement in fluorescence intensity was
greater for SUV containing cholesterol (Fig. 5A, triangles) than for SUV composed of PC alone
(circles). Although StAR influenced the pH dependence of
dansyl-PE in the two forms of SUV only slightly, this influence was
reproducible in opposite directions (Fig. 5B) and required
the same concentrations of StAR at which binding occurred (Fig.
5C). Similar results were obtained with the R182L and L275P
mutants (data not shown). The dimethylamine group in the dansyl
fluorophore titrates between pH 2.0 and 4.0 and no longer absorbs at
340 nm in the protonated state (23). The pK for this
reaction depends on the surrounding lipids; a lower pK corresponds to
increased membrane fluidity, and higher pK corresponds to
increased membrane structure, order, and rigidity (23). The data in
Fig. 5A suggest a pK of <2 for dansyl-PE in a
fluid PC bilayer and a pK between 2.0 and 2.5 in a more
rigid PC/cholesterol membrane. Thus, the binding of StAR increased the pK of dansyl-PE in PC membranes, suggesting that those
membranes had become more rigid (Fig. 5A). This effect is
common for proteins that bind to the surfaces of lipid membranes (24).
By contrast, StAR decreased the pK of dansyl-PE in PC/cholesterol
membranes (Fig. 5A), indicating increased fluidity. This
enhanced fluidity is consistent with aggregation of cholesterol away
from phospholipid-rich regions of the bilayer where dansyl-PE would
reside. This result would be predicted thermodynamically if StAR binds
preferentially to and thus stabilizes cholesterol-rich regions.
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Fig. 6.
Binding of wild-type N-62 StAR to SUV
composed of DMPC. A, binding at 35 °C and pH 3.5 as
a function of N-62 StAR concentration to SUV composed of DMPC
(squares) or 1:1 DMPC/cholesterol (triangles).
B, binding of 0.5 µM N-62 StAR at pH 3.5 as a
function of temperature. C, temperature dependence of
membrane fluidity as indicated by laurdan GP. Laurdan was incorporated
into SUV composed of DMPC (squares) or 1:1 DMPC/cholesterol
(triangles), which were then mixed with (open
symbols) or without (closed symbols)
0.5 µM N-62 StAR. GP was calculated from laurdan emission
spectra (excitation wavelength = 350 nm). D, binding of
N-62 StAR to SUV composed of DMPC as a function of temperature. The
solid line and squares represent
increasing temperature; the dotted line and
open squares represent decreasing
temperature.
1 (most disordered) to +1 (most ordered) (21). Laurdan readily
detected the normal DMPC phase transition near 22 °C, whereas
vesicles composed of 1:1 DMPC/cholesterol remained ordered throughout
the temperature range (Fig. 6C). The addition of N-62 StAR
had little effect on the fluidity of DMPC SUV containing cholesterol,
but in the absence of cholesterol N-62 StAR raised the phase transition
temperature 5 °C. These data indicate that StAR stabilizes the gel
state of the bilayer, thus corroborating the observations in Fig.
5.
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Fig. 7.
Effect of electrostatic charge.
A, binding of wild-type (squares), L275P
(open triangles), and R182L (inverted
triangles) N-62 StAR proteins to SUV composed of 82:18
PC/cardiolipin at pH 3.5. B, laurdan GP as a function of
temperature in SUV composed of 82:18 PC/cardiolipin before
(closed triangles) and after (open
triangles) the addition of StAR (0.5 µM).
C, binding of wild-type StAR to SUV composed of 82:18
PC/DPPG (solid circles) and 70:30 PC/DPPG
(open circles) at pH 3.5.
View larger version (19K):
[in a new window]
Fig. 8.
Dependence on pH. A, binding
of N-62 StAR as a function of pH to vesicles composed of DMPC
(squares; 0.5 µM StAR), DMPC/cholesterol
(triangles; 0.5 µM StAR) and PC/cardiolipin
(inverted triangles; 0.17 µM StAR).
B, effects of pH on fluidity of membranes as measured by
laurdan in vesicles composed of PC/cardiolipin (circles), PC
(inverted triangles), and DMPC
(squares) with (open symbols) or
without (closed symbols) 50 mol % cholesterol.
C, S.D. of fluorescence emission intensity from a Gaussian
curve with increasing concentrations of wild-type StAR mixed with SUV
composed of PC (squares), PC/cardiolipin
(inverted triangles), 82:18 PC/DPPG
(diamonds), and 70:30 PC/DPPG (circles).
-
*) and 222 nm (n-
*)
positions, which is typical of
-helical proteins. In contrast, N-62
StAR shows relatively weak signals at 215-218 nm, corresponding to parallel
-sheets, or at 225-230 nm, corresponding to antiparallel
-sheets (30). In the presence of PC at pH 4.0, the far UV CD spectrum of N-62 StAR showed an increased signal at 218 nm, suggesting an increase in
-sheet; this pattern was only minimally changed in
the presence of cholesterol (Fig.
9A). At pH 3.5, the spectrum in PC without cholesterol was essentially the same as at pH 4.0, but in
the presence of cholesterol the curve shows an increase in the
-sheet contribution, about equal to that of
-helix (Figs. 9,
A and B). Thus, the presence of cholesterol
fosters the pH-dependent molten globule transition
described previously in aqueous buffers (14).
View larger version (27K):
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Fig. 9.
Circular dichroism spectroscopy.
A, far UV CD spectra of 3.5 µM N-62 StAR in 50 µM PC at pH 4.0 in the absence (dotted
curve) and presence of cholesterol
(dotted/dashed curve) and at pH 3.5 in
the absence (dashed curve) and presence of
cholesterol (solid curve). B, a closer
view of the far UV CD spectra shown in A, focusing on the
region from 205 to 222 nm. C, far UV CD spectrum of N-62
StAR in 50 µM 82:18 PC/cardiolipin at the indicated pH
values. D, [ ]228 versus pH
derived from the complete spectra in C.
-sheet structures (31). Thus, cardiolipin strongly favors the association of N-62 StAR
with a PC membrane. As the pH is lowered to 2.5 or raised to 5.0, the
CD signal increases, suggesting a less tight association with the
membrane; at pH 3.0-2.5 the
-sheet structures continue to
predominate, but at pH 5.0 there is a slight increase in the
-helical contribution. A plot of [
]228
versus pH shows the pH dependence with a minimum at pH
3.5-4.0 (Fig. 9, C and D), consistent with a
partially open molten globule conformation, as found previously in
aqueous buffers (14).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Institutes of Health (NIH) Grants R01 DK37922 and U54 HD 34449 (to W. L. M.), NIH Grant K01 DK02762 (to H. S. B.), and National Science Foundation Grant MCB 9904597 (to J. D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence may be addressed. E-mail:
wlmlab@itsa.ucsf.edu.
** To whom correspondence may be addressed. E-mail: john_bell@byu.edu.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M100903200
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ABBREVIATIONS |
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The abbreviations used are: StAR, steroidogenic acute regulatory protein; ACTH, adrenocorticotropic hormone; OMM, outer mitochondrial membrane; IMS, intramembranous space; dansyl-PE, dansyl-phosphatidylethanolamine; PC, egg phosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; DPPG, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol; SUV, small unilamellar vesicles; GP, generalized polarization; Chol, cholesterol.
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