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INTRODUCTION |
Vitamin A is an essential micronutrient required for growth,
differentiation, reproduction, and normal vision. The selective movement of retinoids throughout the body is a subject of intense interest due to these pleiotropic effects. Dietary vitamin A leaves the
enterocyte on chylomicrons in the form of retinyl esters and is
delivered to the liver in the chylomicron remnant. Hepatic processing
results in either release of retinol bound to the extracellular retinol-binding protein
(RBP)1 or storage in stellate
cells as retinyl esters. The released retinol is transported to target
tissues bound to RBP where it is internalized and converted to its
active forms. Within cells, cellular retinol-binding proteins (CRBPs)
are thought to facilitate retinoid metabolism by presenting ligand to
specific metabolic enzymes. The precise roles of extracellular and
intracellular retinoid-binding proteins in ligand transport and
targeting are nevertheless not defined.
It has been shown in vitro that retinoids are capable of
partitioning into the hydrophobic phase of membranes and that transfer between lipid vesicles or to intracellular binding proteins can occur
via diffusional processes (1, 2). The degree to which this spontaneous
diffusional movement accounts for retinoid trafficking in a cellular
context is controversial. An alternative theory suggests that the
directed channeling of retinol to membrane lipid or protein
compartments occurs via its specific binding proteins. For example, an
early report that examined the kinetics of retinol transfer from RBP to
liposomes suggested that retinol transfer occurred by collisional
interactions between RBP and membranes (3). In addition, retinol entry
into cells may involve uptake via a specific cell-surface RBP receptor.
A recent study suggests that the RBP receptor and CRBP-I within cells
are able to interact directly, resulting in a channeling of the
hydrophobic ligand from the extracellular RBP to the intracellular
CRBP-I (4).
Extensive studies directed at the functional role of the CRBPs have
shown that particular forms of these binding proteins are also able to
interact with specific metabolic enzymes. This family of binding
proteins includes cellular retinol-binding protein (type I),
distributed widely throughout the body, and cellular retinol-binding
protein type II, restricted to the enterocyte and the neonatal
hepatocyte (reviewed in Refs. 5 and 6). These two binding proteins have
56% sequence identity, they both bind retinol and retinaldehyde with
high affinity, and their crystallographic structures are virtually
superimposable (5, 6). Holo-CRBP-I and -CRBP-II are both able to serve
as substrate for the esterification of retinol by the microsomal enzyme
lecithin retinol acyltransferase (LRAT). Despite these similarities,
apoCRBP-I has been shown to inhibit LRAT, whereas apoCRBP-II does not
(7). The ability of LRAT to differentiate between these structurally
similar proteins and between the apo and holo forms implies that subtle
conformational differences may dictate differential recognition of
these binding proteins by enzymes and further suggests that functional
differences between CRBP-I and CRBP-II may exist. Similar
discrimination between apo and holo-CRBP-I has been observed with the
microsomal NADP-dependent retinol dehydrogenase (8). The
molecular determinants by which enzymes discriminate between binding
proteins are as yet unknown.
Several of the enzymes involved in cellular vitamin A metabolism are
membrane-associated. It is therefore possible that in addition to
interacting with the enzymes themselves, the binding proteins may also
be interacting with a lipid component in the bilayer. However, only
limited information on the role of these proteins in trafficking
retinoids from and/or between membranes is available. Previous studies
have demonstrated that partitioning of retinol from CRBP-I to model
membranes can occur (9, 10). Transfer from CRBP-II to membranes has
also been reported, but kinetic measurements were not made (10). Here,
using changes in intrinsic protein tryptophan fluorescence, we are able
to directly examine and compare the kinetics and mechanism of transfer
of retinol from CRBP-I and CRBP-II to phospholipid membranes. We demonstrate that the two proteins transfer ligand to model membranes at
significantly different rates and via distinctly different mechanisms.
Transfer from CRBP-I may involve and require effective collisional
interactions with membranes, whereas a diffusional process primarily
mediates transfer from CRBP-II. In addition, two assays were employed
to examine directly the interaction of CRBPs with model membranes. In
both systems the two proteins interacted with membranes to markedly
different degrees, and these differences substantiate the disparities
observed in the retinoid transfer assays. The differences in membrane
interaction may help account for the differential recognition of CRBP-I
and CRBP-II by metabolic membrane-bound enzymes and may relate to
differences in the selective trafficking of retinol in the cell.
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EXPERIMENTAL PROCEDURES |
Materials--
All-trans-retinol,
all-trans-retinaldehyde, and cytochrome c (type
VI, horse heart) were from Sigma. Egg phosphatidylcholine (EPC), egg
phosphatidylethanolamine (EPE),
N-(7-nitro-2,1,3-benzoxadiazol-4-yl) (NBD) egg
phosphatidylcholine, brain phospha- tidylserine (PS), bovine heart
cardiolipin (CL), and
N-(5-dimethylaminonaphthalene-1-sulfonyl)-sn-glycero-3-phosphoethanolamine (DPE) were obtained from Avanti Polar Lipids (Alabaster, AL). All other
reagents were of the highest grade available. Rat apoCRBP-I and CRBP-II
were purified using a recombinant expression system as described
previously (11). Holoprotein was prepared by addition of equimolar
ligand (in ethanol, final ethanol <0.01% v/v), and saturation
was monitored by following the increase in retinol fluorescence
(
ex 350 nm).
Vesicle Preparation--
Small unilamellar vesicles (SUVs) were
prepared by sonication and ultracentrifugation as described (12). The
typical SUVs used in these studies were composed of 100% EPC. To
examine the effect of membrane charge, 10 mol % of either PS or CL was
incorporated in place of an equimolar amount of EPC. The SUVs were
prepared in 40 mM Tris-Cl, 100 mM NaCl, pH 7.4. For CL-containing SUVs the buffer included 1 mM EDTA and
for the studies involving changes in ionic strength the vesicles were
prepared in 40 mM Tris-Cl, pH 7.4. For the cytochrome
c displacement studies, the vesicles were composed of 64 mol
% EPC, 10 mol % EPE, 25 mol % CL, and 1 mol % DPE and were prepared
in 20 mM Tris-Cl, 0.1 mM EDTA, pH 7.4. The
concentration of phospholipid was determined by quantitation of total
inorganic phosphate (13), and concentrations of vesicles containing CL
were corrected for the two phosphate groups per CL molecule.
Measurement of Equilibrium Partitioning--
The molar
equilibrium distribution of retinol between CRBPs and EPC SUVs
(Kp) was estimated by quantifying the increase in intrinsic tryptophan fluorescence of the proteins upon release of
ligand to SUVs (14) as shown in Equation 1.
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(Eq. 1)
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where Fo and Fi are
relative fluorescence intensities in the absence or presence of
[SUV]i. Equation 1 can be rewritten as Equation 2.
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(Eq. 2)
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where
F is the relative difference in fluorescence
at a given molar ratio of lipid to protein,
Fmax is the maximal fluorescence intensity
change, and Kp is the slope of the resultant line
(15). Holo-CRBP-I or -CRBP-II (1 µM in 40 mM
Tris-Cl, 100 mM NaCl, pH 7.4) was titrated with increasing
amounts of EPC SUV, and partitioning was monitored by measuring the
increase in tryptophan fluorescence (
ex 290 nm) using an
SLM-8000 spectrofluorometer. Relative fluorescence emission intensities
at 340 nm were used in Equation 2 to calculate Kp values.
Transfer of Retinoids from CRBPs to Phospholipid
Vesicles--
The Kp values for CRBP-I and CRBP-II,
estimated as above, were necessary to determine the minimum amount of
lipid necessary to ensure a
1:1 ratio of donor:acceptor binding
capacity in the retinol transfer assays so that unidirectional transfer rates could be assessed (16, 17). Thus, transfer from CRBP-I was
examined at a lipid:protein molar ratio of
500:1, and transfer from
CRBP-II was examined at a lipid:protein molar ratio of
100:1. By
surpassing the Kp values, all transfer assays were done at lipid levels that ensured distribution of ligand to membranes, and therefore, rate comparisons between proteins are valid.
Transfer of retinol or retinaldehyde from CRBPs to SUVs was monitored
by two different transfer assays. First, a fluorescence resonance
energy transfer assay was used to monitor the transfer of retinol from
CRBP-II to EPC vesicles containing the fluorescent quencher NBD-PC (3.5 mol %). Briefly, 2 µM holo-CRBP-II was mixed with
increasing concentrations of SUVs, and a time-dependent
decrease in retinol fluorescence (excitation 350 nm, emission filter
450 ± 35 nm) was observed upon transfer of retinol to the
NBD-containing SUVs (9). Transfer from holo-CRBP-I to NBD-containing
vesicles was not detectable using this method due to the large inner
filter quenching of retinol fluorescence by NBD at the SUV levels
required for unidirectional transfer. As a result, a second assay was
developed to monitor changes in protein fluorescence upon transfer of
the ligand from protein to membranes.
The protein fluorescence assay makes use of the decrease in intrinsic
protein fluorescence of the CRBPs that occurs upon ligand binding (14).
This quenching of holoprotein fluorescence is relieved when ligand is
released from the binding site (9). Thus, upon mixing holoprotein (1 µM retinol-CRBP-I or 2 µM retinol-CRBP-II) with EPC SUVs, release of ligand from the protein is monitored via a
time-dependent increase in protein fluorescence (excitation 287 nm, emission filter 355 ± 60 nm). For each condition used, a
rate was obtained by averaging the values from
5 separate transfer data sets. An example of a single transfer data set is shown in Fig.
1. The molar excess of acceptor lipid
necessary to ensure unidirectional transfer was determined by the
partition coefficients estimated as described above, and the final
ratios used are indicated in each figure legend.

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Fig. 1.
Retinol transfer from CRBP-I to EPC SUVs
monitored by tryptophan fluorescence. Using a stopped-flow
spectrofluorometer, the mixing of 1 µM holo-CRBP-I with
500 µM EPC:CL (90:10) SUVs results in a
time-dependent increase in tryptophan fluorescence
( ex 287 nm, emission filter 355 ± 60 nm). The data
were obtained as described under "Experimental Procedures." A
representative data set from a single transfer assay is shown.
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For the transfer assays, proteins and lipids were typically in 40 mM Tris-Cl, 100 mM NaCl, pH 7.4, and transfer
was monitored at 25 °C unless noted otherwise. For studies of the
effects of ionic strength, NaCl was added to the SUV and protein
samples prior to mixing. Both the retinol and tryptophan fluorescence assays utilized a Stopped-Flow Spectrofluorimeter DX 17MV (Applied Photophysics Limited, UK) to monitor the time-dependent
changes in fluorescence, and the data were fit to the
Marquant-Levenberg nonlinear regression algorithm using software
provided with the instrument. All curves were well fit by a single
exponential function.
CRBP Interaction with Membranes Estimated by Protection of
Cytochrome c Quenching--
The binding of cytochrome c to
acidic membranes can be monitored using the resonance energy transfer
between a dansyl-labeled lipid and the heme moiety of cytochrome
c (18, 19). The dansyl fluorescence of DPE-SUV is quenched
upon binding of cytochrome c. The ability of CRBPs to bind
membranes was monitored by determining the degree to which these
proteins inhibit this cytochrome c quenching, as described
previously (20). In a final volume of 2 ml, 0-2 µM
retinaldehyde-CRBP-I was added to 12.5 µM SUV
(EPC:EPE:CL:DPE, 64:10:25:1) in 20 mM Tris-Cl, 0.1 mM EDTA, pH 7.4 (retinaldehyde was used as the ligand as it
does not fluoresce and therefore does not interfere with the dansyl
fluorescence spectrum). After a 5-min equilibration, cytochrome
c was added (0.75 µM final), and the mixture
was equilibrated an additional 2 min prior to monitoring fluorescence
emission at 520 nm (excitation, 335 nm; slit width set at 2 nm for
excitation and 16 nm for emission).
Adsorption of CRBP-I and CRBP-II to the Phospholipid/Water
Interface--
Lipids used in the monolayer experiments were prepared
by dissolving a known quantity in chloroform and storing the lipid solution under argon at
20 °C. Interface experiments employed a
KSV 5000 monolayer apparatus enclosed in a humidified cabinet and
7.2-cm circular Teflon adsorption wells equipped with injection ports
and magnetic stirrers (21). The wells were filled with 50 ml of
phosphate-buffered saline, pH 7.0, containing 0.05% EDTA, and the
buffer surface was cleaned by vacuum aspiration. EPC monolayers were
spread at the air/buffer interface to the desired pressure by dropwise
addition of the chloroform/lipid solution from a Hamilton syringe and
were left for 30 min to ensure complete evaporation of the organic
solvent. Known amounts of apoCRBP-I or apoCRBP-II were then injected
into the subphase to yield a final concentration of 6 × 10
3 g/liter (0.4 µM), and the change in
pressure versus time was automatically recorded using a
platinum Wilhelmy plate suspended from an electrobalance. The precision
of pressure measurements was 0.01 mN/m.
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RESULTS |
Equilibrium Partition Coefficients--
In order to examine the
kinetics of unidirectional retinol transfer to phospholipid vesicles,
we first determined the equilibrium partition coefficients for retinol
between CRBP-I or CRBP-II relative to model phospholipid vesicles as
described under "Experimental Procedures." As expected, for both
CRBP-I and CRBP-II, fluorescence emission at 340 nm increased upon
addition of SUV; a representative experiment is shown in Fig.
2. Titrating in increasing concentrations of SUV and transforming the fluorescence data (inset, Fig.
2) resulted in calculated Kp values of 300 ± 50 for CRBP-I and 24 ± 9 for CRBP-II. These apparent partition
coefficients suggest that CRBP-I has a higher retinol binding affinity
than does CRBP-II. Previous analysis of the relative affinities of these two proteins for retinol have yielded conflicting results. Fluorometric titration studies indicated that CRBP-I and CRBP-II have
similar Kd values for retinol (
10 nM)
(22, 23), whereas NMR analyses have suggested that CRBP-I may have as
much as 100-fold greater affinity for retinol than does CRBP-II (11). The present partition data indicate that CRBP-I and CRBP-II differ in
their affinity for retinol by approximately an order of magnitude, in
that 12-fold more SUV is required to obtain equivalent ligand dissociation from CRBP-I relative to CRBP-II.

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Fig. 2.
Equilibrium partitioning of retinol between
CRBPs and phospholipid vesicles. Fluorescence intensity
( ex 290 nm, em 340 nm) of 1 µM holo-CRBP-I (A) or -CRBP-II (B)
in 40 mM Tris, 100 mM NaCl, pH 7.4, in the
presence of increasing concentrations of egg phosphatidylcholine
vesicles. The change in absolute F340 upon
addition of SUV was applied to Equation 2, and the resultant
linearization (inset) allowed for calculation of
Kp as described under "Experimental Procedures."
The data shown are representative of three separate
trials.
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Transfer of Retinol from CRBPs to Membranes--
Transfer of
retinol from CRBP-I and CRBP-II to SUVs was examined using two
different fluorometric assays as described under "Experimental
Procedures." In the first method, transfer of ligand from the protein
to the NBD-SUVs was monitored by the time-dependent decrease in retinol fluorescence caused by NBD quenching. As the Kp for CRBP-I dictated a minimum of 300-fold molar
excess lipid to ensure unidirectional transfer, inner filter quenching of retinol fluorescence by the NBD fluorophore precluded the use of
this assay with holo-CRBP-I. However, holo-CRBP-II did not require such
high lipid:protein ratios, and the NBD-based transfer assay yielded
transfer rates of 0.08 ± 0.01 s
1 using a 100-fold
excess of lipid relative to protein and 0.07 ± 0.01 s
1 using 200-fold excess lipid.
In the second method, transfer of ligand to membranes was monitored by
increases in binding protein tryptophan fluorescence, in the absence of
any additional fluorophore. As the absolute amount of lipid did not
interfere with tryptophan fluorescence measurements, mechanisms and
rates of transfer of retinol from both CRBP-I and CRBP-II could be
compared directly. The results showed that transfer rates for retinol
from CRBP-I to SUVs were, at minimum, 6-fold faster than rates from
CRBP-II (0.46 ± 0.07 s
1 versus 0.08 ± 0.005 s
1, Fig. 3).
Importantly, the results for CRBP-II using this tryptophan fluorescence
assay were virtually identical to those obtained with the retinol-NBD
energy transfer method (at a 100-fold molar excess lipid,
k = 0.08 ± 0.01 s
1, and at 200-fold
molar excess lipid, k = 0.07 ± 0.004 s
1, Fig. 3). Thus, rates of retinol transfer from CRBP-II
were identical when monitored by either the quenching of retinol
fluorescence or the increase in protein fluorescence.

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Fig. 3.
Effect of acceptor membrane concentration on
the rate of transfer of retinol from CRBPs. The rate of retinol
transfer from holo-CRBP-I (1 µM, ) or -CRBP-II (2 µM, ) was determined as a function of the
concentration of acceptor egg phosphatidylcholine vesicles (EPC-SUV) by
monitoring the change in CRBP tryptophan fluorescence as described
under "Experimental Procedures." The average from three separate
experiments ± S.D. is shown except for CRBP-II with 1000 µM SUV in which one experiment is shown. For CRBP-II at
the three lowest SUV levels, the standard deviation was minimal and
therefore not visible at the given scale.
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Effect of Vesicle Concentration and Charge on Retinol Transfer to
Membranes--
The transfer of a hydrophobic ligand from a protein to
a membrane can occur via several different mechanisms. There can be effective collisional interactions between protein and membrane lipids,
resulting in ligand transfer. There can be release of the ligand to the
aqueous milieu, diffusion through the aqueous phase and then
association onto the membrane, and finally there can be a combination
of both collisional and diffusional events. In order to discern between
these transfer mechanisms, the concentration and charge of acceptor
vesicles were varied. Fig. 3 shows the results obtained when a constant
concentration of holoprotein was mixed with increasing concentrations
of EPC-SUV. Increasing the concentration of acceptor membrane did not
alter the rate of transfer of retinol from holo-CRBP-II, whereas an
increase in acceptor lipid increased the transfer rate from
holo-CRBP-I. These results suggest that transfer of retinol from CRBP-I
includes effective collisions between protein and lipid, whereas
transfer from CRBP-II does not appear to involve such interactions.
The potential differences in retinol transfer mechanisms were further
explored by changing the surface charge density of the acceptor
vesicles, since alterations in acceptor membrane properties can only
influence the ligand transfer rate in the case of a collisional mechanism (24, 25). Indeed, increasing the net negative charge on
phospholipid vesicles increased the retinol transfer rate from CRBP-I
but not from CRBP-II (Fig. 4). Transfer
from CRBP-I to vesicles containing 10 mol % phosphatidylserine was
3-fold faster than transfer to neutral vesicles, and transfer to
vesicles containing 10 mol % cardiolipin was 4-fold faster. In
contrast, transfer of retinol from CRBP-II was relatively unaffected by
surface charge density, indicating that transfer from CRBP-II is not
affected by the anionic character of the acceptor membranes. Taken
together, the acceptor concentration and charge data suggest that these homologous binding proteins transfer retinol to model membranes via
distinctly different mechanisms. CRBP-I may utilize collisional interactions with the membrane to effect retinol transfer, whereas retinol transfer from CRBP-II is likely to occur by aqueous diffusion of the dissociated ligand. The large enhancement of retinol transfer rate to acidic vesicles suggests that electrostatic interactions between CRBP-I and membranes may be involved in the retinol transfer mechanism.

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Fig. 4.
Effect of acceptor vesicle charge density on
the rate of transfer of retinol from CRBPs. The rate of transfer
of retinol from holo-CRBP-I (1 µM, hatched
bars) or holo-CRBP-II (2 µM, open bars)
to acceptor vesicles containing 10 mol % of the negatively charged
phospholipids phosphatidylserine (EPC:PS) or cardiolipin (EPC:CL) was
monitored as in Fig. 1. Protein:lipid molar ratios were 1:500 for
CRBP-I and 1:100 for CRBP-II. The average from at least three separate
experiments ± SD is shown. **, p 0.02 for
CRBP-I versus CRBP-II.
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Transfer of Retinaldehyde Versus Retinol from CRBPs to
Membranes--
CRBP-I and CRBP-II can bind both retinol and
retinaldehyde with high affinity. The two proteins have equal affinity
for retinaldehyde (11, 22), but CRBP-I appears to have a greater
affinity for retinol than does CRBP-II (see above and Ref. 11). As both
retinol and retinaldehyde serve as substrates for key metabolic
enzymes, we compared the relative rates of transfer of these ligands to phospholipid membranes. Transfer of retinaldehyde from CRBP-II to
EPC-SUVs was approximately 4-fold faster than transfer of retinol (on
average 0.29 s
1 for retinaldehyde and 0.07 s-1 for retinol, Fig.
5A). Similarly, transfer of
retinaldehyde from CRBP-I was 5-fold faster at the lowest lipid ratio
examined (2.2 and 0.41 s
1, respectively, Fig.
5B). Therefore, under these conditions both proteins
transfer retinaldehyde to EPC membranes at a significantly faster rate
than they transfer retinol. Increasing the concentration of acceptor
membranes increased the rate of retinaldehyde transfer from CRBP-I but
not from CRBP-II, indicating that for both proteins the mechanism of
transfer of retinaldehyde is similar to that of retinol (Fig. 5).

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Fig. 5.
Retinol versus retinaldehyde
transfer from CRBPs to phosphatidylcholine vesicles. The rate of
transfer of ligand from 2 µM retinaldehyde-CRBP-II ( ),
2 µM retinol-CRBP-II ( ) (A) and 1 µM retinaldehyde-CRBP-I ( ), 1 µM
retinol-CRBP-I ( ) (B) was monitored to increasing
concentrations of egg phosphatidylcholine vesicles as in Fig. 1. The
average from three separate experiments ± S.D. is shown. **,
p 0.03 and *, p 0.05 for
retinol versus retinaldehyde.
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Effect of Ionic Strength on Retinol Transfer--
When diffusional
movement through the aqueous phase is involved in the transfer of a
hydrophobic ligand, increases in the ionic strength of the milieu can
decrease its solubility and thereby decrease transfer rates
logarithmically (26). If transfer occurs via effective collisions, such
decreases in rate are not anticipated. Therefore, we examined the
effects of ionic strength on transfer of retinol from the CRBPs by
measuring the rate of transfer in the presence of increasing
concentrations of NaCl (Fig. 6). Retinol transfer rates from CRBP-I increased significantly with an increase in
ionic strength of the buffer. Approximately 85% of the total increase
was reached by 0.5 M NaCl, and the rate at 1 M
NaCl was more than 3-fold greater than that at 0 M NaCl. These results are analogous to those
obtained for the transfer of fatty acid from heart fatty acid-binding
protein to membranes, which is also postulated to occur by collisional
interactions (25, 27, 28). Transfer rates from CRBP-II to membranes
also increased in response to changes in ionic strength, although to a
far lesser extent, with a difference of only 10% between 0 and 1 M NaCl (Fig. 6). Although these results for CRBP-II are not
consistent with the expected decrease in transfer rate for an aqueous
diffusion-mediated process, they nevertheless demonstrate substantial
differences between the two CRBPs. Circular dichroism spectra showed
that the molar ellipticity of the CRBPs did not change between 0.1 M NaCl and 0.5 M NaCl (data not shown), and
therefore gross tertiary unfolding cannot account for the ionic
strength effects seen in Fig. 6.

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Fig. 6.
Effect of ionic strength on retinol transfer
from CRBPs. Transfer of retinol from holo-CRBP-I (1 µM, ) or holo-CRBP-II (2 µM, ) was
measured as a function of NaCl concentration as described under
"Experimental Procedures." Acceptor phospholipid concentrations
were 500 µM for CRBP-I and 200 µM for
CRBP-II. Average rates from four experiments ± S.D. are
shown.
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CRBP Interaction with Membranes Assessed Using Cytochrome c Binding
Assays--
Cytochrome c has been shown to interact with
acidic membranes in vitro under conditions of low ionic
strength (29, 30). We took advantage of this interaction by assessing
whether preincubation with CRBPs modulates the fluorescence energy
transfer between cytochrome c and acidic membranes
containing 1 mol % of the fluorophore DPE as described under
"Experimental Procedures." Retinaldehyde was used as the ligand in
these assays as it does not fluoresce under these spectroscopic
conditions, whereas retinol does. As expected, cytochrome c
addition resulted in a concentration-dependent quenching of
the dansyl fluorescence (Fig.
7A). Fig. 7B
demonstrates that preincubation of the vesicles with increasing amounts
of CRBP-I-retinaldehyde was effective in preventing subsequent
cytochrome c binding. Preincubation with 0.5 µM holo-CRBP-I resulted in a 48% increase in dansyl
fluorescence over that seen in the absence of CRBP-I. In contrast,
preincubation with CRBP-II had no effect on dansyl fluorescence. CRBP-I
thus has a greater affinity for model membranes than does CRBP-II, in
that it can prevent the binding of cytochrome c and the
resultant quenching of dansyl fluorescence, whereas CRBP-II cannot
protect against cytochrome c binding. These data are
consistent with the results from the transfer assays, which suggested a
membrane interaction-dependent ligand transfer mechanism
for CRBP-I. Similar experiments with the apoproteins yielded somewhat
inconsistent results (data not shown), perhaps reflective of subtle
differences in the ability of apo and holo forms of the CRBPs to
interact with membranes.

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Fig. 7.
Effect of CRBPs on cytochrome c
binding to anionic membranes. A, 12.5 µM vesicles containing 1 mol % DPE (EPC:EPE:CL:DPE,
64:10:25:1) were incubated with increasing concentrations of cytochrome
c for 2 min prior to monitoring dansyl emission as under
"Experimental Procedures." B, vesicles were preincubated
in the absence or presence of increasing concentrations of
retinaldehyde-CRBP-I ( ) or retinaldehyde-CRBP-II ( ). After 5 min,
0.75 µM cytochrome c was added and allowed to
incubate for 2 min prior to acquiring the fluorescent spectrum as
described under "Experimental Procedures." Results are expressed as
the percent relative fluorescence intensity, where 100% represents the
relative fluorescence intensity of DPE-SUV in the presence of
cytochrome c alone. Results are the average of four
experiments ± S.D.
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Adsorption of CRBP-I and CRBP-II to the Phospholipid/Water
Interface--
The interfacial adsorption rate and exclusion pressure
for CRBP-I and CRBP-II were determined by injecting these proteins beneath EPC monolayers spread at increasing initial pressures (pi) and determining the initial rate and final
equilibrium change in surface pressure (
P). Following
injection of the CRBPs into the buffer subphase, there was an immediate
exponential increase in interfacial pressure (Fig.
8), indicating adsorption and insertion of CRBP-I molecules into the phospholipid monolayer. Rate constants for
the initial adsorption of the CRBPs, calculated from the slope of the
linear regression of time versus ln(P), decreased
with increasing initial surface pressure (Table
I). At initial pressures up to and
including 15 mN/m, the adsorption rates of CRBP-I were 2-3-fold faster
than CRBP-II; at 20 mN/m the adsorption rates were similar. Equilibrium
binding of CRBP-I to the phospholipid interface, as evidenced by the
final
P, decreased with increasing pi
(Fig. 8, inset). Extrapolation of the pi
P curves to 0 indicated that CRBP-I could no longer
penetrate the surface at pi >28.8 mN/m, whereas
CRBP-II could not penetrate the surface at pi >25.9
mN/m. These results indicate that CRBP-I interacts more strongly with
the EPC monolayer than does CRBP-II. The absolute values of the
exclusion pressures obtained here are lower than those similarly
determined for the human plasma apolipoproteins, which are in the range
of 29-34 mN/m (31-36), indicating that the CRBPs have lower intrinsic
surface activity compared with the apolipoprotein family of
lipid-binding proteins.

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Fig. 8.
Binding of CRBP-I and CRBP-II to the
phospholipid/water interface. CRBP-I (6 mg/liter) was injected
into the subphase beneath EPC monolayers spread at 5-6 mN/m, and the
surface pressure was monitored until it reached a stable plateau.
Inset, determination of the interfacial exclusion pressure
of CRBP-I and CRBP-II. CRBP-I was injected beneath EPC monolayers
spread at 5-25 mN/m, and the final equilibrium change in surface
pressure, P, was plotted against the initial pressure,
pi. Extrapolation of the pi P curves to 0 yielded the exclusion pressure given under
"Results."
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Table I
Rate of adsorption of CRBPs to the phospholipid/water interface
Known amounts of apoCRBP-I or apoCRBP-II were injected into EPC
monolayers spread on a KSV monolayer apparatus at varying initial
pressures (pi). The initial rate of protein
adsorption was determined by noting changes in the interfacial pressure
as described under "Experimental Procedures." ND, not determined.
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 |
DISCUSSION |
Several studies of the intracellular retinol-binding proteins have
suggested that these proteins may play an important regulatory role in
trafficking ligand to microsomal metabolic enzymes (5, 37). Integral to
understanding the molecular basis of retinoid movement, therefore, is
examining these potential interactions between binding proteins and
membrane components. In this study we examined the ability of CRBP-I
and CRBP-II to transfer retinol to phospholipid membranes as a means of
modeling in vivo transfer to membranes and/or membrane-bound
enzymes. By using changes in intrinsic tryptophan fluorescence as well
as retinol fluorescence, we were able to determine the kinetics and
mechanism of release of native ligand from the proteins to model
membranes. The
6-fold faster retinol transfer rates from CRBP-I
compared with CRBP-II does not reflect the estimated 12-fold greater
affinity of CRBP-I for this ligand. As Kd is an
equilibrium function reflecting the ratio of ligand dissociation and
association rates, a discrepancy in magnitude between the relative
equilibrium binding constants and the unidirectional ligand transfer
rates may imply that retinol dissociation from the two proteins is
occurring via different mechanisms and/or that retinol association
rates are an order of magnitude higher for CRBP-II. The kinetic studies
presented here indicate mechanistic differences in retinol transfer
from CRBP-I versus CRBP-II to membranes. It is likely that
the rates observed for ligand transfer from CRBP-II reflect spontaneous dissociation of ligand from the protein, whereas for CRBP-I the mechanism by which retinol leaves the protein appears to involve an
interaction with the acceptor membrane. The fact that the rate of
increase in tryptophan fluorescence upon retinol release from CRBP-II
is identical to the rate of decrease of retinol fluorescence upon
insertion into the NBD-labeled SUVs supports the interpretation of the
transfer rates as a measure of ligand dissociation from the binding pocket.
The present studies show that transfer of retinol from CRBP-I and
CRBP-II to membranes does occur. Moreover, it was found that transfer
rates from CRBP-I are dependent on acceptor membrane concentration,
implying that transfer may occur via protein-membrane "collisions".
These results are in contrast with two previously published studies. An
earlier fluorometric study of retinol transfer rates from CRBP-I to
model membranes found transfer rates from retinol CRBP-I to be
independent of the acceptor vesicle concentration (9). Importantly,
however, the lipid:protein ratios examined in that study ranged from
50:1 to 300:1. As those ratios do not exceed the retinol partition
coefficient between CRBP-I and model membranes (determined here to be
300:1), unidirectional transfer from protein to membranes was not
being examined under those conditions, and evidence for a collisional
mechanism would not be expected. A different study noted that the
fluorescence spectrum of retinol bound to 1 nmol of CRBP-I did not
change in the presence or absence of 25 µg of microsomal protein,
suggesting that transfer of retinol to membranes from CRBP-I does not
occur under those conditions (7). Assuming liver microsomes are
composed of 30% lipid by weight (38) and using 800 as the average
molecular weight of a phospholipid, the conditions employed in this
latter study translate to a lipid:protein molar ratio of 9:1, far below
the partition value and thus below the amount of membrane necessary to
detect release of retinol from CRBP-I.
Retinol transfer from CRBP-II to membranes was independent of acceptor
membrane concentration or charge, suggesting that transfer from CRBP-II
occurs via diffusion of the ligand through the aqueous phase.
Nevertheless, experiments examining the effects of ionic strength on
transfer did not clearly support a diffusional transfer mechanism.
Increases in the ionic strength of the solution would decrease the
aqueous solubility of retinol, and a logarithmic decrease in transfer
rate is expected when that transfer process involves aqueous diffusion.
For CRBP-II, however, a small increase in rate was obtained with
increasing salt. The explanation for this discrepancy is not known at
this point. The changes may be due to slight alterations in the protein
structure upon increasing NaCl (39) at levels undetectable by CD
measurements, which are particularly insensitive to
-sheet
structure. The substantial increase in retinol transfer rate with ionic
strength for CRBP-I argues against a diffusion-mediated mechanism and
may be a combination of protein structural effects and effects on the
membrane surface. For example, increases in ionic strength can effect
both bilayer structure and shield surface charges, two parameters
likely to be important in the collisional mechanism proposed here (27). The large increase in transfer rate from CRBP-I is similar to ionic
strength effects observed for the transfer of fatty acid from heart
fatty acid-binding protein to phospholipid vesicles, which is likely to
occur during collisional interactions between protein and membranes
(25, 27, 28). Overall, these data demonstrate large differences between
the two binding proteins in the modulation of retinol transfer by ionic
strength and therefore support the differences observed for retinol
transfer as a function of vesicle composition and concentration.
For both CRBPs, transfer of retinaldehyde to membranes occurred at a
faster rate than transfer of retinol. Although it is difficult to
correlate the transfer rates obtained in this model system with
physiological processes in an absolute sense, the differences for
CRBP-II may have an impact on retinoid flux through the intestine. In
the enterocyte, CRBP-II obtains retinol from the hydrolysis of dietary
retinyl esters or obtains retinaldehyde from cleavage of the more
abundant
-carotene. CRBP-II-retinaldehyde undergoes reduction to
retinol by a microsomal reductase. The CRBP-II-retinol obtained from
dietary esters or via reduction is esterified by microsomal LRAT.
In vitro determinations demonstrate that LRAT and the
reductase have similar Vmax values in intestinal mucosal microsomes (40). The data in Fig. 5A suggest that
within the cell the reductase may obtain its retinaldehyde substrate from CRBP-II at a greater rate than LRAT obtains its retinol substrate and that despite similar maximal velocities, net flux through the
reductase may therefore be greater. As the product of the reductase
reaction must next be esterified by LRAT, the results suggest that in
the enterocyte the esterification by LRAT, and not the reduction by
reductase, may be rate-determining in processing
-carotene-derived
vitamin A.
In addition to the kinetic evidence suggesting that CRBP-I is able to
interact effectively with lipid vesicles during the process of retinol
transfer, the protection against cytochrome c binding
directly demonstrates that CRBP-I is membrane-interactive. In contrast,
CRBP-II is not able to prevent the subsequent binding of cytochrome
c, indicating that it cannot interact with membranes as
effectively as CRBP-I. These observations are supported by the
monolayer exclusion data that demonstrate that CRBP-I is more surface-active than CRBP-II. Both the rate of adsorption and
interfacial exclusion pressure are greater for CRBP-I than for CRBP-II,
which may provide an explanation for the increased rate of transfer from CRBP-I, as the association of CRBP-I with membranes is both faster
and stronger than the association of CRBP-II. For CRBP-II it appears
that the degree of membrane interaction is insufficient to obtain
regulation of the ligand transfer rate by membrane properties, a
hallmark of collisional ligand transfer.
Results from these model systems cannot only be extended to cellular
lipid components but may also extend to the mechanism of transfer from
these proteins to charged protein components, e.g. enzymes,
within cellular membranes. The data indicate that the molecular
interaction of CRBP-I and CRBP-II with cellular membranes and
membrane-bound enzymes may be markedly different. Transfer of retinol
to membrane enzymes could involve direct protein interaction with the
membrane, direct interaction with the enzyme, or a combination of both.
The fact that holo-CRBP-II can present ligand to microsomal enzymes (7)
but does not effectively interact with model membranes in ligand
transfer suggests that interactions between CRBP-II and membrane-bound
enzymes may be governed by direct protein-protein interactions. In
contrast, the evidence here for collisional transfer of retinoids from
CRBP-I suggests that interaction between CRBP-I and membrane enzymes
could include recognition of a lipid component, allowing for specific
and selective discernment of the binding protein by metabolic enzymes
in particular membrane environments.
The cellular retinol-binding proteins are members of the intracellular
fatty acid-binding superfamily. The structure of these proteins
consists of 10 strands of anti-parallel
-sheet capped by two short
-helices. This family of binding proteins displays flexibility in
the helical cap region, and it has been hypothesized that these helices
represent a portal cap for the ligand binding pocket (41-47). This
laboratory has demonstrated that distinct members of the fatty
acid-binding protein (FABP) family can transfer fatty acid to model
membranes via direct interactions with membranes (reviewed in Ref. 48).
Specifically, the helix-turn-helix domain of intestinal FABP was shown
to be critical for the collisional transfer of fatty acid to membranes,
and mutagenesis studies have implicated the amphipathic
-helix-I of
heart-type FABP as important in dictating electrostatic interactions
with membranes (20, 25). Amphipathic helices are known to be involved
in protein-membrane interaction via their ability to orient proteins at
the polar/nonpolar interface of a membrane (49, 50). Crystallographic
data for CRBP-I show that
-I of CRBP-I is also amphipathic, with
Asn-15, Glu-17, Glu-18, and Arg-21 oriented toward the bulk solvent
phase and Phe-16, Tyr-19, Leu-20, Ala-22, and Leu-23 oriented toward the portal region of the protein (Fig.
9). Neither the second
-helix of
CRBP-I nor either of the helices of CRBP-II demonstrate such
amphipathicity. Thus, by analogy to the FABPs, the amphipathic
-I of
CRBP-I may be involved in the membrane interactions demonstrated here.
Furthermore, monolayer affinity and exclusion pressure are related to
the number and amphipathicity of
-helices. That CRBP-I has a higher
exclusion pressure than CRBP-II may reflect the fact that neither of
the helices in CRBP-II are amphipathic. In addition, the fact that
neither of the CRBPs have exclusion pressures as high as the
apolipoproteins (31, 32, 34-36) may reflect the paucity of the helices
in both the CRBPs. Further studies will be required to clarify the role
of these specific structural domains in the functions of CRBP-I.

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Fig. 9.
Ribbon representation of
-I of holo-CRBP-I. This figure highlights the
-I segment of holo-CRBP-I as determined by Winter et al.
(41) and was prepared using the software package Hyperchem (Autodesk).
Hydrophilic amino acid residues are shown in dark gray and
hydrophobic residues are in light gray.
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