From the Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada
Received for publication, June 19, 2002, and in revised form, October 18, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amphitropic proteins are regulated by reversible
membrane interaction. Anionic phospholipids generally promote membrane
binding of such proteins via electrostatics between the negatively
charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT)
that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of
the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not
zwitterionic membranes, increasing protein positive charge and
hydrophobicity. The binding and insertion into lipid vesicles of a
synthetic peptide containing the three glutamates was
pH-dependent with an apparent pKa
that varied with anionic lipid content. Glutamate to glutamine
substitution eliminated the pH dependence of the membrane interaction,
and reduced anionic membrane selectivity of both the peptide and the
whole CT enzyme examined in cells. Thus anionic lipids, working
via surface-localized pH effects, can promote membrane binding by
modifying protein charge and hydrophobicity, and this novel mechanism
contributes to the membrane selectivity of CT in vivo.
Proteins that interact reversibly with cell membrane lipids
usually have selectivity for negatively charged phospholipids (1, 2).
Some of these proteins show specificity for a particular anionic
phospholipid. For example, protein kinase C binds preferentially to
phosphatidylserine (3); MARCKS (4) and proteins with PH domains
(5) bind selectively to various phosphoinositides. However, many
proteins exhibit non-selectivity with respect to the anionic
phospholipid by a simple electrostatic interaction between clusters of
basic residues on the protein and negatively charged lipid head groups
(6). This binding affinity may be regulated by changes in membrane
anionic lipid content, but more often by modification of the charge on
the protein by a mechanism referred to as an "electrostatic switch"
(7). Phosphorylation of a basic patch on such proteins as MARCKS (7),
Src (8), and ARNO (9) neutralizes positive charge.
Alternatively, calcium binding to acidic residues in the C2 domain of
protein kinase C and phospholipase A2 increases positive charge (10).
Hisactophilin exhibits a variation on this theme, in which the protein
charge may be modulated by cytosolic pH change (11). In this work, we
provide an example in which increased anionic lipid composition modulates protein charge and hydrophobicity by influencing its protonation state and thereby increases membrane affinity.
CTP:phosphocholine cytidylyltransferase
(CT)1 catalyzes a key
rate-limiting step in PC synthesis and contributes to maintenance of
cell membrane PC homeostasis (12). When the relative membrane PC
content is altered, CT could respond by recognition of ensuing changes
in the physical features of PC-deficient or PC-overloaded membranes,
including changes in the negative surface charge density (13). Thus,
how CT responds to changes in surface charge has great bearing on the
control of membrane phospholipid compositional homeostasis. There are
three homologous mammalian CT isoforms, A well characterized membrane-binding domain present in all CT isoforms
(domain M) consists of a long amphipathic
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
1, and
2 (12). Most
of the biochemical characterization, including the work in this study,
has been done with CT
. CT
activity is regulated by reversible
membrane binding, which involves both electrostatic and hydrophobic
interactions (12, 13). It binds poorly to pure PC membranes in
vitro, and its binding affinity increases in proportion to the
negative surface charge of the membrane (14, 15). Importantly, its
translocation to membranes in living cells also increases as a function
of anionic lipid content (16-18). Membrane translocation is also
accompanied by dephosphorylation of its C-terminal domain, but this
event is subsequent to membrane binding (19), and the effects of
phosphorylation status on membrane partitioning can be overcome by
raising the anionic lipid content of the membrane (20). Based on
in situ imaging with fluorescent antibodies or with
GFP-tagged CT
, the enzyme is predominantly nuclear in many cells
(reviewed in Ref. 12) and translocates to the nuclear envelope upon
stimulation with exogenous fatty acids (12, 21). However, in other
cells and contexts CT
appears to be cytoplasmic and ER-bound (12, 22-24). The reason for these cell-dependent differences in
CT
localization is unresolved.
-helix (Fig.
1A and Refs. 25-27). Domain M
can be subdivided further into: (i) an N-terminal polybasic region
(subdomain N, residues 237-255), (ii) a central, net negative region
containing three 11-mer repeats (the VEEKS subdomain, residues
256-288), and (iii) a C-terminal aromatic-rich region terminating in a
predicted bend (Fig. 1A). Peptides corresponding to either
the entire domain M, subdomain N, or the VEEKS subdomain bind
selectively to anionic lipids (27). The non-polar face of domain M
helix creates a ~120o wedge containing 18 aliphatic or
aromatic side chains. The polar face is rich in acidic side chains. One
of the interfacial strips is exclusively basic, and the other is a
mixture of acidic and basic residues (Fig. 1, B and
C).
View larger version (29K):
[in a new window]
Fig. 1.
Domain Structure of CT, highlighting the
amphipathic domain M peptides used in this study. A,
domain structure of rat-CT (29). B, subdomain N peptide
NMR structure (25). Only the helix-forming residues 242-268 are
displayed. C, VEEKS-repeat peptide structure is a merge of
the NMR structures of overlapping peptides, Pep-8K (providing residues
256-267) and a 22-mer (providing residues 268-288) (25). Structures
are displayed using RasMol (58). Backbone and hydrophobic side chains
are yellow, basic side chains are violet, acidic
and polar side chains are red. Side chains mutated in the
study are in ball-and-stick representation.
Our goal is to characterize the determinants in domain M responsible for CT selectivity toward anionic lipids. Using a VEEKS subdomain peptide, we previously showed that mutating to alanine the three serines interrupting the nonpolar face increases peptide hydrophobicity and reduces (but does not eliminate) the selectivity for anionic lipids (27). The N-terminal portion of domain M is the most highly conserved region of domain M (28), and has the highest concentration of positive charge. Here we have probed the contribution of these basic amino acids to the electrostatic interaction with anionic phospholipids by en bloc substitution of 5 or 8 interfacial Lys/Arg with glutamine (Fig. 1B). We found that the membrane affinity was progressively reduced upon progressive elimination of peptide positive charge. The electrostatic component of the membrane binding was virtually eliminated upon removal of positive charge from both interfaces flanking the hydrophobic face of the peptide.
CT binding to anionic lipid vesicles is enhanced as the pH is lowered
from 7.4 to 6.3 (15). One hypothesis for this effect is that lowering
the pH protonates the weakly acidic side chains in domain M,
specifically the three interfacial glutamates, thereby neutralizing
their negative charge and enhancing peptide hydrophobicity. We proposed
(25) that the probability of protonation of these glutamates would be
higher at the interface of an anionic membrane versus a
zwitterionic membrane, because of the attraction of protons at the
negative surface (Fig. 2). In this way,
ironically, the positioning of three interfacial glutamic acid side
chains could contribute to the selectivity for anionic phospholipids.
To test this hypothesis we compared the binding characteristics of a
wild-type VEEKS repeat peptide with a mutant analog, in which the
interfacial glutamates were replaced with glutamine (Fig.
1C). We found that the mutant peptide had reduced dependence
on the anionic lipid content for binding, and that binding was
pH-insensitive. These results imply that the interfacial glutamates
contribute to the selectivity for anionic lipid. The same mutations
were generated in the whole enzyme. The glutamine-substituted enzyme
had higher affinity for membranes and, like the mutant peptide, was
less dependent on high membrane surface charge for binding.
|
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- Cell culture materials, restriction enzymes, and PCR primers were from Invitrogen. All other chemicals were reagent grade or better.
Synthesis of Peptides--
All peptides were acetylated and
aminated on their N and C termini. The syntheses of the 33-mer peptides
corresponding to the wild-type subdomain N sequence (residues 236-268)
and the VEEKS repeat sequence (residues 256-288) of rat CT (29)
were described previously (27). All other peptides were synthesized by
Dr. Krystyna Piotrowska at the University of British Columbia Peptide
Service Laboratory on an ABI model 431A synthesizer using Fmoc
chemistry. The peptides were >98% pure after HPLC purification using
a VYDAC C18 column. Their masses, determined by MALDI-mass spectroscopy, were within <2 daltons of their calculated mass, confirming the correct sequences. After lyophilization to remove the
bound trifluoroacetic acid, the peptides were dissolved in water to a
working stock concentration of 0.3-1 mM as confirmed by
comparison to the fluorescence of a tryptophan standard.
Lipids-- Egg PC and egg PG were from Northern Lipids (Vancouver, B.C.) or Avanti (Alabaster, Alabama). Stocks were quantitated (30) and checked for purity by TLC as described (26). Small unilamellar vesicles (SUV) were prepared by sonication as described (27).
Preparation of Buffers with Varying Ionic Strength and pH-- Ionic strength of samples was varied by addition of 1-100 mM phosphate buffer stocks at pH 7.0 and addition of NaCl. Ionic strength was calculated using Equation 1,
![]() |
(Eq. 1) |
Lipid Vesicle Filtration Binding Assay-- SUVs of various compositions were incubated with peptide (10 µM) in phosphate buffer, pH 7.0, at 20 °C. Vesicle-bound peptide was trapped by centrifugation through Microcon-100 filters (Amicon; Beverly, MA) at 3000 × g for 10-30 min until one-half to three-fourths of the original volume had filtered (27). Samples of the filtrate were diluted 2-fold in phosphate and/or NaCl to equalize ionic strength, and adjusted to pH 10 with NaOH. The free peptide concentration was analyzed by derivatization with fluorescamine (0.4 mM, Sigma Chemical) (31). For binding assays with the VEEKS-repeat peptides, samples of the filtrate were diluted 1:1 with methanol, and the peptide concentration was measured via tryptophan fluorescence (excitation, 280 nm; emission, 345 nm) to increase sensitivity by a factor of ~5. For each analysis, standard curves were conducted with known concentrations of the appropriate peptide. A dimensionless partition coefficient (Kx) was calculated using samples where the percent-bound peptide ranged between 16 and 70% unless otherwise stated, using Equation 2,
![]() |
(Eq. 2) |
Fluorescence Assay of Peptide Insertion--
SUVs of various
compositions were incubated with peptide (3 µM) at
20 °C in phosphate buffer as described for at least 5 min prior to
spectral acquisition. Tryptophan fluorescence spectra were acquired as
described previously (27). Tyrosine fluorescence of Pep-5KQ (15 µM) was monitored at 304 nm (excitation, 280 nm). The
increase in peptide fluorescence at 304 nm in the presence of vesicles
was normalized to F304 of peptide in buffer. All spectra were acquired at 20 °C on an SLM 4800C or PTI model QM-1
spectrofluorometer. Spectra were smoothed, and the contribution of the
lipid was subtracted. To calculate Kx values
from fluorescence measurements, the ratio of bound/total peptide was
estimated from the fluorescence measurements by (F Fo)/(Fmax
Fo), where F = fluorescence
increase or blue shift of sample containing peptide + lipid;
Fo = fluorescence value of lipid-free peptide;
Fmax = fluorescence value at saturating lipid contents.
Circular Dichroism--
CD spectra were acquired at 20 °C as
described previously (27). Peptides (30 µM) were mixed
with lipid vesicles or trifluoroethanol for at least 5 min prior to
spectral acquisition. Spectra were smoothed and the contribution of the
buffer and/or lipid was subtracted. The CD values were converted to
mean residue molar ellipticity (; deg cm2
dmol
1), and the percent helix was estimated from
222 nm as described (27).
Construction and Expression of Wild-type and Mutant CTs--
To
generate a mutant CT containing glutamines at codons 257, 268, and
279, we designed the following mutagenic primers: CL1, 5'-cGGATCCaaATCGATAGATCTcatccagaagtggcaggagaagtCCCGGGagttcattggaagt-3'; CL2;
5'-cGGATCCAGATCTATCGATttctcctgcactttctgcacaaattctttcgacttttcctgcacatctttcacttt-3'. The mutagenic nucleotides are in bold and the engineered
restriction sites, BamHI, BglII, ClaI,
and SmaI (uppercase) created silent mutations. Two PCR
reactions were performed using Pfu polymerase with wild-type
rat CT
cDNA (32) inserted into the SalI site of pBSKS
(+) as a template. The CL1 mutagenic primer was paired with the vector
reverse T7 primer, and the CL2 mutagenic primer was paired with the
vector forward T3 primer to generate PCR products of 910 and 430 bp,
respectively. These PCR products were cut with ClaI
and SalI and inserted into the appropriate sites of pBSKS (+). The accuracy of the resulting constructs was confirmed by sequencing. The two PCR fragments were joined at the ClaI
site to generate CT-3EQ. CT-3EQ was moved to the expression vector pAX142 (33) using SalI.
Expression and Membrane Partitioning of CT-WT and CT-3EQ in COS
Cells--
COS-1 cells were cultured and transfected with pAX-142
constructs as described (34) except that the seeding density was 1 × 106 cells/10-cm dish, and the cells were
glycerol-shocked for 2.5 min following rather than preceding treatment
with 100 µM chloroquine for 3 h at 37 °C. Cells
were transfected with 3 µg of pAX142-CT-WT per 10-cm dish for
20 h and 10 µg of pAX142-CT
-3EQ for 60 h to achieve
equivalent expression levels. To enrich cells with oleic acid they were
incubated for 1 h at 37 °C with media containing 1 mM sodium oleate (Sigma Chemical) and 0.5-10 mg
ml
1 BSA (fatty acid free; Calbiochem) to achieve OA/BSA
molar ratios of 133:6.6. The oleate was prepared as a 10× sonicated
stock in phosphate-buffered saline and was co-sonicated with the BSA
prior to addition to cells. At the highest OA:BSA ratio, the viability of the cells after 1 h was >90%.
The transfected cells were harvested with phosphate-buffered saline
containing 2.5 mM EDTA, and homogenized by sonication for
2 × 15 s at 4 °C in 0.4 ml 10 mM Tris, pH
7.4, 1 mM EDTA, 3 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM
dithiothreitol. The protein concentration of the homogenates was ~5
mg ml1 (35). Electrophoresis and Western blotting using
an antibody against the N-terminal 17 amino acids of CT
was as
described (36). After adding K2HPO4 to a final
concentration of 0.2 M, the homogenates were centrifuged at
100,000 × g for 1 h at 4 °C to generate a
particulate and soluble fraction. The particulate fraction was
resonicated in homogenization buffer/0.2 M
K2HPO4 as above. Aliquots of both fractions
were assayed for CT activity for 15 min under optimum conditions of
substrates (15) and lipid activator (200 µM egg PC/oleic
acid (1:1)).
Determination of the Oleic Acid Content of COS Cell Particulate
Fraction--
The lipids in the particulate fractions were extracted
(37) and analyzed for phospholipid phosphorus content (30). Fatty acid,
cholesterol, and triacylglycerol were separated by TLC on precharred
silica H plates (Analtech) using hexane/diethyl ether/acetic acid
(60:40:1). The TLC plates were scanned and densitometry was performed
with Scion-Image software by reference to standards of oleic acid,
cholesterol, and triolein, which were spotted on each TLC plate. Linear
regression analysis of the plots of density versus nmol of
standard lipid between 0-20 nmol gave r = 0.93-0.995. The mol fraction oleic acid was calculated as mol fatty acid/(mol PL + mol cholesterol + mol triacylglycerol + mol fatty acid).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Design of Mutant Peptides--
The contribution of the interfacial
lysines and glutamates to membrane binding were analyzed using peptides
in which the motifs are best represented. The role of the interfacial
basic strip was investigated using peptides corresponding to amino
acids 236-268 of rat CT (29). The membrane interactions of the
wild-type version of this peptide have been previously characterized
(27), and its structure in complex with SDS micelles has been solved (25). It consists of a continuous
-helix between residues 242-268, linked via a ~50o bend to a loosely coiled N terminus
(25). Fig. 1B shows the positioning of amino acids that were
targeted for mutation. In the mutant peptide 5KQ, five basic amino
acids on the right-hand interfacial zone were changed to glutamines:
Arg-245, Lys-248, Lys-252, Lys-259, and Lys-266. These residues are
generally conserved in animal CTs. Assuming protonation of His-241, the
substitutions changed the net charge from +4 to
1. In the mutant
peptide 8KQ, an additional three lysines on the opposite face, Lys-250,
-254, and -261, were changed to glutamine, to generate a peptide with a
net charge of
4. In the wild-type and 8KQ peptide F263 was substituted with tryptophan to facilitate monitoring of fluorescence.
The three interfacial glutamates (Glu-257, Glu-268, Glu-279) were
mutated to glutamine using the VEEKS subdomain peptide (residues 256-288; Fig. 1C). The membrane binding behavior and
secondary structure of the wild-type version of this peptide have been
extensively studied (26, 27). The NMR-derived structure of a 22-mer
peptide containing the second and third VEEKS repeats in complex with SDS micelles is a continuous -helix (25). The 3E to 3Q mutation changes the net charge from
2 to +1. This peptide also contains 6 interfacial basic residues that likely contribute to electrostatic binding.
The substitutions did not alter the propensity of the peptides for
helix formation, as determined by CD in 50% trifluoroethanol, a
solvent that promotes internal H-bonding (data not shown). The helical
contents estimated from the molar ellipticity at 222 nm were similar:
60% for Pep-8K, 73% for Pep-5KQ and 56% for Pep-8KQ; 69% for Pep-3E
and 62% for Pep-3EQ. All peptides were predominantly random coil in
water, except for Pep-8KQ, which adopted mostly -structure in water
(data not shown).
Loss of Interfacial Basic Residues Diminishes Peptide Affinity for
Anionic Membranes by Inhibiting the Electrostatic Attraction--
The
interactions of subdomain N peptides with SUVs were monitored by a
direct vesicle-filtration binding assay, by CD, and by fluorescence
changes. Thus, the binding event can be correlated with conformational
changes and bilayer insertion processes. The progressively weakened
response to PG/PC (1:1) lipid vesicles at 22 mM ionic
strength upon removal of 5 or 8 interfacial lysines is evidenced by all
three methods in the sets of parallel binding curves shown in Fig.
3. Weakened membrane binding (Fig.
3A) is accompanied by reduced -helix formation (Fig.
3B) and by weaker insertion (Fig. 3C). The
bilayer insertion of wild-type peptide and Pep-8KQ was monitored via
the blue shift in the fluorescence (330:350 nm) of a tryptophan located
in the nonpolar face of the amphipathic helix. This change reached a
plateau at a lipid/peptide ratio between 30-50 for the wild-type
peptide. By contrast, no change in the fluorescence of Pep-8KQ was
observed below a lipid/peptide ratio of 100. For Pep-5KQ, which was not
engineered with a tryptophan, we monitored an increase in fluorescence
at 304 nm, indicative of the movement of Tyr-240, the lone fluorophore
in this peptide, into a more hydrophobic environment. A lipid/peptide
ratio of
100 was required for maximum change of the F304
for Pep-5KQ. These data suggest that both strips of interfacial lysines
contribute to peptide binding. In addition they provide the first
evidence for membrane insertion of the lone aromatic residue in the
bend at the N terminus of domain M.
|
We next probed the electrostatic nature of the interaction of WT, 5KQ,
and 8KQ peptides by measuring their partitioning between aqueous and
lipid phases as a function of mole percent anionic lipid and medium
ionic strength. The effect of vesicle anionic lipid content is shown in
Fig. 4A. The binding of the
peptides to pure PC vesicles was too weak to obtain a reliable estimate of their molar partition coefficients, Kx
(sensitivity limit of the assay was Kx 1 × 104). The Kx of the
wild-type peptide increased at least 2 orders of magnitude when the
mol% PG was increased from 10 to 100% (Fig. 4A). The
response to increasing anionic lipid content was progressively muted
upon substitution of the 5 or 8 lysines. This is in keeping with an
elimination of the electrostatic component of the binding. The effect
of ionic strength is shown in Fig. 4B. Using 50% PG vesicles, the Kx for the wild-type peptide was
reduced ~50-fold as the ionic strength was raised from 11 mM to 0.75 M. At the lowest ionic strength,
substitution of 5 or 8 lysines reduced the Kx
values 10- and 15-fold, respectively. The affinity of Pep-5KQ and 8KQ
was lowered an additional ~3-fold by raising the ionic strength to 44 mM. These data show that the ionic strength required to
eliminate the electrostatic component of the binding reaction is
greatly reduced for the peptides missing 5 or 8 of the interfacial lysines.
|
The Apparent pKa for Membrane Binding of the Wild-type
VEEKS-repeat Peptide Is a Function of the Anionic Lipid
Content--
CT binding to PG/PC (2:3) vesicles is induced over a pH
range from ~7.1 to 6.5 (15), suggesting that a protonation event in
domain M drives binding. We explored the effect of vesicle charge
density on the pH-dependent binding of the wild-type
peptide, Pep-3E (Fig. 1C), using tryptophan fluorescence
blue shifts as a measure of membrane binding. Binding to vesicles
containing 5-35 mol% PG was enhanced as the pH was lowered from 7.5 to 4.25 (Fig. 5A). The
protonation state of the PG is not affected over this pH range (38)
thus the effect is due to a change in the protonation state of the
peptide. Increasing the anionic lipid content of the vesicles increases
the differential between the bulk pH and membrane surface pH, as
protons are attracted by the negative membrane surface. This
should result in an increase in the apparent pKa
for binding shown in Equation 3,
|
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
Substitution of Three Interfacial Glutamates with Glutamine
Eliminates the VEEKS Peptide Binding Dependence on pH--
The
interfacial glutamates of the VEEKS peptide provide likely sites for
protonation, as this would neutralize their negative charge and thus
promote pH-dependent lipid interaction. A prediction of
this model is that Pep-3EQ, as a mimic of the protonated form of the
wild-type peptide (Pep-3E), should not require a reduction in pH for
membrane binding to acidic vesicles. The data shown in Fig.
6 validate this prediction. Fluorescence
and CD spectra of Pep-3E in the presence of 30 mol% PG vesicles were
pH-dependent with apparent pKa
values of 5.7 (fluorescence) or 5.9 (CD), whereas the spectra of
Pep-3EQ were nearly equivalent over a pH range of 7.5-4.9. The binding
of Pep-3EQ to 10 and 20% PG vesicles, which was less than 100% under
these conditions, was also pH independent (data not shown). Since there
was no evidence of protonation using conditions where membrane binding
is incomplete, the pH independence of Pep-3EQ is not merely because
enhanced hydrophobicity increases the affinity so as to mask the
effects on binding of protonation at another (non-interfacial) site.
Rather, these data indicate unequivocally that the interfacial
glutamates are the sites of protonation.
|
Substitution of Three Interfacial Glutamates to Glutamine Lowers
the Anionic Lipid Requirement for Peptide Binding--
The elimination
of negative charge upon substitution of glutamate with glutamine should
increase peptide affinity by creating more favorable electrostatic
interactions and by enhancing peptide hydrophobicity. Both should be
reflected by decreased dependence on membrane negative charge density
and decreased sensitivity to medium ionic strength. In Fig.
7 the mol% PG was varied, and vesicle
interactions were probed via tryptophan fluorescence blue shifts. In
110 mM ionic strength medium the wild-type peptide (Pep-3E)
did not interact with vesicles containing <35% PG, whereas Pep-3EQ
interacted with vesicles containing only 10 mol% PG. In fact, the
small blue shift for Pep-3EQ in the presence of 100% PC vesicles was
significant. CD analysis of the peptides using the same conditions
revealed parallel changes in helical content; a lower membrane negative
charge density was required to induce -helical content in the 3EQ
peptide as compared with the wild-type Pep-3E (Fig. 7). Fluorescence
blue shift analyses were used to obtain partition coefficients for
binding of Pep-3E and 3EQ to PC vesicles containing 0, 20, 30, and 50%
PG at 22 mM ionic strength (Fig. 10B). The
results clearly show reduced anionic lipid selectivity and increased
hydrophobicity for the 3EQ peptide.
|
The effect of the E to Q substitutions on peptide hydrophobicity was
also investigated by monitoring the fluorescence blue shift as a
function of medium ionic strength. Fig. 8
shows that the binding of Pep-3EQ to PG/PC (3:7) vesicles is less
sensitive to ionic strength than the wild-type 3E peptide. Increasing
ionic strength decreases the negative surface potential at the
membrane, reducing both electrostatic interactions with peptide and
interfacial changes in apparent pKa (see
Equations 3 and 4 in the legend to Fig. 5). Whereas at 200
mM salt the wild-type peptide fluorescence ratio is the
same as the lipid-free peptide, Pep-3EQ shows significant ionic
strength-independent binding. Thus, a key effect of the substitution of
the 3E to 3Q is an increase in peptide hydrophobicity. Interestingly,
the binding of the wild-type Pep-3E increased at high ionic strength
(>500 mM). This may be due to charge neutralization of the
interfacial glutamates by the sodium counterion.
|
Substitution of three interfacial glutamic acid residues with glutamine
reduces the selectivity for anionic lipid and enhances partitioning
into cell membranes. To test whether the three interfacial glutamates
contribute to anionic lipid selectivity of CT in cells, we engineered
the 3Glu Gln substitution in the CT cDNA and expressed wild-type and mutant protein in COS cells. The wild-type and mutant cDNAs were expressed at very high (100×) levels, effectively
swamping out the endogenous activity. We adapted transfection
conditions to achieve equivalent expression of the mutant and wild-type
CTs (see "Experimental Procedures" and Fig.
9, inset). The specific activity of the mutant CT was equivalent to wild-type enzyme when assayed in the cell homogenate (Fig. 9, inset), suggesting
that the folding of the enzyme was unperturbed by the mutation. To examine the effects of the mutation on membrane partitioning and selectivity for anionic membranes we incubated cells with various ratios of BSA/OA to elevate the anionic lipid content of cellular membranes (Fig. 9). This is a standard protocol for promoting reversible membrane binding of CT, as assessed by sedimentation, digitonin permeabilization, or in situ immunofluorescence
(16, 17, 21, 34, 39). There is evidence that fatty acids can serve as
regulators of CT activity and membrane association in vivo.
CT activity correlates positively with elevated fatty acid content in
lung microsomes during developmental maturation (18) and the fatty acid
content in CT-associated lipidic particles in lung cytosol following
glucocorticoid administration (41). Moreover, up-regulation of fatty
acid synthesis in CHO cells overexpressing a regulator of fatty acid
synthesis genes enhanced CT activity, and inhibition of fatty acid
synthesis in these cells with cerulenin reversed the effect on CT
(21).
|
The oleic acid content of the cell particulate fraction from the BSA/OA-treated cells ranged from 0 to 28 mol% of total lipid (Fig. 9). The distribution of CT-3EQ between soluble and particulate fractions was altered in a manner reminiscent of the membrane affinity changes of the mutant peptide. In control cells CT-WT was distributed ~13% in the membrane fraction. Membrane-associated CT increased gradually with increasing oleic acid content, and then jumped to ~65% of total as the mol% oleic acid increased between 8 and 16 mol%. In contrast, CT-3EQ partitioned ~50% in the membrane fraction of control cells, and increased to 80% in cells containing 17 mol% oleic acid. The data in Fig. 9 are similar to those obtained with wild-type and mutant peptides in Fig. 7. The higher affinity of CT-3EQ for membranes from control cells in Fig. 9 is expected, as cellular membranes contain acidic lipid (typically 20 mol%), unlike the pure PC lipid vesicles that served as the reference lipid for the experiment in Fig. 7.
Several additional experiments confirmed that the increased
partitioning of CT-3EQ into the particulate fraction was the result of
enhanced membrane affinity and not aggregation into an insoluble form.
The partitioning of both CT-WT and CT-3EQ into the soluble fraction was
dependent on the homogenate volume and the medium ionic strength (data
not shown). Particulate CT-3EQ, like CT-WT, was released into the
soluble fraction upon solubilization of the membrane phospholipid with
Triton X-100 (data not shown), implying a membrane association. In all
experiments, the particulate/soluble ratio of CT-3EQ was greater than
that of CT-WT. Together these data suggest that the 3EQ mutation
enhanced membrane partitioning.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we have characterized two strategies used by CT as the membrane negative charge sensors. The first, a strategy commonly used by amphitropic proteins, consists of strips of lysines flanking both sides of the nonpolar face of the amphipathic helix, which provide the electrostatic drive. The second, a novel strategy, is the selective protonation of three interfacial glutamates at the acidic lipid surface, which eliminates charge repulsion and elevates hydrophobicity.
Contribution of the Interfacial Lysines to the Binding--
The
data in Fig. 4, A and B indicate that successive
elimination of the interfacial lysines in the subdomain N peptide
results in elimination of the electrostatic component of binding. That there is a small effect of ionic strength and PG content on the binding
of Pep-8KQ suggests that the two remaining lysines and/or histidine at
the N terminus that were not substituted with glutamine participate in
the charge interaction with membranes. The decrease in the binding of
the Pep-5KQ and 8KQ is the result of the reduction in peptide positive
charge and not an altered binding mechanism, since the binding of the
mutant peptides is also accompanied by -helix formation and
insertion into the bilayer. The Lys
Gln substitutions did not
increase the hydrophobicity of this peptide (Fig. 4B), in
contrast to the Glu
Gln substitutions in the VEEKS peptide.
How much binding energy does each lysine contribute to the
electrostatic interaction? The ion pairing of lysines with negatively charged lipid head groups in 0.1 M salt has been estimated
to contribute 1 kcal/mol based on analysis of the adsorption of simple unfolded polybasic peptides of variable positive charge (42). The free
energies associated with the binding of the 3 subdomain N peptides to
PC/PG (1:1) vesicles at 22 mM ionic strength were calculated from Kx values obtained from
fluorescence measurements shown in Fig. 3C and from the
filtration assay shown in Fig. 4A. The relationship between
the number of peptide lysines and the G is linear for
both assays (Fig. 10A), and
indicates a
G for removal of 8 lysines of 2 kcal/mol
(data derived from the filtration assay) and 2.65 kcal/mol (data
derived from the fluorescence assay). Thus the electrostatic
contribution per lysine at a low ionic strength of 22 mM is
only 0.25 to 0.32 kcal/mol. The
G associated with
charge neutralization of Pep-8K by raising the ionic strength from 0.1 to 1 M is only 1.1 kcal/mol (values derived from the data
in Fig. 4B) or 1.65 kcal/mol (values derived from analogous fluorescence binding analyses, data not shown). Thus, the contribution of each lysine at 100 mM ionic strength is computed to be
only 0.14-0.2 kcal/mol. All these analyses reveal that the
contribution per lysine is much less than the 1 kcal/mol associated
with a purely electrostatic attraction. Because CT peptides are not
only adsorbed but inserted into the bilayer, an energetic cost of
dehydrating the charged lysines decreases the net electrostatic
component (43).
|
Pep-5KQ, 8KQ, and the wild-type VEEKS peptide (Pep-3E) have net charges
of 1,
4, and
2 respectively, yet they demonstrate electrostatic
responses to acidic membranes (Figs. 4, 7, and 8). This could be
explained if peptide secondary structures segregate the acidic from the
basic residues. Moreover, the electrostatic interactions of wild-type
and mutant peptides were maximal at ionic strengths less than 20 mM. This observation suggests that the acidic residues in
the polar face of the amphipathic helix (see Fig. 1, B and
C) do not come into close contact with the negatively
charged bilayer. Otherwise higher ionic strength would serve to mask
their negative, repulsive charge, and the binding would show an optimum
at medium ionic strength (e.g. 100 mM). This is
a feature of the binding of some other membrane surface interacting
peptides containing mixed charges (11, 44). If peptide folding into an
-helix precedes surface attraction and insertion, this would place
strips of basic charge flanking the hydrophobic wedge, and would
position the negatively charged polar face away from the membrane
surface (Fig. 1B). In this model the
-helical conformer
of the peptide would form in solution, and the membrane would serve as
a trap for this conformer. The alternative model, that the peptide is
attracted electrostatically to the surface in an open, unfolded form
and folds into a helix on the surface, seems less likely.
Contributions of Glutamates 257, 268, and 279 to Membrane
Binding--
The Glu Gln substitution in the VEEKS peptide
increased the hydrophobicity of the peptide, as evidenced by increased
affinity for vesicles with low or zero negative charge (Figs. 7 and
10B) and increased ionic strength resistance (Fig. 8). The
increased hydrophobicity of the glutamine-substituted domain M was also apparent when we examined the distribution of CT-WT and CT-3EQ between
membrane and soluble fractions of transfected cells. The increased
partitioning of CT-3EQ into the cellular particulate fraction was a
result of its increased membrane affinity rather than its aggregation
into an insoluble form since CT-3EQ had the same specific activity as
CT-WT, suggesting a native fold. Moreover, the partitioning of CT-3EQ,
like CT-WT, was influenced by the anionic lipid content of the cell
membranes. This would be unlikely if it were an insoluble aggregate.
How much binding energy is associated with the Glu Gln
substitution? The substitution of the three glutamates with glutamine should theoretically increase the interfacial hydrophobicity by 4.3 kcal/mol (45). Protonation of the three glutamates should increase the
interfacial hydrophobicity by 6 kcal/mol (45). The partition
coefficients and derived
G values for the interaction of
peptides 3E and 3EQ with 0-50% PG vesicles clearly show the increase
in the hydrophobic component upon charge neutralization of the three
glutamates (Fig. 10B). Although the binding of Pep-3EQ to
100% PC vesicles yielded a reliable Kx of
(1.6 ± 0.5) × 104 by the filtration binding
assay and (1.9 ± 0.4) × 104 by the fluorescence
binding assay, the binding of Pep-3E to PC vesicles was below the
detection limit of both assays. Thus we cannot directly obtain the
increase in the hydrophobic component of binding. Extrapolation to 0%
PG of the data describing Pep-3E binding in Fig. 10B might
predict a
G associated with the Glu
Gln mutation
of 4-6 kcal/mol. However, we feel the extrapolation is not reliable
since the shape of this line at low mol% anionic lipid is unknown. The
anionic lipid dependence is influenced not only by the electrostatic
attraction of the interfacial lysines, but also by protonation of the glutamates.
These considerations provide an explanation for why glutamates have evolved at interfacial positions on the CT amphipathic helical membrane binding domain. If polar uncharged amino acids such as glutamine occupied these positions, the CT- membrane interaction would be too strongly hydrophobic to allow regulation by small changes in anionic lipid content. If lysines occupied these positions the electrostatic interaction might be too strong at low anionic lipid contents. By contrast, the interfacial glutamates ensure that CT is not bound to cell membranes until the anionic lipid content increases well above the homeostatic norm.
The binding scenario we envision is that domain M, in a transient
-helical conformation, is electrostatically attracted to enriched
anionic lipid surfaces via the basic strip on one interface. The
glutamates on the opposite interface become protonated in the low pH
milieu at the anionic membrane surface (Fig. 2). Protonation eliminates
the charge repulsion and increases the hydrophobicity of the domain,
thus overcoming the barrier preventing insertion of the hydrophobic
face of the amphipathic helix. Once inserted, the glutamic acids remain
protonated during the protein residence time. Thus, the anionic
membrane traps the protonated form. Therefore, there are two factors
that increase the apparent pKa of the
glutamates. The first is the negative surface potential that lowers the
surface pH. The second is the lower dipole moment of the interfacial
zone of the membrane. These two factors may account for the observed apparent pKa values in Fig. 5B.
Lipid interactions driven by protonation of protein acidic side chains
have emerged as a regulatory principle in in vitro studies
with other membrane proteins and peptides, where the lipid interaction
is promoted by lowering the pH. Protonation drives a conformational
change to a molten globule form prior to lipid insertion of such
proteins as colicin A (46), cytochrome c (47), a
trichosanthin toxin (48), and apolipophorin III (49). Low pH at the
surface of anionic membranes was proposed to promote this protonation
event. Other studies with peptides analogs of hemagglutinin fusion
proteins (50, 51) or bacteriorhodopsin membrane-spanning peptides (52)
have identified cases where protonation of glutamates at low bulk pH
promotes amphipathic -helix formation and membrane insertion into
zwitterionic PC vesicles. A correlation between anionic lipid content
and the activity of several amino acid transporters in
Escherichia coli (53) and yeast (54) was postulated to
involve surface potential modulation of the pKa
values of acidic residues on the transporters involved in substrate transport.
A role for anionic lipid in modulating the protonation state of a model membrane-interacting peptide emerged from studies with myristoylated model hexapeptides (55). A tryptophan residue adjacent to the C-terminal residue was inserted into PG/PC (1/1) vesicles at a higher bulk pH than observed for insertion into PC vesicles. This was attributed to protonation of acidic residues in the low interfacial pH at the surface of anionic lipid membranes. Our study has gone a step further in establishing this feature of anionic lipids in vivo by demonstrating that the engineering of mutations at the candidate glutamic acid sites that mimic their protonated form can greatly alter the affinity of an amphitropic protein for membranes in a living cell. Acidic residues are found in proximity to the anionic membrane-binding domains of such amphitropic proteins as Src (8) and MARCKS (4), which interact via a polybasic motif, and Dna A (56) and vinculin (57), which interact via an amphipathic helix. Whether protonation of these residues provides a mechanism for anionic membrane selectivity in these or other proteins remains to be established.
In summary we have identified two membrane negative charge sensors
featured in domain M of CT; the interfacial basic strips contribute an
electrostatic driving force for negatively charged surfaces, whereas
the interfacial glutamates, like the three serines interrupting the
non-polar face (27), reduce the binding affinity for zwitterionic
membranes. The surface of anionic, but not zwitterionic membranes
induces a modification of domain M (protonation of the glutamates),
thereby increasing its net positive charge and hydrophobicity. We
demonstrated that the interfacial glutamates serve as functional determinants of the CT membrane affinity in living cells, and this
suggests that we have unraveled a bona fide mechanism for regulating the lipid selectivity of CT and for maintaining the anionic
phospholipid/PC ratio at its functional optimum in cells.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Heiko Heerklotz for helpful comments on this article.
![]() |
FOOTNOTES |
---|
* This work was supported by Canadian Institute for Health Research Grant 12134.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.
Present address: Dept. of Biochemistry, University of British Columbia.
§ Jointly appointed to the Dept. of Chemistry, Simon Fraser University. To whom correspondence should be addressed. Tel.: 604-291-3709; Fax: 604-291-5583; E-mail: cornell@sfu.ca.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M206072200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CT, CTP:phosphocholine cytidylyltransferase; PC, phosphatidylcholine; PG, phosphatidylglycerol; SUV, small unilamellar vesicles; CD, circular dichroism; BSA, bovine serum albumin; OA, oleic acid; GFP, green fluorescent protein; WT, wild type; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Buckland, A. G., and Wilton, D. C. (1999) Biochim. Biophys. Acta 1483, 199-216 |
2. | Johnson, J. E., and Cornell, R. B. (1999) Mol. Membr. Biol. 16, 217-235[CrossRef][Medline] [Order article via Infotrieve] |
3. | Newton, A. C., and Johnson, J. E. (1998) Biochim. Biophys. Acta 1376, 155-172[Medline] [Order article via Infotrieve] |
4. | Wang, J., Arbuzova, A., Hangyas-Mihalyne, G., and McLaughlin, S. (2001) J. Biol. Chem. 276, 5013-5019 |
5. | Lemon, M. A., and Ferguson, K. M. (2000) Biochem. J. 350, 1-18[CrossRef][Medline] [Order article via Infotrieve] |
6. | Buser, C. A., Kim, J., McLaughlin, S., and Peitzsch, R. M. (1995) Mol. Membr. Biol. 12, 69-75[Medline] [Order article via Infotrieve] |
7. | McLaughlin, S., and Aderem, A. (1995) Trends Biochem. Sci. 20, 272-276[CrossRef][Medline] [Order article via Infotrieve] |
8. | Murray, D., Hermida-Matsumoto, L., Buser, C. A., Tsang, J., Sigal, C. T., Ben-Tal, N., Honig, B., Resh, M. D., and McLaughlin, S. (1998) Biochemistry 37, 2145-2159[CrossRef][Medline] [Order article via Infotrieve] |
9. | Santy, L. C., Frank, S. R., Hatfield, J. C., and Casanova, J. E. (1999) Curr. Biol. 9, 1173-1176[CrossRef][Medline] [Order article via Infotrieve] |
10. | Murray, D., and Honig, B (2002) Mol. Cell 9, 145-154[Medline] [Order article via Infotrieve] |
11. | Hanakam, F., Gerisch, G., Lotz, S., Alt, T., and Seelig, A. (1996) Biochemistry 35, 11036-11044[CrossRef][Medline] [Order article via Infotrieve] |
12. | Cornell, R. B., and Northwood, I. C. (2000) Trends Biochem. Sci. 25, 441-447[CrossRef][Medline] [Order article via Infotrieve] |
13. | Cornell, R. B. (1998) Biochem. Soc. Trans. 26, 539-544[Medline] [Order article via Infotrieve] |
14. | Cornell, R. B. (1991) Biochemistry 30, 5873-5880[Medline] [Order article via Infotrieve] |
15. | Arnold, R. S., and Cornell, R. B. (1996) Biochemistry 35, 9917-9924[CrossRef][Medline] [Order article via Infotrieve] |
16. | Cornell, R. B., and Vance, D. E. (1987) Biochim. Biophys. Acta 919, 26-36[Medline] [Order article via Infotrieve] |
17. |
Wang, Y.,
MacDonald, J. I. S.,
and Kent, C.
(1993)
J. Biol. Chem.
268,
5512-5518 |
18. |
Weinhold, P. A.,
Rounsifer, M. E.,
Williams, S. E.,
Brubaker, P. G.,
and Feldman, D. A.
(1984)
J. Biol. Chem.
259,
10315-10321 |
19. |
Houweling, M.,
Jamil, H.,
Hatch, G. M.,
and Vance, D. E.
(1994)
J. Biol. Chem.
269,
7544-7551 |
20. |
Wang, Y.,
and Kent, C.
(1995)
J. Biol. Chem.
270,
17843-17849 |
21. |
Legace, T. A.,
Storey, M. K.,
and Ridgeway, N. D.
(2000)
J. Biol. Chem.
275,
14367-14374 |
22. |
Lykidis, A.,
Baburina, I.,
and Jackowski, S.
(1999)
J. Biol. Chem.
274,
26992-27001 |
23. |
Delong, C.,
Qin, L.,
and Cui, Z.
(2000)
J. Biol. Chem.
275,
32325-32330 |
24. |
Ridsdale, R.,
Tseu, I.,
Wang, J.,
and Post, M.
(2001)
J. Biol. Chem.
276,
49148-49155 |
25. | Dunne, S. J., Cornell, R. B., Johnson, J. E., Glover, N., and Tracey, A. (1996) Biochemistry 35, 11975-11984[CrossRef][Medline] [Order article via Infotrieve] |
26. | Johnson, J. E., and Cornell, R. B. (1994) Biochemistry 33, 4327-4335[Medline] [Order article via Infotrieve] |
27. | Johnson, J. E., Rao, N. M., Hui, S. W., and Cornell, R. B. (1998) Biochemistry 37, 9509-9519[CrossRef][Medline] [Order article via Infotrieve] |
28. | Friesen, J. A., Liu, M., and Kent, C. (2001) Biochim. Biophys. Acta 1533, 86-93[Medline] [Order article via Infotrieve] |
29. | Kalmar, G. B., Kay, R. J., Lachance, A., Aebersold, R., and Cornell, R. B. (1990) Proc. Natl. Acad. Sci. 87, 6029-6033[Abstract] |
30. |
Bartlett, G. R.
(1959)
J. Biol. Chem.
234,
466-468 |
31. | Castell, J. V., Cervera, M., and Marco, R. (1979) Anal. Biochem. 99, 379-391[Medline] [Order article via Infotrieve] |
32. | MacDonald, J., and Kent, C. (1993) Prot. Express Purif. 4, 1-7[CrossRef][Medline] [Order article via Infotrieve] |
33. | Cornell, R. B., Kalmar, G. B., Kay, R. J., Johnson, M. A., Sanghera, J. S., and Pelech, S. L. (1995) Biochem. J. 310, 699-708[Medline] [Order article via Infotrieve] |
34. |
Walkey, C. J.,
Kalmar, G. B.,
and Cornell, R. B.
(1994)
J. Biol. Chem.
269,
5742-5749 |
35. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
36. | Veitch, D. V., Gilham, D., and Cornell, R. B. (1998) Eur. J. Biochem. 255, 227-234[Abstract] |
37. | Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917 |
38. | Tocanne, J., and Teissie, J. (1990) Biochim. Biophys. Acta 1031, 111-142[Medline] [Order article via Infotrieve] |
39. | Terce, F., Record, M., Tronchere, H., Ribbes, G., and Chap, H. (1992) Biochem. J. 282, 333-338[Medline] [Order article via Infotrieve] |
40. | Evans, R. W., Williams, M. A., and Tinoco, J. (1987) Biochem. J. 245, 455-462[Medline] [Order article via Infotrieve] |
41. | Mallampalli, R., Salome, R., Li, C. H., VanRollins, M., and Hunninghake, G. (1995) J. Cell. Physiol. 162, 410-421[Medline] [Order article via Infotrieve] |
42. | Kim, J., Mosior, M., Chung, L. A., Wu, H., and McLaughlin, S. (1991) Biophys. J. 60, 135-148[Abstract] |
43. | Murray, D., Ben-Tal, N., Honig, B., and McLaughlin, S. (1997) Structure 5, 985-989[Medline] [Order article via Infotrieve] |
44. |
Murray, D.,
McLaughlin, S.,
and Honig, B.
(2001)
J. Biol. Chem.
276,
45153-45159 |
45. | Wimley, W. C., and White, S. H. (1996) Nat. Struct. Biol. 3, 842-848[Medline] [Order article via Infotrieve] |
46. | van der Goot, F. G., Gonzalez-Manas, J. M., Lakey, J. H., and Pattus, F. (1991) Nature 354, 408-410[CrossRef][Medline] [Order article via Infotrieve] |
47. | Pinheiro, T., Elove, G. A., Watts, A., and Roder, H. (1997) Biochemistry 36, 13122-13132[CrossRef][Medline] [Order article via Infotrieve] |
48. | Xia, X-F., and Sui, S-F. (2000) Biochem. J. 349, 835-841[Medline] [Order article via Infotrieve] |
49. | Weers, P. M., Kay, C. M., and Ryan, R. O. (2001) Biochemistry 40, 7754-7760[Medline] [Order article via Infotrieve] |
50. |
Zhelev, D.,
Stoicheva, N.,
Scherrer, P.,
and Needham, D.
(2001)
Biophys. J.
81,
285-304 |
51. | Dubovskii, P. V., Li, H., Arseniev, A. S., and Akasaka, K. (2000) Protein Sci. 9, 786-798[Abstract] |
52. | Hunt, J. F., Rath, P., Rothchild, K. J., and Engelman, D. M. (1997) Biochemistry 36, 15177-15192[CrossRef][Medline] [Order article via Infotrieve] |
53. | Cerbon, J., and Luna, C. H. (1991) Int. J. Biochem. 23, 161-167[Medline] [Order article via Infotrieve] |
54. | Calderon, V., and Cerbon, J. (1992) Biochim. Biophys. Acta 1106, 251-256[Medline] [Order article via Infotrieve] |
55. | Leenhouts, J. M., van den Wijngaard, P. W. J., de Kroon, A. I. P. M., and de Kruijff, B. (1995) FEBS Lett. 370, 189-192[CrossRef][Medline] [Order article via Infotrieve] |
56. | Yamaguchi, Y., Hase, M., Makise, M., Mima, S., Yoshimi, T., Ishikawa, Y., Tsuchiya, T., and Mizushima, T. (1999) Biochem. J. 340, 433-438[CrossRef][Medline] [Order article via Infotrieve] |
57. | Johnson, R. P., Niggli, V., Durrer, P., and Craig, S. W. (1998) Biochemistry 37, 10211-10222[CrossRef][Medline] [Order article via Infotrieve] |
58. | Sayle, R., and Milner-White, E. J. (1995) Trends Biochem. Sci. 20, 374-376[CrossRef][Medline] [Order article via Infotrieve] |
59. | McLaughlin, S. (1977) Curr. Top. Membr. Transport 9, 71-144 |