 |
INTRODUCTION |
How a polytopic membrane protein inserts into the membrane and
adopts its native conformation remains one of the major unresolved questions of biology. During membrane assembly, integral membrane proteins must interact with other proteins and with the lipid bilayer
itself, resulting in their proper conformational maturation. The role
phospholipids play in forming a membrane bilayer and a hydrophobic
environment for membrane protein folding and assembly is well accepted
(1, 2); however, the role specific phospholipids play in assisting or
directing folding of membrane proteins has not been extensively
explored. In order to determine the role that individual phospholipids
play in the folding of membrane proteins in Escherichia
coli, we are using the lactose permease (LacY)1 as a model system.
LacY is one of the most intensively studied integral membrane proteins
for which there is both extensive structural information (3, 4) and
monoclonal antibodies (mAbs) directed against several extramembrane
epitopes whose recognition depends on secondary or tertiary structural
organization (5, 6). The organization of extramembrane and
transmembrane domains of LacY is characteristic of the major
facilitator superfamily of transport proteins (7), making results
obtained from studies on LacY applicable to a variety of other
transport proteins.
Employing a novel blotting technique (Eastern-Western) in which
proteins are exposed to phospholipids bound to a solid support during
renaturation from SDS in the standard Western blotting procedure, we
previously presented the following experimental evidence (8, 9) that
the phospholipid phosphatidylethanolamine (PE) acts as a non-protein
molecular chaperone in the folding of LacY: (i) LacY appears to fold
in vivo with the assistance of PE as assessed by recognition
by a conformation-sensitive mAb directed against periplasmic loop P7
(Phe242-Gly254), which is flanged by
transmembrane domains VII and VIII and defines epitope 4B1 (Fig. 1);
(ii) LacY assembled initially in vivo in the presence of PE
either retains native-like conformation of this epitope throughout or
has sufficient "conformational memory" to reform this epitope after
SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis
in the absence of added lipid; (iii) after SDS-PAGE and Western
blotting, no phospholipid can be detected associated with LacY (less
than one molecule of PE per 75 LacY monomers); (iv) LacY initially
assembled in vivo in the absence of PE can form the
native-like conformation of epitope 4B1 during refolding from SDS in
the presence of specifically PE but not phosphatidylglycerol, CL, or
phosphatidylcholine (PG, CL, and PC, respectively); (iv) extensive
denaturation of LacY using urea-SDS eliminates epitope 4B1 in LacY
originally assembled in PE-containing membranes and prevents its
refolding in the presence of PE; (v) the requirement for PE in the
folding of LacY has been demonstrated in an in situ assembly
system using in vitro protein synthesis coupled with
in vitro phospholipid synthesis in the presence of membranes
initially lacking PE; (vi) PE is not required either prior to or
concomitant with membrane insertion but is required in a late step of
conformational maturation to attain native structure. Thus, PE corrects
in vitro a LacY folding defect caused by either in
vivo or in vitro assembly in PE-deficient membranes,
but once this epitope is formed in vivo, PE is not required
to maintain its conformation. Based on these results, we proposed as a
general principle that phospholipids can act as molecular chaperones of
non-protein origin that specifically mediate the folding of
multispanning polytopic membrane proteins, thereby extending the
definition of chaperones to other biomolecules in addition to proteins.
What are the minimum requirements of a phospholipid to facilitate
membrane protein folding? In this study, we establish a specific
requirement for a nonmethylated aminophospholipid of natural chirality
and preference for lamellar organization for refolding of LacY into its
native conformation in the region defined by epitope 4B1. The proper
conformation of epitope 4B1 is required for full function of LacY as a
transporter (5).
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EXPERIMENTAL PROCEDURES |
Materials--
For natural occurring phospholipids, no fatty
acid composition is indicated, although the acyl chains are generally
16 carbons or longer and fully saturated at the
1-sn-position and unsaturated at the
2-sn-position. For all synthetic diacyl-phospholipids, the
indicated fatty acids are the same in both positions of the glycerol
backbone unless otherwise indicated, and the stereochemical configuration of the glycerophosphate backbone and the head groups are
the natural configuration unless otherwise indicated. Unless otherwise
noted, unsaturated fatty acids are of the natural cis configuration. Monounsaturated fatty acids are
9, and diunsaturated fatty acids are
9,12. E. coli PE, bovine heart CL, bovine
brain phosphatidylserine (PS), egg yolk PC, egg yolk lyso-PE, and
1-oleoyl-sn-glycero-3-phosphoethanolamine (lyso-(C18:1)PE)
were supplied by Sigma. E. coli PG was purchased from
Matreya, Inc., and the N-methyl ((C18:1)PMME) and N,N'-dimethyl ((C18:1)PDME) derivatives of PE
came from Avanti Polar Lipids, Inc. rac-(C16:0)PS
(rac referring to the configuration of glycerophosphate in
the backbone) and (C16:0)PS were purchased from Sigma. (C12:0)PE,
(C16:0)PE, (C18:0)PE, (C18:1)PE, (C18:1
9trans)PE, (C18:2)PE, (C16:0)PG, and (C18:1)PG were purchased from Avanti Polar
Lipids; both PG derivatives were rac with respect to the glycerol head group. Cyclic antibiotic Ro09-0198 isolated from Streptovertcillium that specifically interacts with PE has
been described elsewhere (10). PE plasmalogen and
1-palmitoyl,2-oleoyl-sn-glycero-3-phospho-D-serine (P-D-S) were kindly provided by Drs. F. Paltauf (Technische
Universitat Graz, Graz, Austria) and R. Epand (McMaster University,
Hamilton, Ontario, Canada), respectively. Antibodies directed against a continuous epitope formed by periplasmic domain P7 (mAb 4B1) and a
discontinuous epitope consisting of cytosolic domains C8 and C10 (mAb
4B11) were generously provided by Dr. H. R. Kaback (UCLA). Nitrocellulose sheets (pore size 0.45 µm) for immunoblotting were purchased from Schleicher and Schuell. Immobilon-P (polyvinylidene difluoride) sheets were obtained from Millipore Corp. Silica gel thin
layer chromatography plates were purchased from EM Science and Merck.
The ECL kit was obtained from Amersham Pharmacia Biotech.
Bacterial Strains, Plasmids, and Growth Conditions--
Strains
carrying the pss93::kan null allele
cannot synthesize PE and require either growth medium containing 50 mM MgCl2 for viability or a functional
plasmid-borne copy of the pssA gene (plasmid pDD72,
temperature-sensitive for replication) to restore wild type
phospholipid composition (11). Plasmid pT7-5 (12) carries the intact
lacY gene under control of both lacOP and the T7
RNA polymerase promoter (p(T7)) and was used for in vivo
high level expression of LacY. Cells were grown in LB-rich medium, and
ampicillin (100 µg/ml) was included in the growth medium for maintenance of plasmid pT7-5. Since replication of plasmid pDD72 is
temperature-sensitive and mutants lacking PE require MgCl2 for viability, all strains (mutant and wild type) were grown at 30 °C in 50 mM MgCl2. Overexpression of the
lacY gene on plasmid pT7-5 directed by the lacOP
promoter was carried out in mutant and wild type strains grown
exponentially in the presence of 1 mM isopropyl
-thiogalactoside. Strain JA200/pPSD2b carrying a copy of the
psd gene (encodes phosphatidylserine decarboxylase) and a
tetR marker on a multicopy number plasmid was
grown in LB medium containing 25 µg/ml tetracycline (13) and was used
to prepare membranes enriched in phosphatidylserine decarboxylase.
Enzymatic Preparation of D-(C16:0)PS--
E.
coli strain JA200/pPSD2b was used to prepare a Triton X-100
membrane extract containing amplified levels of phosphatidylserine decarboxylase as described previously (13). Phosphatidylserine decarboxylase is specific for the L-isomer
(sn-glycero-3-phosphate isomer) of PS and is not inhibited
by the D-isomer (13). A solution of 1.4 mM
rac-(C16:0)PS and 8.6 mM Triton X-100 in 0.5 ml
of 0.1 M potassium phosphate (pH 7.0) was supplemented with
sufficient crude phosphatidylserine decarboxylase (0.2 units) to
convert all of the L-PS to PE as judged by pilot
experiments using L-(C16:0)PS as substrate. Phospholipids
were extracted from the reaction mixture with chloroform/methanol/water
(1:2:0.5), and PE was separated from D-(C16:0)PS by silica
gel thin layer chromatography as described below.
Membrane preparation and SDS-PAGE--
Inside-out membrane
vesicles, the source of membrane protein in all experiments, were
prepared as described previously (14), using a French press to break
cells at 8,000 p.s.i. in the presence of 20 mM
MgCl2. Protein was determined by the BCA assay according to
the manufacturer's suggestions. Inside-out membrane vesicles were
adjusted to the concentration of the SDS gel-loading buffer of 2.8%
SDS, 10% glycerol, 100 mM dithiothreitol and heated at 37 °C for 15 min and centrifuged to remove any insoluble material prior to being subjected to SDS-PAGE in 12.5% polyacrylamide as described previously (8).
Eastern-Western Blotting--
Phospholipids (0.1-0.2 mg) were
first developed on silica gel thin layer chromatography plates using
chloroform/methanol/water/30% ammonium hydroxide (120:75:6:2, v/v/v/v)
(15) and were blotted to either nitrocellulose or polyvinylidene
difluoride sheets (Eastern blotting) as follows. The plate was dried
thoroughly and then dipped in the blotting solvent of isopropyl
alcohol/0.2% aqueous CaCl2/methanol (40:20:7, v/v/v) for
20-30 s (16). First a nitrocellulose or polyvinylidene difluoride
sheet and then a 3MM glass microfiber filter sheet (Whatman) were
placed over the face-up plate, and the assembly was pressed for 30 s with a heating block at 130-150 °C applied to the filter. The
sheet was then air-dried and used in the Western blotting protocol. The
presence of phospholipids on polyvinylidene difluoride sheets was
verified as described previously (16). In order to make a complex
between phospholipid and antibiotic Ro09-0198, a stock solution (50 mg/ml) in 50% Me2SO, 50% water (v/v) was diluted to 0.1 mg/ml in water. Whatman No. 4 filter paper was first immersed in this
solution and then was sandwiched between the area of phospholipid on
the above solid support and a glass plate for 10 min.
Nitrocellulose or polyvinylidene difluoride sheets with preblotted
phospholipids were immersed in distilled water for 10 s. Developed
SDS-PAGE gels were pre-equilibrated in Western blotting cathode buffer
(see below) for 10 min before electroblotting the proteins to solid
support to reduce but not completely remove SDS, which is important for
the recovery of epitope recognition. The helical structure (secondary
structure) of many membrane proteins is maintained in SDS, but the
helix-helix interactions (tertiary structure) are largely disrupted.
The optimal amount of SDS remaining in the gel must be empirically
determined by varying immersion time and methanol concentration; too
low a methanol concentration (<10%) will not facilitate removal of
SDS from the gel and proteins, and too high a concentration can lead to
aggregation of membrane proteins in the gel matrix. The gel was
overlaid with the area containing LacY lined up with the patch of
phospholipid on the face-up solid support. The use of prestained
SDS-PAGE protein standards (Bio-Rad wide range) is helpful in aligning
the gel and the solid support. Electroblotting for 90 min was carried out using a Milliblot-SDE semidry electroblotting apparatus under the
following conditions: constant voltage power supply with initial current density of 2.5 mA/cm2 of gel area. The following
buffers were used: anode buffer 1 (0.3 M Tris-HCl, 10%
methanol, pH 10.4), anode buffer 2 (25 mM Tris-HCl, 10%
methanol, pH 10.4); and cathode buffer (25 mM Tris-HCl, 40 mM glycine, 20% methanol, pH 9.4). The semidry blotting
system appears to be essential to the success of the process. The
sheets were then blocked overnight with 5% bovine serum albumin in TBS buffer (10 mM Tris-HCl, pH 7.4, 0.9% NaCl) containing
0.05% Nonidet P-40 at 4 °C. The sheets were washed once with
TBS/Nonidet P-40 buffer for 15 min and incubated for 1 h in either
mAb 4B1 or mAb 4B11 at a final dilution of 1:10,000 in the TBS/Nonidet
P-40 buffer. The sheets were washed three times for 15 min each with
TBS buffer/Nonidet P-40 buffer. The washed sheets were incubated for
1 h with peroxidase-labeled anti-mouse antibody from the ECL
detection kit (Amersham Pharmacia Biotech) diluted 1:10,000 in
TBS/Nonidet P-40 buffer followed by three washes with TBS/Nonidet P-40
buffer and once with TBS buffer only. The sheets were incubated for 1 min with ECL detection reagents (Amersham Pharmacia Biotech) mixed 1:1
and immediately exposed to x-ray film for 10 s, 45 s, or 5 min.
Nitrocellulose sheets were stripped of the first antibody and reprobed
with a different antibody as follows. The immunostained and developed
sheets were washed twice with TBS-Nonidet P-40 at room temperature and
submerged in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubated at
50 °C for 30 min in a sealed plastic bag with agitation. The sheets
were washed twice in TBS/Nonidet P-40 and treated with ECL detection
reagents as described above followed by exposure to x-ray film to
ensure full removal of the first antibody. The sheets were washed twice again with TBS/Nonidet P-40 and blocked by immersing in 5% bovine serum albumin in TBS/Nonidet P-40 buffer overnight at 4 °C. Finally, immunodetection was performed with the indicated antibody as described above.
 |
RESULTS |
Effect of Lamellar Versus Non-lamellar Forming Phospholipids on
LacY Refolding--
We previously demonstrated (8) that LacY assembled
in vivo in PE-containing membranes, but not in PE-deficient
membranes, subjected to SDS-PAGE and Western blot analysis is
recognized by a conformation-specific mAb 4B1 directed against the
continuous epitope 4B1 (Fig. 1) within
the periplasmic domain P7 (8). However, if E. coli PE or
total E. coli phospholipids were blotted to the solid
support (Eastern blot) prior to transfer of proteins from the
polyacrylamide gel, the LacY from PE-deficient cells regained
recognition by mAb 4B1 (8). Thus, LacY lacking recognition by mAb 4B1
can be induced to form epitope 4B1 if partially denatured and then
renatured in the presence specifically of PE. We next wished to refine
our understanding of the collective physical properties and individual
chemical properties of phospholipids necessary to assist in the
refolding of LacY improperly assembled in vivo in membranes
lacking PE.

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Fig. 1.
Topological model of LacY.
Rectangular boxes represent the transmembrane
domains joined by hydrophilic domains on the periplasmic or cytoplasmic
side of the inner membrane of E. coli (49). mAb 4B1 has been
shown to recognize a conformation-sensitive, continuous,
PE-dependent epitope (4B1) in the periplasmic hydrophilic
loop P7 (5, 8); the highlighted amino acids are part of this epitope.
mAb 4B11 has been shown (6) to recognize a conformation-sensitive,
discontinuous, PE-independent epitope (4B11) formed by the last two
cytoplasmic loops, C8 and C10 (6, 9). Formation of this epitope is
dependent on membrane insertion of LacY (9).
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As described under "Experimental Procedures," membrane preparations
of LacY expressed in PE-deficient strain AD93 were subjected to
Eastern-Western blotting using PE's with different fatty acid compositions followed by a determination of the degree of proper refolding of LacY as indicated by recognition by mAb 4B1 and the intensity of the immunoreactive LacY band after Western blotting. Fig.
2 illustrates a few examples of the
results from many experiments summarized in Table
I. Recognition by mAb 4B1 was restored
with E. coli-derived PE (primarily C16 and C18 fatty acids
saturated at 1-sn and unsaturated at the 2-sn) or
synthetic PEs with C16 or longer saturated fatty acids at both
positions. PEs with unsaturated fatty acids at both acyl positions or
lyso-PE (any fatty acid composition) did not support proper refolding;
(C12:0)PE was effective but at a reduced level.

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Fig. 2.
Effect of PE derivatives on the refolding of
epitope 4B1. LacY from PE-deficient cells was subjected to
Eastern-Western blotting either without phospholipid (None)
or the indicated phospholipid. E. coli refers to PE derived
from E. coli. In binary mixtures, PE and PG were in an equal
molar ratio. In A, two sets of samples (6 and 12 µg) and
in B one sample (12 µg) of total membrane protein was
subjected to SDS-PAGE and analyzed using mAb 4B1.
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Phospholipids with fully saturated fatty acids normally adopt a bilayer
(lamellar) organization irrespective of hydration, pH, ionic strength,
or divalent cation nature and concentration (17). Among unsaturated
fatty acid-containing phospholipids, only PE adopts the non-bilayer
inverted hexagonal II phase (HII) at physiological
temperatures, pH values, and salt concentrations, while divalent
cations induce the HII phase for CL. PEs with combined long
chain and more extensively unsaturated fatty acids such as PE
plasmalogen adopt the HII phase almost exclusively.
Lysophospholipids generally adopt a micellar organization.
Phospholipids that adopt the bilayer phase exclusively can stabilize
HII-preferring lipids (i.e. unsaturated PE) in
an overall bilayer organization in mixed binary systems (18). The
proportions of bilayer lipid required to achieve this can vary
substantially (usually > 20%), with 30 mol % of PG mixed with
(C18:1)PE being lamellar in its physical state. To distinguish between
the potential contributions of individual phospholipid structure and
the physical state of the lipid, we further investigated whether this
difference between saturated and unsaturated phospholipid in supporting
refolding might be explained by different phase-promoting properties of
these lipids.
Consistent with the physical state of PE being an important determinant
for renaturation, those forms of PE that can readily assume a bilayer
or lamellar structure (19) at room temperature (Fig. 2 and Table I,
saturated fatty acids) supported renaturation, while those derivatives
that tend to form micellar structures or the non-bilayer
HII arrangement did not support renaturation. However,
HII-forming lipids in binary mixtures with a lamellar or
bilayer-forming lipid such as PG supported renaturation of LacY
irrespective of the fatty acid composition of PG. The same was true for
lyso-PE, but a higher proportion of PG was required probably to convert
micellar to lamellar organization. PE plasmalogen, which also highly
favors the HII phase over the lamellar phase, required a
higher mol % of PG to be effective. There may be some advantage of
saturated fatty acids over unsaturated fatty acids, since binary
mixtures with (C18:1)PG were not as effective as with (C16:0)PG in
supporting renaturation by (C18:1) or (C18:2)PE. Although
(18:1
9trans)PE by itself was not as effective as
(C18:0)PE, it did support significant refolding, especially when
compared with (18:1
9cis)PE, which was only effective in
the presence of PG. The basis for the reduced effectiveness of
(C12:0)PE in the absence of PG is not apparent, but the short acyl
chains may not provide a large enough hydrophobic domain to efficiently
accommodate LacY.
Effect of Degree of Amine Methylation on Refolding--
It has
been established that LacY specifically requires a phospholipid with a
free primary amine (such as PE or PS) to support active transport in an
in vitro reconstituted proteoliposome system (29). Thus,
both in vivo (14) and in vitro in the absence of
PE or PS, LacY only carries out facilitated diffusion of substrates. PC, which is not present in E. coli, does not substitute for
PE in supporting active transport, and we have previously shown that PC
will not support proper refolding of LacY originally assembled in
PE-deficient membranes (8). To gain more insight into the requirement
of the phosphoamino group in the refolding of LacY, the renaturation of
partially denatured protein was performed in the presence of
progressively methylated derivatives of PE (Table I). As methylation
increased or the availability of an ionizable proton decreased (PMME to
PDME to PC), the effectiveness of the derivatives in supporting
refolding of LacY decreased, with PC being completely ineffective. The
nature of the fatty acid chains appears not to be a factor, since none
of the derivatives of PC were effective. Although the PMME and PDME
derivatives used were C18:1, both have been determined to favor the
lamellar state (21). Unlike the ability of PG to rescue non-lamellar
derivatives of PE, this lipid could not induce PC to properly refold
LacY even when at 85 mol %. Therefore, either an ionizable amine or a
small amine-containing head group is required for the renaturation process.
Even more surprising was the finding that subjecting LacY originally
assembled in the presence of PE, and therefore containing epitope 4B1,
to the Eastern-Western procedure in the presence of PC resulted in loss
of epitope 4B1 (Fig. 3, lanes
7-9 versus lanes 4-6).
Therefore, PC appears to act as an antagonist in the maintenance of
native LacY structure. This unexpected finding may explain why native
LacY reconstituted in the presence of PC lacks full function (29, 30)
(i.e. only carries out facilitated but not active transport
of substrate). Also shown in Fig. 3 (lanes 1-3) is an
increase in the apparent yield of epitope 4B1 when LacY originally
assembled in the presence of PE is subjected to Eastern-Western
blotting in the presence of E. coli-derived PG/CL. This
result is similar to a previous observation that total E. coli phospholipids were more effective than PE alone (8) although PG/CL cannot in themselves support refolding of LacY assembled in the
absence of PE (Ref. 8 and Fig. 2B, lane 3).

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Fig. 3.
Negative effect of PC on epitope 4B1.
LacY from PE-containing cells was subjected to Eastern-Western blotting
without phospholipid (lanes 4-6), with E. coli-derived PG/CL (in a molar ratio of 20:1; lanes
1-3), or with egg yolk PC (lanes
7-9). In each set of three samples, 3, 6, and 12 µg of
total membrane protein was subjected to SDS-PAGE and analyzed using mAb
4B1.
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Defining the Head Group Specificity of PE--
Consistent with the
above requirement (29, 30) of either PE or PS for reconstitution of
full LacY function in vitro, refolding of LacY (assembled in
membranes lacking PE) in the presence of bovine brain PS led to
recovery of epitope 4B1 (Table II and
Fig. 4B, lanes
3 and 4). Therefore, PS is as effective as PE in
reconstituting epitope 4B1. These results are also consistent with the
lack of active transport mediated by LacY in cells lacking PE (14) but with the ability to actively transport lactose in mutants in which 95%
of the PE has been replaced by PS (31).

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Fig. 4.
Effect of PE-specific antibiotic Ro09-0198 on
the refolding of epitope 4B1. LacY from PE-deficient cells was
subjected to Eastern-Western blotting without phospholipid
(A, lanes 4-6; B,
lanes 1 and 2), with E. coli-derived PE (A, lanes 1-3),
or with bovine brain PS (B, lanes 3 and 4). Ro09-0189 was applied as indicated prior to Western
blotting. In each set of samples, 3, 6, and 12 µg (A) or 6 and 12 µg (B) of the total membrane protein was submitted
to SDS-PAGE followed by analysis using mAb 4B1.
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The importance of the amino group of PE for the proper refolding of
LacY was further tested by shielding the phosphoethanolamine head group
of PE by the peptide antibiotic Ro09-0198, which accommodates the head
group of PE specifically and does not interact with other phospholipids
including PS (10). No refolding of LacY was observed when solid support
was preblotted with PE treated with Ro09-0198 (Fig. 4A,
lanes 1-3 versus lanes
7-9). In a control experiment, the specificity of Ro09-0198
for PE and its lack of interference with antibody detection were
demonstrated by the lack of effect of the antibiotic on PS-assisted
refolding of LacY (Fig. 4B, lanes 5 and 6) and on the detection of LacY assembled in
PE-containing cells (data not shown). Stripping of mAb 4B1 as described
under "Experimental Procedures" and reprobing with mAb 4B11
verified the presence of ample amounts of LacY in all lanes (data not
shown). mAb 4B11 recognizes a discontinuous epitope defined by
cytoplasmic domains C8 and C10 (Fig. 1) with the same efficiency
whether or not assembly occurred in the presence of PE (9).
Effectiveness of PS Analogues on Refolding--
The ability of PS
analogues to promote proper refolding of LacY was investigated (Table
II). Bovine brain PS and L-(C16:0)PS were able to support
refolding, but L-(C18:1)PS was only able to support
refolding in binary mixtures with PG although PS normally adopts a
lamellar organization irrespective of fatty acid composition (17). The
unnatural diastereomers P-D-S and D-PS were
also not able to support renaturation unless in binary mixtures with PG (Fig. 5); rac-PS did support
renaturation but with about 50% of the efficiency when compared with
the natural diastereomer. Although we were not able to investigate the
dependence on fatty acid composition extensively, the lack of
effectiveness of P-D-S appears not to be due to its fatty
acid composition, since it is similar to that of bovine brain PS, which
does support refolding. These differences between diastereomers are
quite surprising as is the difference between the saturated and
unsaturated derivations of PS. These results may be due to subtle
differences in the physical properties between diastereomers and
between PSs with different fatty acid compositions as discussed
below.

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Fig. 5.
Effect of PS derivatives on refolding of
epitope 4B1. LacY from PE-deficient cells (12 µg of the total
membrane protein) was subjected to Eastern-Western blotting without
phospholipid (lane 1) or with
L-(16:0)PS (lane 2),
D-(16:0)PS (lane 3), binary mixtures
of 50% D-(16:0)PS and 50% (C16:0)PG (lane
4), P-D-S (lane 5), or
50% P-D-S and 50% (C16:0)PG (lane
6). Detection was with mAb 4B1.
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 |
DISCUSSION |
The aim of the present study was to explore in more detail the
specific properties of the head group, acyl chains, and physical state
of phospholipids that support proper refolding of LacY. Although we do
not understand the physical and chemical properties of the lipids and
proteins once bound to a solid support in the Eastern-Western blotting
procedure, several clear relationships from our studies can be made
between the requirements to be effective in refolding of LacY and the
physical and chemical properties in solution of phospholipids that
support refolding: (i) a positively charged head group preferably with
a small or ionizable amine; (ii) a fatty acid composition either singly
or in binary mixtures that favors an overall bilayer or lamellar
organization in solution; and (iii) a distinct preference for naturally
occurring diastereomers of the aminolipid in single component systems
or the presence of lamellar forming phospholipids of natural chirality
in binary mixtures with an unnatural diastereomer of the aminolipid.
These relationships clearly extend our previous conclusions supporting a distinct biological specificity for PE in phospholipid-assisted folding of LacY both in vivo and in vitro that
goes well beyond simply providing a nonspecific detergent-like or
two-phase environment for refolding of LacY.
Since the requirement for naturally occurring PE in the refolding of
LacY had been established both in vivo and in
vitro, the finding that unsaturated PE as opposed to fully
saturated PE cannot facilitate refolding was surprising. This finding
raised the question of whether the phase properties of phospholipids were a factor in the refolding process. The refolding effectiveness of
the various PE derivatives singly or in binary mixtures with PG is
consistent with the importance of the overall macrostructure of the
lipid phase and strongly suggests that refolding of LacY is dependent
an overall lamellar macrostructure.
Several reports have documented a preference or specificity for a
particular physical organization of phospholipids. Protein translocation across the E. coli inner membrane lacking PE
could be restored by (C18:1)PE (non-lamellar) but not (C14:0)PE or
(C18:1)PC (both lamellar) (32). (18:1
9cis)PE
(non-lamellar) but not (18:1
9trans)PE (lamellar)
significantly enhanced the respiratory control ratio of
ubiquinol-cytochrome c reductase and ATP-induced membrane
potential of the H+-ATPase (33). The photochemical function
of rhodopsin (34) and the ATPase activity of E. coli SecA
protein (50) are enhanced by non-lamellar PE derivatives. However,
mammalian Ca2+-ATPase activity (35) is stimulated
significantly more by lamellar phase- than HII
phase-forming derivatives of PE. Finally, lipopolysaccharide derivatives that tend to organize in nonbilayer structures were better
facilitators of folding of PhoE protein in situ as it
transits the inner membrane of E. coli (36).
PS, which is only found in trace amounts in wild type E. coli, may present a more complex special case. Unlike PE, PS
contains two chiral centers, one at the 2-sn-position and
the other in the serine moiety. Consequently D-PS and
L-PS are diastereomers and not enantiomers much like the
glycerol-based glycolipids that have multiple chiral centers in the
glycerol and sugar moieties. L-(C18:1)PS did not support
refolding although it presumably favors a lamellar organization and
contains a primary amine. Furthermore, the unnatural diastereomers at
either chiral center did not support refolding, but all derivatives of
PS were effective in binary mixtures with PG. Previous results have
noted different effects of chirality between mixtures versus
single systems. Maximal activation of protein kinase C requires the
synergistic action specifically of PS (natural diastereomer),
Ca2+, and diacylglycerol. This activation displays no
stereoselectivity or strict chemical specificity within the head group
in the absence of diacylglycerol; i.e. P-L-S,
P-D-S, and even PG are equally effective but at a reduced
level in the absence of diacylglycerol (37). 2H and
31P NMR studies have revealed small differences in the head
group conformations of P-L-S and P-D-S (38). It
was suggested that such structural differences might affect packing
interactions and alter the effective area individual lipids occupy in
respective single component bilayers of these diastereomers (39) that
is not apparent in binary mixtures. Finally, the natural
1,2-sn-diastereomer of the glycolipid monoglucosyl
didodecylglycerol has a higher propensity to form the lamellar phase
than the unnatural 2,3-sn-diastereomer (40) that adopts the
HII phase. Moreover, the 1,2-rac mixture of this
glycolipid displayed phase properties very similar to the natural
diastereomer, indicating that mixtures of diastereomers are not simply
additive in their final physical properties. All of these studies
including our results support as biologically important the physical
properties of the phospholipid macrostructure as well as the chemical
properties of individual phospholipid species in biological processes.
The failure of PC to assist proper refolding of LacY assembled in the
absence of PE is not surprising, since PC cannot support active
transport of LacY in an in vitro reconstituted system (29, 30). However, the fact that PC actively denatures or destabilizes epitope 4B1 during refolding of LacY originally assembled in the presence of PE now explains why native LacY does not display full function in PC-containing lipid vesicles. This result should be taken
as a general caution in designing experiments involving membrane-associated proteins in which endogenous phospholipids are
replaced by phospholipids not normally present in vivo.
Still puzzling is why anionic phospholipids in the absence of PE do not
support full function in reconstituted systems even for LacY originally
assembled in the presence of PE (14, 29) and still maintaining epitope
4B1 (Fig. 3, lanes 1-3). Although previous
evidence (8, 9) strongly suggests that retention of epitope 4B1 after
subjecting native LacY to Western blotting is not due to residual PE,
the presence of epitope 4B1, although required, may not be sufficient
to support full LacY function in active transport in the absence of PE.
Our results cannot rule out that PE per se is required for
either maintenance of proper structures of domains other than 4B1 or is
directly involved in net turnover of the permease required for
energy-dependent active transport of substrate. The
molecular basis for this PE requirement might be direct involvement in
the mechanism of coupling of uphill movement of substrate with downhill
proton movement. The ionizable phosphoamino group of PE may be located
in close proximity to functionally important residues such as
Glu269 of transmembrane helix VIII (41) or the salt bridge
(Asp240-Lys319) between transmembrane helices
VII and X (42) and could alter their pKa values,
thus affecting net movement of substrate (4).
There is growing evidence that phospholipids can facilitate the folding
of membrane proteins or proteins that interact with membranes as part
of their biogenesis. CL may selectively influence refolding of
rhodanase as it passes through the mitochondrial membrane without the
aid of protein chaperones (43-45). OmpA, an outer membrane protein of
E. coli, was unfolded in 8 M urea without detergent, and refolding was initiated by rapid dilution of urea in the
presence of PC vesicles that were required for regeneration of its
native structure (46). Recent reports of specific effects of PG on
folding of E. coli periplasmic DegP protein (47) and of the
glycolipid portion of lipopolysaccharide on the assembly of E. coli outer membrane protein PhoE (36, 48) indicate that the
phenomenon of lipid-assisted folding may be widespread and general
among proteins that come in contact with membranes during their
assembly. Heightened awareness of lipid-assisted folding of proteins
hopefully will stimulate other investigators in designing experiments
to investigate this process in many other systems.