(Received for publication, July 25, 1994; and in revised form, October 24, 1994)
From the
Experiments with mutant Escherichia coli cells lacking
phosphatidylethanolamine (PE) as a membrane component (DeChavigny, A.,
Heacock, P. N., and Dowhan, W.(1991) J. Biol. Chem. 266,
5323-5332) were carried out to establish whether or not PE is
necessary for full function of the lac permease in
vivo. The V for active transport of both
lactose (in cells lacking
-galactosidase, lacZ) and the
unhydrolyzable lactose analog,
methyl-
-D-galactopyranoside (TMG), by mutant cells
lacking PE was reduced 5-10-fold relative to cells containing PE,
while the K
for the uptake of both
substrates was the same in both types of cells. The low rate of TMG and
lactose uptake by PE-deficient cells was unaffected by the presence of
a protonophore (uncoupler) and for TMG uptake was on the order of the
greatly reduced rate of uptake in uncoupler-treated cells containing
PE. The rate of entry of lactose into lacZ
derivatives of both types of cells, as a measure of facilitated
diffusion, was nearly the same. The K
for
lactose (lacZ cells) and TMG transport in PE-deficient cells
was unaffected by the presence of an uncoupler which had a small effect
on V
. In PE-containing cells these kinetic
parameters for TMG transport were reduced by an uncoupler to the level
found with PE-deficient cells while an uncoupler reduced lactose uptake
by PE-containing (lacZ) cells to below measureable levels.
Inverted membrane vesicles made from both types of cells could be
loaded with TMG, but energizing TMG-loaded vesicles by ATP only induced
rapid, uphill, permease-dependent efflux of TMG from PE-containing
vesicles. The decrease in apparent active transport activity of cells
with no PE was not due to a change in membrane permeability, to a
reduced
µ
(proton electrochemical
gradient) across the cell membrane, or to a reduced level of
membrane-associated lac permease protein. These results
suggest that in the absence of PE the lac permease cannot
couple substrate uptake to
µ
in
order to effect accumulation of substrate and as a result only carries
out facilitated diffusion.
Transport of -galactosides into Escherichia coli is catalyzed by the product of the lacY gene, the lac permease. In the presence of a proton electrochemical gradient
(
µ
The function, membrane
topology, and assembly of the lac permease has been the
subject of numerous detailed investigations utilizing biochemical,
physical chemical, and molecular genetic
approaches(2, 4) . However, only a limited number of
reports address the role of the native phospholipid environment in the
assembly and function of this transporter. In most studies on the lipid
requirement of transport systems, the carrier has been extracted from
the membrane (5) followed by reconstitution of the transport
system into liposomes of different phospholipid compositions.
Reconstituted lac permease showed a near absolute dependence
of active transport, but not downhill facilitated diffusion, on
proteoliposomes containing the amino-based zwitterionic phospholipids
PE or phosphatidylserine(6, 7) . However, limitations
of this in vitro approach are that differences observed
between the various phospholipids employed might be due to variability
in the incorporation efficiency of the transport protein into
proteoliposomes of different compositions, in the ability of
proteoliposomes to maintain an artificially imposed
µ
, in the ability to form sealed
proteoliposomes, and in proteoliposomal internal volumes. The
extraction and reconstitution procedures can also affect native
conformation of reconstituted proteins. Even mild approaches to
altering phospholipid composition such as fusion of membrane vesicles
and liposomes (8) and nonspecific lipid transfer protein-based
methods (9) have the disadvantage that endogenous lipids
remain present in the membranes and the natural lipid asymmetry of the
membrane may not be maintained. Finally, in all in vitro reconstituted systems influence of dynamic metabolism on
regulation and function is not present or testable. What is generally
lacking in this area are extensive studies of these carriers in
vivo in membranes with systematically controlled variations in
phospholipid composition.
Limited data on the effect of alterations in phospholipid metabolism on the in vivo properties of the lac permease system have been reported. Accumulation of phosphatidylserine to about 20% of the total phospholipid of E. coli in a temperature-sensitive mutant of the psd gene (encoding phosphatidylserine decarboxylase) had little effect on the properties of the endogenous lac permease(10) , which is consistent with the in vitro data(6, 7) . The use of the existing conditional lethal mutants in E. coli phospholipid metabolism is complicated by the necessity of separating the pleiotrophic effects brought about by cell growth arrest or cell death from the specific effects on transport systems. The recent development of a series of mutants in which dramatic changes in phospholipid composition can be made under conditions which allow for cell growth and viability has opened up the possibility of more systematic studies on the role of specific phospholipids and phospholipid composition in cell processes in vivo(9, 11, 12, 13) .
In order to initiate studies on the in vivo role of phospholipids in lac permease assembly and function, we utilize in this report strains carrying the pss93::kan null allele (normally encodes phosphatidylserine synthase) which renders the cell devoid of the zwitterionic amino-based phospholipids phosphatidylserine and PE (14) . Our results support the previously reported in vitro requirement for PE in the functioning of the lac permease particularly in proton-coupled active transport. A preliminary report of this work has appeared(15) .
Transport by IMV was determined as described
previously (23) with minor modifications. For assay, IMV were
diluted into 0.5 ml of 100 mM HEPES/KOH buffer (pH 7.5)
containing 15 mM MgCl, 250 mM sucrose, 20
mM KCl to make a final concentration of 2.5 mg of protein/ml.
The assay mixture was supplemented with 10 mM phosphoenolpyruvate and 0.05 mg/ml phosphoenolpyruvate kinase. The
IMV were incubated for 10 min at room temperature with 0.4 mM radioactive TMG (5 µCi/ml). ATP (3 mM final
concentration) was then added and samples (50 µl) were transferred
to stop solution (0.15 ml of 10 mM HgCl
) at
various times followed immediately by the addition of 1 ml of ice-cold
wash buffer (100 mM HEPES/KOH (pH 7.5) containing 20 mM MgCl
and 0.2 mg of poly-L-lysine (M
= 10, 500)). The resulting flocculant
suspensions were collected on 0.1-µm Gelman Super-100 filters,
washed with 5 ml of ice-cold wash buffer (no polylysine), and dried.
The radioactivity trapped on the filters was counted by liquid
scintillation spectrometry. In control experiments with
P-labeled IMV, it was found that IMV do not pass through
the filters under the above conditions.
where pK is the negative logarithm of the
apparent ionization constant of DMO (approximately 6.32 at 25 °C),
pH
is the extracellular pH, DMO
is the
intracellular concentration of DMO, DMO
is the
extracellular concentration of DMO. The intracellular water volume was
taken as 6.34 µl/mg of cell protein(26) . The
pH is
the difference between pH
and pH
(alkaline
inside). The above method was also used to verify that 50 µM FCCP fully de-energized cells at extracellular pH 5.5 and 150
mM potassium ion.
Figure 1:
Uptake of TMG by
PE-containing and PE-deficient cells. The uptake of TMG (0.1
mM) was determined as a function of time by either strain
AD93/pDD72 (, pss
) or strain AD93
(
, pss), without (A) or with (B)
plasmid pT7-5/lacY as described under
``Experimental Procedures.'' Dependence of the reciprocal of
the initial rate of TMG uptake (V
) on the reciprocal
of the TMG concentration was determined for strain AD93/pDD72 (C) and strain AD93 (D).
Figure 2:
Uptake of lactose by PE-containing and
PE-deficient cells in a lacZ background. A, strain
AD932 (lacZ) harboring either both plasmids pDD72 and
pT7-5/lacY (, pss
) or
plasmid pT7-5/lacY alone (
, pss) was
assayed for its ability to take up lactose (0.4 mM) as a
function of time as described under ``Experimental
Procedures.'' B, strain AD932 either with (
, pss
) or without (
, pss) plasmid
pDD72 was assayed for its ability to take up lactose as a function of
lactose concentration and displayed as in Fig. 1, C and D.
Maintenance of membrane impermeability to lac permease
substrates was verified by measuring ONPG hydrolysis by whole cells.
Cells of strain AD93 (lacY lacZ
) with and without plasmid pDD72 showed
similar high rates of ONPG hydrolysis by internal
-galactosidase.
However, cells of strain AD931 (lacY lacZ
)
showed the same low rate of ONPG hydrolysis either with or without
plasmid pDD72, thus ruling out an increase in membrane permeability
resulting in exit of substrate through passive diffusion as an
explanation of the low rate of active transport and low level of
accumulation by PE-deficient cells.
The addition of the uncoupler
FCCP had no effect on either the low rate of uptake or the level of
accumulation of TMG by PE-deficient cells and was on the order of the
residual FCCP-independent uptake by PE-containing cells (Fig. 3); the level of uptake observed in the presence of FCCP
was significant over the control level in uninduced cells. In
PE-containing cells FCCP reduced the V for TMG
uptake to 8 nmol/min mg of protein (versus 40 nmol/min mg of
protein, Fig. 1C) with no effect on K
while neither V
nor K
were affected by FCCP in PE-deficient cells (data not shown).
Similarly, the kinetics of lactose uptake in PE-deficient lacZ cells was little affected by FCCP (data not shown) which reduced
the V
from 2.1 ± 0.13 to 0.83 ±
0.06 nmol/min mg of protein with no change in K
(0.5 ± 0.1 mM). The steady-state level of lactose
accumulation in PE-deficient cells was also unaffected by FCCP (Fig. 4) but was drastically reduced in PE-containing cells;
cells carrying a plasmid borne copy of lacY
gene were used to rapidly reach a steady-state level of
accumulation. Unlike the measurement of TMG kinetic parameters in the
presence of FCCP, a quantitative determination of these parameters for
lactose uptake in FCCP-treated PE-containing cells (lac Z) was
not possible in the 0.1-2 mM range probably consistent
with the high K
(>10 mM)
characteristic of facilitated diffusion in wild type de-energized
cells(22, 29) ; no similar data is available on the
effect of de-energization on the K
for TMG uptake,
but our data indicates there is no effect on K
.
De-energization of both strains by FCCP under the above conditions was
confirmed by measurement of the chemical gradient of protons across the
membrane with the weak acid DMO at pH 5.5 and 150 mM potassium
ion(30) .
Figure 3:
Effect of FCCP on TMG uptake by
PE-containing and PE-deficient cells. Strain AD93 either with (pss,
,
) or without (pss,
,
) plasmid pDD72 was used to measure uptake
of TMG (0.4 mM) either in the absence (
,
) or
presence (
,
) of 50 µM FCCP as described under
``Experimental Procedures.'' As a control, TMG uptake was
monitored in strain AD93 (
) without prior induction by
IPTG.
Figure 4:
Effect
of FCCP on lactose uptake by PE-containing and PE-deficient cells in a lacZ background. Strain AD932/pT7-5/lacY either
with (pss,
,
) or without (pss,
,
) plasmid pDD72 was used to
measure uptake of lactose (0.4 mM) as a function of time
either in the absence (
,
) or presence (
,
) of
50 µM FCCP as described under ``Experimental
Procedures.''
Some transport systems are driven by the
pH component of the
µ
at an
external pH of 5.5 while at an external pH of 7.5 all transport systems
are driven by the membrane potential component of
µ
(33) . The measurement
of TMG uptake by cells under the conditions of the
pH
determination (i.e. at pH
of 5.5) showed a
similar reduction in TMG uptake by PE-deficient cells (data not shown);
therefore, the potential to utilize
pH in place of the membrane
potential at pH 5.5 did not alter transport in cells lacking PE.
Figure 5:
Downhill lactose transport by
PE-containing and PE-deficient cells. Strain AD93 (lacZ) containing both plasmids pDD72 and
pT7-5/lacY (
,
, pss
) or plasmid pT7-5/lacY alone (
,
, pss) was assayed for its ability to
transport lactose (0.4 mM) in the absence (
,
) or
presence (
,
) of 5 mM TDG as described under
``Experimental Procedures.''
Figure 6:
Downhill lactose transport by
PE-containing and PE-deficient cells in the presence of FCCP. Strain
AD93 (lacZ) containing both plasmids pDD72
and pT7-5/lacY (
,
, pss
) or plasmid pT7-5/lacY alone (
,
, pss) was assayed for its ability to
transport lactose (0.4 mM) in the absence (
,
) or
presence (
,
) of 50 µM FCCP as described under
``Experimental Procedures.''
Figure 7:
ATP-driven efflux of internal TMG from IMV
prepared from PE-containing and PE-deficient cells. IMV were prepared
from strain AD93 harboring either both plasmids pDD72 and
pT7-5/lacY (,
, pss
) or plasmid pT7-5/lacY alone (
, pss) and assayed for TMG efflux from
preloaded vesicles as described under ``Experimental
Procedures.'' ATP (3 mM, final concentration) was added
at the arrow. Valinomycin and nigericin (4 µg each per mg
of membrane protein) were added prior to ATP in one experiment for IMV
prepared from pss
cells
(
).
Figure 8:
Western blots of membranes from
PE-containing and PE-deficient cells. Membranes were prepared from the
cells of strain AD93 (induced with IPTG) either with (pss) or without plasmid pDD72 (pss)
and were subjected to SDS-PAGE (12.5, 25, and 50 µg of protein/lane
from left to right) and to Western blot analysis using anti-lacY permease antibody and
S-labeled Protein A as
described under ``Experimental Procedures.'' The uninduced
lane represents membranes from strain AD93/pDD72 grown in the absence
of IPTG. The major band at 33 kDa represents the lac permease.
Figure 9: Alkali fractionation of extracts from PE-containing and PE-deficient cells. Total French press lysates (T) from the same strains as described in Fig. 8were alkali-fractionated (0.1 N NaOH) into pellet (P) and supernatant (S) fractions as described under ``Experimental Procedures.'' Pellets were also retreated with 0.2 N NaOH and the remaining pellets (P*) examined. The samples were subjected to SDS-PAGE and Western blot analysis as described in Fig. 8. Analyses were carried out also on membranes isolated from French press lysates from strain AD93/pDD72 uninduced with IPTG(-) or strain AD93/pDD72/pT7-5/lacY induced (+) for lac permease expression from the T7 RNA polymerase promoter on plasmid pT7-5/lacY. The major band at 33 kDa represents the lac permease.
Wilson (6, 7) reported the first detailed observations on the phospholipid requirement for activity of the E. coli lac permease establishing that the transporter specifically requires a phospholipid with a free primary amine (such as PE or phosphatidylserine) for active transport (i.e. energy dependent) in reconstituted proteoliposomes. Phosphatidylcholine could not substitute for PE and monomethyl- and dimethyl-PE were progressively less effective. In a similar study Page et al.(34) showed that the phospholipid environment in which the lac permease is reconstituted greatly affects its activity. Binding of substrate (required for both facilitated and active transport) was not affected by the phospholipid environment but reactions involving net turnover of the permease (required for active transport) were dependent on both the nature of the phospholipid head group and the fluidity of the lipid core; again a strong dependence on PE was noted, but these authors could not verify the ability of phosphatidylserine to substitute for PE. We have extended these studies to determine whether a requirement exists for PE in the in vivo functioning of the lac permease by analyzing transport function in a mutant strain with a null allele of the pss gene either with (PE-containing) or without (PE-deficient) a plasmid-borne copy of the pss gene(14) .
Our results demonstrate
that the rate of apparent active transport (V)
of lactose in a lacZ background and its analog TMG in a lacZ
background is reduced at least
5-10-fold in PE-deficient cells relative to cells with a normal
level of PE with no effect of the lipid composition on the K
for uptake. However, the entry of lactose (at
subsaturating concentrations) into lacZ
cells, as an index of facilitated (downhill) diffusion mediated
by the lac permease under fully energized conditions, was not
affected by the absence of PE. The sensitivity of active transport in
PE-containing cells to an uncoupler and the resistance of transport
activity to an uncoupler in PE-deficient cells confirms the
µ
-linked nature of active
transport by lac permease in a wild type lipid environment and
suggests that the carrier may only be able to mediate
µ
-independent facilitated
diffusion in cells lacking PE. The level of uptake of TMG in
PE-deficient cells (with or without FCCP) was on the order of the
residual level of uptake by PE-containing cells in the presence of
uncoupler, again consistent with lack of active transport in
PE-deficient cells. Loading of inverted membrane vesicles with TMG,
also as an indicator of carrier-mediated facilitated diffusion, was
unaffected by the lack of PE, but TMG efflux driven by
µ
, as an indicator of active
transport, only occurred with PE-containing membrane vesicles. The
decreased rate of transport measured under conditions of active
transport (absence of FCCP) in cells containing no PE was not due to
the suppression of lac operon expression, to an increase
proton permeability of the membrane, or to a reduction in the level or
an alteration in subcellular location of the lac permease.
These results indicate that the in vivo function of the lac permease in the active, energy-coupled mode is dependent
on PE as observed in vitro(6, 7, 34) and that the permease in the
absence of PE cannot effectively couple substrate uptake against a
concentration gradient with the cotransport of a proton down a proton
gradient. This conclusion is further supported by the lack of
inhibition by an uncoupler of the low steady-state level of both TMG
and lactose uptake by PE-deficient cells. However, even in the
de-energized state, the apparent K
for lactose
uptake in lacZ PE-deficient cells does not increase
dramatically as has been reported for PE-containing cells (22, 31 and
this work) which suggests a significant difference in the interaction
of the permease with its second substrate, a proton or
µ
; our inability to measure
significant lactose uptake in the presence of FCCP in PE-containing
cells is consistent with a significant change in the kinetic parameters
upon de-energizing of the membrane.
There are several reports
suggesting that other membrane-associated transport proteins show
preference for PE when studied in vitro. PE had a stimulatory
effect on the V but not the K
for transport mediated by the reconstituted branched-chain amino
acid carrier of E. coli(8) . A PE requirement has been
shown for the melibiose transport protein of E. coli(35) as well for the sodium-dependent leucine transport
system in Pseudomonas aeruginosa(36) . A similar
requirement for amino acid carriers has been reported for Bacilli species (37) which, like E. coli and Pseudomonas species, have high levels of PE. The lac permease of E. coli catalyzes active
-galactoside
transport when expressed in the Gram-positive organism Corinebacterium glutamicum which lacks PE(38) .
However, the membranes of this organism are rich in mono- and
diglucosyldiglyceride glycolipids, the former of which shows
stimulatory effects similar to those of PE on the in vitro function of amino acid carriers(8) .
On the basis of
the distribution of glycolipids and PE among Gram-positive and
Gram-negative bacteria, it has been suggested that these two types of
lipids have similar structural functions in membranes(39) .
These glycolipids share a common physical property with PE in their
ability to form the nonbilayer structures referred to as the reversed
hexagonal (H) phase(40) . Cardiolipin in the
presence of divalent metal ions can also form such a phase which has
been postulated as the reason mutants lacking PE require divalent
cations, have increased cardiolipin levels, and have an absolute
requirement for cardiolipin which is not shown by PE-containing cells (14, 41, 42) .
The molecular basis for the
PE requirement of the above solute transporters is unknown. One might
expect that such carriers would be dependent on the membrane
environment first for proper assembly into the phospholipid bilayer and
second for the conformational changes required for translocation of
substrates. The insensitivity of the carrier to uncouplers and the
inability to effect accumulation of substrate in energized PE-deficient
cells might suggest that the carrier is defective in the release of a
proton on the interior of the cell which would be necessary to carry
out active transport coupled to
µ
. The permease in the absence of
PE may be locked in a low K
form, in contrast to
the normal case where the K
is inversely
proportional to the
µ
(22, 29, 31) .
The inability of the permease in the absence of PE to cycle between a
low K
form (protonated) on the outside of the cell
and a high K
form (unprotonated) on the inside of
the cell would explain the lack of active transport with retention of
high affinity for lactose and insensitivity to uncouplers.
The apparent defect in active transport may be related to misassembly of the permease in membranes lacking PE. Little is known about the mechanism or factors which govern the assembly of polytopic membrane proteins, except the observation that cytoplasmically oriented hydrophilic loops of such proteins tend to carry a net positive charge (43) . Preliminary experiments employing the expression of lacY-phoA chimeric fusions indicate that these fusion proteins do not properly orient into membranes lacking PE(44) . Future studies following the assembly of such polytopic protein chimers into membranes with different phospholipid composition may reveal a previously unrecognized dependence on specific phospholipids for both the assembly and function of membrane proteins as was observed in the dependence on anionic phospholipids of protein translocation across the cytoplasmic membrane of E. coli(9, 11, 12, 13) .