From the Department of Chemistry and Biochemistry,
University of Bern, Freiestrasse 3, 3012 Bern, Switzerland and
¶ ZLB Bioplasma AG, Wankdorfstrasse 10, 3000 Bern
22, Switzerland
Received for publication, December 8, 2000, and in revised form, February 9, 2001
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ABSTRACT |
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Exposure of Semliki Forest virus 1 to mildly
acidic conditions results in conformational changes of the viral spike
proteins, which in turn leads to a pore formation across its membrane.
The ability to form a pore has been ascribed to the ectodomain of the
Semliki Forest virus (SFV) E1 spike protein. To elucidate whether the
E1 protein per se is sufficient for low
pH-dependent pore formation, we expressed E1 in
Escherichia coli in an inducible manner using the pET11c
expression system. The data obtained clearly showed that the E1 protein
was expressed in the bacterial cell membrane and that exposure of
E. coli expressing the SFV E1 protein to low pH (<6.2)
resulted in a permeability change of the membrane. Thus, we conclude
that the E1 protein of SFV per se is sufficient to promote
pore formation under mildly acidic conditions.
The entry of a virus into a host cell is an essential step in the
chain of events leading to infection. A multitude of viruses use the
endocytotic pathway to access host cells. As a model for the entry of
enveloped animal viruses into cells, the Under mildly acidic conditions (pH 5.8) the spike proteins undergo an
irreversible conformational change that results in the dissociation of
the E1/E2/E3 complex, the formation of an E1 homotrimer and the
exposure of a fusion peptide on the E1 protein (7). This conformational
change also leads to the formation of a pore, which causes an
alteration in the permeability of the virion membrane or of a cell
membrane expressing the spike proteins (10-14). It has been suggested
that this acid-induced pore formation plays a crucial role in the
penetration and uncoating process of SFV (15).
Several experiments have shown that pore formation is dependent on the
ectodomain of the E1 spike protein (14, 16, 17).
It has been speculated that the E1 protein per se would be
sufficient for triggering acid-induced pore formation. So far, all
attempts to express isolated E1 protein on the surface of eukaryotic
cells have failed. The E1 protein was synthesized but retained in the
endoplasmic reticulum, since efficient transport of the glycoproteins
to the plasma membrane requires heterodimerization of E1 with E2/E3
(18). Therefore, to further investigate the role of E1 during pore
formation, we decided to express E1 in the prokaryotic host
Escherichia coli.
Our data clearly demonstrate that the E1 protein, although lacking a
signal sequence, is transported to the plasma membrane of E. coli. Furthermore, we have shown that E1 is capable of modifying the membrane permeability of E. coli in a
pH-dependent manner, leading to pore formation.
Construction of the SFV E1 Expression Plasmid--
DNA
manipulations were performed by the use of standard cloning procedures
(19). Polymerase chain reaction was used to amplify the
SFV1 E1 gene from pSKm-E1'.
This plasmid contains the entire E1 gene and was derived by subcloning
a 1951-bp SpeI-EcoRV fragment from pSP6-SFV4,
which contains the full-length cDNA sequence of SFV (20, 21).
The primers were designed to introduce a start and an additional stop
codon for translation. To facilitate cloning of the E1 gene into the
expression vector pET11c (Stratagene AG, Amsterdam, Netherlands), the oligonucleotides encoded a unique NdeI and
BamHI restriction site at their 5' and 3' end, respectively.
Since the E1 gene contains an intragenic NdeI restriction
site, the 3' primer was designed to introduce a silent point mutation
(A>G) that eliminated this NdeI cutting site.
The 1340-bp E1 polymerase chain reaction fragment was purified
using a polymerase chain reaction purification kit (Qiagen AG, Basel,
Switzerland), digested with the restriction enzymes BamHI and NdeI, and ligated into the pET11c
vector. The ligation mixture was used to transform competent XL-1
blue E. coli cells and the resulting colonies
screened by digestion of the isolated plasmid DNA with the appropriate
restriction enzymes. The plasmid containing the E1 gene (pET11c-E1) was
then used to transform BL21(DE3) E. coli cells.
Construction of the pET11c_E1/23_E2/432 Plasmid--
Most of the
E2 protein sequence was cut out from the plasmid pSp6-SFV4 (21) using
the restriction enzymes BglI and SgrAI. The ends of the resulting fragment of 1262 bp were polished with Klenow
enzyme and mung bean nuclease. The plasmid pMSEH1_E1/23_phoA (Nyfeler
et al.)2 was
digested with PstI and SalI, resulting in a
5371-bp fragment whose ends were blunted, too. This fragment was
ligated with the 1262-bp-long fragment encoding the "E2" protein
(432 amino acids, no signal sequence), and the resulting plasmids were
transformed into XL-1 blue. Plasmids derived from growing
colonies were analyzed for correct orientation of the insert. The
correctly oriented plasmid was designated as pMSEH1_E1/23_E2/432. The
region encoding for the "E1/"E2 fusion protein (1385 bp; 467 amino
acids) was cut out with XbaI and HindIII and
subcloned into a pET11c vector. This newly constructed plasmid
pET11c_E1/23_E2/432 encoded for a fusion protein composed of the 23 N-terminal amino acids of E1, 12 amino acids resulting from the cloning
strategy, and the C-terminal 432 amino acids of the E2 protein.
Growth and Induction of Recombinant Bacteria--
Single
colonies of BL21(DE3) E. coli cells harboring either
pET11c-E1 or pET11c (control) plasmids were grown in M9 medium, pH 7.4, supplemented with 0.2% glucose, 2 mM
MgSO4, and 0.1 mM CaCl2 (22). After
4 h the cultures were split, half of the cultures were induced
with 50 µM
isopropyl-1-thio-
In another set of experiments bacteria harboring the pET11c-E1 plasmid
were grown to an A600 of ~0.1 before
the pH was adjusted to either 7.4, 6.4, or 5.0, respectively, and the
cells induced by addition of 50 µM IPTG. Bacterial growth
was recorded by measuring the A600.
Expression of E1 was tested in bacteria that had been induced with IPTG
for 3.5 h.
Purification of E. coli Membrane--
Cells were pelleted and
resuspended in 0.10 of the original volume of buffer A (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA pH 8, 1 mM Dithiothreitol), disrupted
with a French press, and centrifuged at 12,000 × g for
5 min. The resulting pellet was washed twice with buffer A and
resuspended in 400 µl of buffer B (10 mM Tris-HCl, pH
9.3, 1 mM Localization of the E1 Protein--
To demonstrate that the E1
protein was inserted into the E. coli plasma
membrane, different control experiments were performed. E. coli expressing the E1 protein were mixed with E. coli
harboring a plasmid pTSGH11 encoding the enzyme IICB of the glucose
transporter (23). Then the cell membranes of this E. coli
mixture were isolated and the proteins analyzed by SDS-PAGE and
subsequent immunoblotting using antibodies against E1 and enzyme IICB,
respectively. Alternatively the enzyme IICB was co-expressed in
pET11cE1 harboring cells using the plasmid pAGB421 (24). Cells
containing both plasmids were selected based on the kanamycin (pAGB421)
and ampicillin resistance (pET11cE1). The presence of both plasmids in
the growing colonies was verified by plasmid isolation and subsequent
restriction analysis. Membranes of E. coli containing both
plasmids were isolated and analyzed for the presence of the two
proteins as described above.
Treatment of Purified Membranes with 8 M
Urea--
Urea treatment has been used to distinguish between
peripheral and integral membrane proteins (25, 26) and to solubilize inclusion bodies. Hence, to determine the localization of the E1
protein, urea was added to the purified membrane fraction and to the
cell debris pellet to a final concentration of 8 M. The samples were incubated at room temperature for 45 min and then centrifuged for 18 min at 300,000 × g. The pellets
were washed with 8 M urea, centrifuged as above, and
resuspended in 200 µl of buffer A. The resulting fractions were
dialyzed against 1% SDS, electrophoretically separated, and
immunoblotted as described above.
Electrophoretic Analysis of Proteins--
The proteins of the
purified membranes and of the pellet consisting of cell debris,
respectively, were separated by SDS-PAGE (10 or 20%) under reducing
conditions. Expression of E1 and enzyme IICB was detected by Western
blot analysis using a polyclonal rabbit anti-SFV and rat anti-enzyme
IICB antibody, respectively, followed by horseradish
peroxidase-conjugated anti-rabbit IgG (Bio-Rad AG, Glostrup,
Switzerland) and anti-rat IgG (Dako, Glattbrugg, Denmark).
Visualization occurred by either using a BM chemiluminescence kit
(Roche Diagnostics Ltd., Rotkrenz, Switzerland) or
diaminobenzidine as substrate for the horseradish peroxidase.
Preparation of Spheroplasts--
E. coli BL21DE3
containing the pET11cE1 plasmid were grown in M9 medium. Expression was
induced by addition of 1 mM IPTG. After 2-3 h of induction
cells were harvested, and spheroplasts were prepared as described by
Osborn et al. (27). Conversion of bacteria to spheroplasts
was monitored by phase contrast microscopy and was generally between 95 and 99%.
Labeling of Spheroplasts with Antibodies and FACS
Analysis--
Spheroplasts were fixed with 1% paraformaldehyde
immediately after isolation, washed, incubated with rabbit anti-SFV
antibodies, washed, subsequently incubated with fluorescein
isothiocyanate-labeled anti-rabbit IgG, and finally subjected to
FACS analysis. FACS analysis was performed on a Becton Dickinson flow
cytometer. To test the integrity of the spheroplasts FACS analysis was
performed in the presence of propidium iodide. For controls anti-SFV
antibodies were replaced with a preimmune serum.
Proteinase K Treatment--
E. coli cells expressing
the pET11cE1 plasmid were converted into spheroplasts as described
above. The spheroplasts were washed and resuspended in
phosphate-buffered saline supplemented with 20 mM glucose
and subsequently incubated with 0.1 mg/ml proteinase K for 20 min on
ice either in the presence or absence 0.4% Triton X-100. Control
samples consisted of spheroplasts kept under the same conditions but in
the absence of proteinase K.
Choline Flux Experiment and pH Optimum--
To test changes in
membrane permeability, cells were loaded with
[14C]choline, and the released radioactivity was
measured. Cells were grown and induced as described above, loaded with
[14C]choline chloride (Amersham Pharmacia Biotech,
Dübendorf, Switzerland) at a concentration of 2 µCi/ml
and incubated for 45 min. Cells were then pelleted, washed three times,
and resuspended in M9 medium (pH 7.5 or 5.85). Aliquots of 1 ml were
taken at different time points and cells separated from the supernatant
either by centrifugation through a silicon oil cushion or by filtration through a 0.2-µm Dyna guard filter (Spectrum Laboratories Inc.). Aliquots form supernatants were mixed with a phenylxylylethane-based scintillation fluid (Beckman AG, Zürich, Switzerland), and
radioactivity was measured in a Kontron MR 300
Cells were grown, preloaded, and washed as described above. Cells were
then resuspended in M9 medium at different pH values and choline efflux
measured after 10-min incubation as described previously.
Expression of SFV E1 Protein in E. coli Cells--
Comparison of
the growth of noninduced bacteria harboring either the pET11c or
pET11c-E1 at various pH (5.4-7.2) showed no significant difference.
However, induction with 50 µM IPTG strongly hampered the
growth of bacteria containing the pET11c plasmid, but not bacteria
containing the pET11c-E1. Under mildly acidic condition (pH 5)
proliferation of induced cells containing the pET11c-E1 was strongly
impeded, whereas at pH 6.4 and 7.4, respectively, growth appeared to be
normal (data not shown). These results would be in agreement with a
functional expression of the E1 protein in the E. coli membrane.
Expression of E1 was detected by analyzing whole cell lysates by
SDS-PAGE and subsequent immunoblotting using a polyclonal rabbit
anti-E1 antibody (data not shown). Separation of the bacterial plasma
membrane from other cell components followed by Western blot analysis
revealed that E1 was not solely localized in the membrane but also
associated with cell wall fragments or other protein aggregates (Fig.
1A). To further determine
whether the E1 protein found in the cell membrane fraction is indeed a
membrane-bound protein and does not represent a contamination by
inclusion bodies, we used 8 M urea (25, 26) to treat the
fraction containing the bacterial membrane as well as the "cell
debris" pellet. SDS-PAGE and subsequent Western blot analysis of the
different fractions showed that the E1 protein remained associated with
urea-treated membranes (Fig. 1B, lane MiUp),
whereas the corresponding supernatant fraction revealed no signal for
E1 (lane MiUu). Treatment of the cell debris pellet
with 8 M urea demonstrated that a significant amount of E1
protein was most probably localized intracellularly in inclusion bodies
or was associated with cell wall components (Fig. 1B,
lane PiUu). These results were further strengthened by
demonstrating the co-localization of the E1 protein and the enzyme IICB
in isolated membranes obtained from either a mixture of E. coli expressing E1 and enzyme IICB, respectively, or membranes isolated from E. coli that expressed both proteins
simultaneously (Fig. 1, C and D).
Incorporation of the E1 protein into the plasma membrane of E. coli was further assessed by FACS analysis of spheroplasts generated from E. coli containing the pET11c-E1 plasmid that
were labeled with rabbit anti-SFV and fluorescein
isothiocyanate-anti-rabbit antibodies. FACS analysis in the presence of
propidium iodide showed that the membranes of the spheroplasts were
intact and non leaky (Fig. 2,
A2 and B2). Hence,
the positive signal depicted in Fig. 2, B1 proves that the
E1 protein faces the extracellular space of the spheroplasts. To
further analyze the orientation of the protein in the membrane,
spheroplasts were exposed either in the presence or absence of Triton
X-100 to proteinase K and the remaining membrane-associated protein
fragments analyzed by SDS-PAGE and Western blot using anti-SFV
antibodies. The results (Fig. 3) clearly
show that a residual fragment of 3 kDa (lanes 2 and
3) was unaffected by the digestion. The size of the fragment and its reactivity with antibodies strongly suggest that it is the
anchoring region (25 amino acids) of the E1 protein. Hence, the E1
protein was correctly oriented in the membrane.
Modification of the E. coli Membrane Permeability at Mildly Acidic
pH--
It has been shown previously that under acidic conditions,
virus spike proteins can alter the host cell membrane permeability by
pore formation (11). Several lines of evidence lead to the assumption
that the SFV E1 protein is responsible for this process (14, 20). To
test this hypothesis we have performed efflux experiments, as described
under "Materials and Methods," by using E. coli
containing either the pET11c-E1, or as controls the pET11c_E1/23_E2/432 or pET11c plasmids, respectively. At neutral pH, the
[14C]choline efflux of E. coli cells
expressing the E1 protein remained unchanged compared with controls. As
depicted in Fig. 4, lowering the pH
(pH = 5.85) resulted in an increase in choline release within the
first 10 min in E. coli expressing E1. In contrast E. coli exposed to mildly acidic pH and harboring either the fusion protein E1/E2 or the pET11c plasmid only showed no or only a marginal increase in choline release compared with pH 7.5.
To test the pH influence on choline efflux, preloaded cells expressing
the E1 protein were resuspended in medium ranging from pH 4 to 7. After
10 min of incubation, choline release was measured. As shown in Fig.
5, choline efflux in E1-expressing cells
was strongly dependent on the pH of the extracellular medium, starting at a pH < 6.2 and reaching a maximum efflux rate at a pH of
~5.2.
The entry of many enveloped animal viruses into cells is mediated
by conformational changes of the viral envelope proteins. These changes
are triggered by binding of the virion to the receptor and/or by low
pH, e.g. within the endosome, leading to fusion of the viral
with the endosomal membrane. This membrane fusion is essential for a
successful infection. In the case of the Semliki Forest virus this
membrane fusion in the acidic milieu of the endosome is catalyzed by
the envelope spike proteins (15). Previous findings indicate that these
spike proteins may be responsible not only for membrane fusion but also
for pore formation across the viral envelope (13, 14) and the membrane
of infected insect cells (10, 11) under slightly acidic conditions.
It was postulated that this pore formation plays a crucial role in the
penetration process of SFV (15), but the question which of the viral
structural proteins plays the key role in this process remained open.
One possible candidate is the small structural membrane protein 6K,
which is present only in small amounts in the viral membrane. Upon
expression in E. coli 6K was capable of increasing the
membrane permeability leading to cell lysis (22). Similar results have been demonstrated for poliovirus protein 3A (28). However, a deletion
mutant of SFV lacking the 6K protein showed unaltered behavior
with respect to low pH-induced pore formation in infected eukaryotic
cells (20), although there was a reduction in virus release (21). This
may indicate that the 6K protein plays a role in the budding process
rather than in pore formation, as suggested by Loewy et al.
(29).
It has been demonstrated previously that low pH-induced pore formation
is dependent on the ectodomain of the viral spike (13). Furthermore,
other findings using various mutant viruses strongly suggest that the
E1 protein plays a crucial role in this process (20). Experiments
showing that pore formation also takes place in the so-called E1
particles, where the E2 ectodomain has been removed by proteolysis (14)
supported this notion. However, since E1 particles still contain the
trans-membrane part of the E2 protein, an involvement of E2 could not
be entirely excluded.
Independent expression of E1 protein on the cell surface of vertebrate
cells has not yet been achieved (the protein is produced, but not
transported to the plasma membrane) (18). Thus, it has not been
possible to prove that the E1 protein per se is sufficient for the formation of pores across the membrane.
In this study we have therefore expressed the E1 protein in E. coli in an inducible manner using the pET11c expression system (30) and showed that these E1 proteins are indeed integrated into the
plasma membrane of E. coli cells in an identical orientation as in SFV or SFV-infected cells. Fig. 3 clearly shows that in spheroplasts exposed to proteinase K the E1 protein is digested. An
E1-derived peptide of ~3 kDa is protected from the proteinase digestion, i.e. is localized inside the periplasmic
membrane. The size of this peptide is in agreement with the expected
size of the anchoring region (2.9 kDa) and strongly supports our
findings that the E1, in the E. coli cytoplasmic membrane,
is correctly oriented.
To investigate whether pH-dependent pore formation occurs,
[14C]choline release assays were performed. An enhanced
choline efflux at pH 6-6.2 and below was found. It was maximal at a pH
around 5.2, which is a pH similar to the one prevailing in endosomes. Lanzrein et al. (10) have reported corresponding results
using SFV-infected insect cells: upon lowering the extracellular pH, efflux of a radiolabeled tracer molecule started at a pH of ~6.2 and
reached a maximal level at a pH of ~5.5. Furthermore, these data are
in accordance with what is known about the pH dependence of the SFV
membrane fusion reaction (5) that in turn is dependent on the
conformational change of the spike proteins.
The fact that exposure of E. coli expressing the E1 protein
to mildly acidic pH results in a change of the membrane permeability, which shows the characteristics of previously described acid-induced pore formation by the virus proteins, further strengthens the notion
that the E1 protein is indeed incorporated into the cell membrane. The
formation of pores at a pH below 6.2 also explains the observations
that the growth of bacteria containing the pET11c-E1 plasmid was
strongly hampered at pH 5, but not at pH 6.4 or 7.4, respectively.
In conclusion, the data presented demonstrate that the E1 per
se is sufficient to form acid-induced pores. Thus, these results confirm the previously proposed hypothesis (15).
However, these data also raise the interesting question how the E1
protein gets inserted into the E. coli cell membrane. In a
regular infection of eukaryotic cells the viral spike proteins are
synthesized as a polyprotein, which is cleaved co-translationally into
the single proteins, and the signal for the insertion of the E1 protein
into the membrane is contained within the 6K protein that precedes E1
on the polyprotein (6). Within the membrane the E1 protein has a type I
orientation (C terminus inside, N terminus outside). The membrane
anchor sequence is located within the C-terminal 25 amino acids with
just two arginines on the inside. With the selected strategy of cloning
the E1 protein coding sequence into the pET11c vector, all of the 6K
sequence was omitted and replaced by a start codon.
Analysis of the E1 sequence (439 amino acids) for both prokaryotic and
eukaryotic signal sequences using the PSORT program (31, 32) predicted
the protein to reside in the cytoplasm. Hence, the cloned DNA
containing the sequence encoding the E1 protein lacks a known signal
that would govern the insertion of the protein into the cell membrane.
To identify the sequences within the E1 protein responsible for protein
insertion into the membrane, further experiments are needed.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
virus Semliki
Forest virus has been extensively studied (1). Once attached, the virus
is internalized via coated vesicles and transferred to the endosome.
Due to the acidic conditions within this organelle, the lipid envelope
of SFV fuses with the endosomal membrane of the target cell (2). This
low pH-induced fusion is mediated by the so called virus spikes (3-5).
Each spike is a heterotrimer, being composed of the type I integral
membrane glycoproteins E1 (50.786 kDa) and E2 (51.855 kDa), plus the
peripheral glycoprotein E3 (11.369 kDa), which is associated with E2
(6). Several functions have been ascribed to the spike proteins;
e.g. the E2 and E3 precursor protein p62 forms a heterodimer
with E1 in the endoplasmic reticulum and is responsible for the
transport of the complex to the plasma membrane (6). The E1 protein is
involved in the acid-induced fusion of the viral and endosomal
membranes (7-9).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (final
concentration; Roche Diagnostics Ltd., Rotkrenz, Switzerland), and bacterial growth was recorded by measuring
A600.
-mercaptoethanol). The supernatant of the
12,000 × g centrifugation was centrifuged at
300,000 × g for 50 min and the pellet resuspended in 1 ml of buffer B, homogenized, and centrifuged at 300'000 g for 60 min.
Finally, the pellet containing the membranes was resuspended in 400 µl of buffer B.
-counter.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of E1 protein detected by Western
blot analysis. A, cultures of BL21(DE3) E. coli cells harboring pET11c-E1 were induced with 1 mM
IPTG or left noninduced as a control. Plasma membranes were isolated
and analyzed as described under "Materials and Methods."
M, membrane fraction, noninduced; Mi,
membrane fraction, induced; P, cell debris pellet,
noninduced; Pi, cell debris pellet, induced;
SFV, isolated, purified virus. B, fractions
containing the bacterial plasma membrane or the cell debris pellet,
respectively, were treated with 8 M urea and subsequently
analyzed as described under "Materials and Methods."
MiUp, pellet of membrane fraction, induced; MiUu,
supernatant of membrane fraction, induced; PiUp, cell debris
pellet, induced; PiUu, supernatant of cell debris pellet,
induced; SFV, isolated, purified virus. C,
immunoblot using anti-SFV as first antibody. Lane 1,
membranes isolated from E. coli harboring the pET11cE1
plasmid. Lane 2, membranes isolated from a mixture of
E. coli expressing the E1 protein and enzyme IICB,
respectively. Lane 3, membranes isolated from E. coli co-expressing E1 and enzyme IICB. Lane 4,
membranes isolated from E. coli expressing enzyme IICB.
D, immunoblot using anti-enzyme IICB as first antibody.
Lanes 1-4 correspond to the lanes in Fig. 3C).
Gray scale pictures were produced from the original
immunoblots using a Hewlett-ackard Scan Jet 4c and the corresponding
software Desk Scan II.
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Fig. 2.
FACS analysis of rabbit anti-SFV-labeled
spheroplasts containing the pET11c-E1 plasmid. Spheroplasts that
had been fixed with paraformaldehyde as described under "Materials
and Methods" were incubated with anti-SFV (B) or as a
control with preimmune rabbit serum (A) followed by
fluorescein isothiocyanate goat anti-rabbit antibody. Prior to
measurement on the flow cytometer, the DNA stain propidium iodide was
added to assess viability of the spheroplasts. As shown in the
histogram, ~20% of the spheroplasts gated in a forward scatter/90°
side scatter plot (not shown) were stained with the anti-SFV antibody
(B versus control A). This staining
was surface-located: no DNA staining was obtained with propidium iodide
as can be seen from the dot plots 2B and 2A which display
simultaneously propidium iodide staining (FL-2) and anti-SFV staining
(FL-1). As a positive control for propidium iodide staining,
spheroplasts permeabilized by mild detergent treatment were used. These
fixed and leaky spheroplasts were readily stainable with propidium
iodide (not shown).
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Fig. 3.
Association of the E1 protein and fragments
thereof with the cytoplasmic membrane. E. coli cells
expressing the E1 protein were converted into spheroplasts (lane
1), subsequently incubated with proteinase K in the absence
(lane 2) or presence of Triton X-100 (lane 3).
Proteins were analyzed by SDS-PAGE followed by immunoblotting.
Gray scale pictures were produced from the original
immunoblots using a Hewlett-Packard Scan Jet 4c and the corresponding
software Desk Scan II.
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Fig. 4.
Acid-induced membrane permeability change in
E. coli expressing E1. A, BL21(DE3)
E. coli cells containing either the pET11c-E1 ( ),
pET11c E1/23 E2/432 (
), or pET11c (
) plasmid were
induced with 1 mM IPTG and subsequently loaded with
[14C]choline for 60 min. The cells were then washed and
incubated in M9 medium at pH 7.5 or 5.85, respectively. Aliquots of the
bacterial suspension were taken at various time intervals, and
radioactivity was measured in the supernatant. The relative efflux
(er) was determined with respect to the total
radioactivity in each sample and the ratio of er
at pH 5.85 over er at pH 7.5 for each time point
was calculated.
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Fig. 5.
pH dependence of choline release.
BL21(DE3) E. coli cells containing either pET11c or
pET11c-E1 were induced with IPTG and loaded with
[14C]choline. Aliquots were adjusted to pH values ranging
from 4 to 7, and the choline release was measured 10 min after
adjustment of the pH. The curve depicts the difference between the
efflux measured with pET11c-E1 and pET11c (background release)
containing cells.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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ACKNOWLEDGEMENTS |
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The two plasmids encoding the enzyme IICB as well as the anti-enzyme IICB antibodies were kindly provided by Prof. B. Erni (Department of Chemistry and Biochemistry, University of Bern, Switzerland).
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FOOTNOTES |
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* This work was supported in part by Swiss National Science Foundation Grant 31-49217.96 (to C. K.).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 Pathology, University Hospital, Schmelzbergstrasse 12, 8091 Zürich, Switzerland.
To whom correspondence should be addressed: Dept. of
Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland. Tel.: 41-31-6314339; Fax: 41-31-6314887;
E-mail: kempf@ibc.unibe.ch.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M011061200
2 F. Käsermann, S. Nyfeler, T. Roten, and C. Kempf, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
SFV, Semliki
Forest virus;
bp, base pair(s);
IPTG, isopropyl-1-thio--D-galactopyranoside;
PAGE, polyacrylamide gel electrophoresis;
FACS, fluorescence-activated cell
sorter.
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