 |
INTRODUCTION |
All membrane proteins must adopt their correct asymmetric
orientations in the membrane in order to function correctly. This is
achieved according to two major known parameters. The hydrophobic stretches within membrane proteins enable the protein to partition into
the membrane and span the bilayer, whereas the hydrophilic regions are
retained at the correct face of the membrane as dictated by the
"positive inside" rule (1). This rule states that regions rich in
positively charged residues are directed to the cytoplasmic face of the
inner membrane of Escherichia coli (2, 3). The basis for the
positive inside rule has not been pursued diligently until recently (4,
5). One hypothesis is that the proton motive force,
µH+
(pmf),1 which renders the
cytoplasm basic and the periplasm acidic, favors a cytoplasmic location
for positively charged amino acids. The electrical component of the
pmf, 
, imposes an "uphill" barrier for positively charged
residues to translocate into the periplasm, whereas negatively charged
residues move downhill with the pmf (2). The pmf has been shown to
drive the insertion of hydrophilic domains of both
sec-dependent (6-8) and sec-independent proteins (9-11).
Evidence for an electrophoretic membrane insertion mechanism has been
reported for mutants of the exported protein proOmpA (12) and for
derivatives of leader peptidase and procoat (4, 13). It was shown that
the pmf promotes the translocation of negatively charged residues
within a short periplasmic loop (4, 13) and an amino-terminal tail (13)
of a membrane protein. In addition, the pmf appears to impede
translocation of an amino-terminal tail when it possesses a positive
charge (13) since the tail inserts more efficiently in the absence of
the potential. The pmf also contributes to the topology of the protein,
since abolishing the pmf causes a partial topology inversion in various
positively charged mutants (4). However, similar studies using the M13 procoat protein showed that a positively charged periplasmic loop inserted slightly less efficiently when the pmf was collapsed (13).
Therefore, it is not clear whether the electrical potential plays a
general role in influencing the membrane topology of proteins.
Another major factor that drives hydrophilic regions of membrane
proteins across the phospholipid bilayer is the hydrophobicity of the
transmembrane segments. It has been shown that the hydrophobic amino
acids within these transmembrane domains are necessary for carboxyl-
and amino-terminal translocation of hydrophilic regions (14, 15).
Although the importance of hydrophobicity for protein translocation is
well known (1, 16), the relative contributions of the hydrophobicity
and the pmf for a type II membrane protein have not been examined.
In this study, the role of the pmf in the translocation of positively
charged residues was tested. Also, the severity of the barrier of
translocating positively charged residues caused by decreasing
hydrophobicity of the inserting membrane protein was tested. In
addition, we examined whether the pmf can play a primary role for
translocation of negatively charged residues across the membrane when
hydrophobicity is reduced. We fused an antigenic tag from leader
peptidase to the carboxyl terminus of procoat, which enabled us to
efficiently immunoprecipitate the proteins with leader peptidase
antiserum (17). We have constructed a series of deletions within both
the relatively hydrophobic signal sequence and the extremely
hydrophobic membrane anchor. We found that translocation of basic
residues within the periplasmic loop of procoat is increasingly
hindered by the pmf as the hydrophobicity was incrementally reduced.
This is in contrast to what is observed with negatively charged
mutants. Here we observed that the pmf becomes increasingly critical
for translocation as the hydrophobicity of the protein was reduced.
This directional reaction to the pmf provides more evidence for an
electrophoretic insertion mechanism and suggests that the electrical
potential may be the basis for the positive inside rule of inner
membrane proteins in agreement with Andersson and von Heijne (4). In
addition, our results suggest that a pH gradient is not necessary for
insertion of the procoat mutants, and the effects of charged
residues on translocation can be attributed to the 
.
 |
EXPERIMENTAL PROCEDURES |
Strains and Plasmids--
E. coli strain MC1061
(
lacX74, araD139,
(ara, leu)7697,
galU, galK, hsr, hsm, strA) was from our laboratory stocks.
XL-1 Epicurean coli (supE44, hsdR17,
recA1, endA1, gyrA46, thi
relA1, lac
) was from Stratagene. TolC
(lacY1 or lacZ4, gal-6, hisG1(Fs),
tolC5, uxaC201,
rpsL8 or rpsL104 or rpsL17, malT1(
R),
mtlA2) was from the E. coli Genetic Stock Center at
Yale University. The pING plasmid (18), which contains the arabinose
promoter and arabinose regulatory elements, was used to express
procoat-lep mutants.
Materials--
Lysozyme, amino acids, phenylmethylsulfonyl
fluoride, and CCCP were from Sigma. Proteinase K was from Boehringer
Mannheim. The Sequenase version 2.0 kit was from U.S. Biochemical Corp. Tran35S-label, a mixture of 85%
[35S]methionine and 15% [35S]cysteine was
from ICN. Materials for Quikchange were from Stratagene. Deoxynucleotides were from Amersham Pharmacia Biotech. Qiagen Midi
Plasmid Purification Kit was from Qiagen. PAGE-purified
oligonucleotides were obtained from Integrated DNA Technologies.
DNA Manipulations--
Plasmid preps were accomplished using the
Qiagen midi kit. Mutagenesis was carried out according to the
Quikchange procedure, with modifications as described (19). Sequencing
was done according to the methods of the Sequenase version 2.0 kit.
Mutants verified by sequencing were transformed into MC1061 using the
calcium chloride method of Cohen et al. (20).
Protease Mapping Assay--
One milliliter of M9 medium
containing 0.5% fructose and 50 µg/ml each amino acid except
methionine was inoculated with 20 µl of an overnight MC1061 cell
culture bearing the plasmid of interest and allowed to grow for 3 h with shaking at 37 °C. Expression of procoat-lep mutants was
induced for 1 h with a final concentration of 0.2% arabinose. The
cells were metabolically labeled with 100 µCi of
Tran35S-label for 1 min and subsequently transferred to
prechilled microcentrifuge tubes. Where indicated, carbonyl cyanide
m-chlorophenyl hydrazone (CCCP) was added 45 s prior to
labeling at a final concentration of 50 µM. The cells
were pelleted for 40 s at 14,000 rpm in a microcentrifuge and then
resuspended in 250 µl of Solution I (Tris acetate, pH 8.2, 0.5 M sucrose, and 5 mM EDTA). Spheroplasts were formed by adding lysozyme to a final concentration of 80 µg/ml and
250 µl of ice-cold water immediately thereafter. After a 5-min incubation, MgSO4 was added to a final concentration of 40 mM. The spheroplasts were harvested by spinning again for
40 s and resuspended in 300 µl of Solution II (50 mM
Tris-HCl, 0.25 M sucrose, 10 mM
MgSO4). The spheroplasts were split into 3 aliquots. The first aliquot received no protease. 7.5 µl of 20 mg/ml proteinase K
was added to the other two aliquots, and additionally, 30 µl of 10%
Triton X-100 was added to the third aliquot. The spheroplasts were
incubated on ice for 1 h and then the proteinase K was inactivated with 5 mM phenylmethylsulfonyl fluoride (final
concentration). 250 µl of ice-cold 20% trichloroacetic acid was
added, and immunoprecipitations were carried out (21). The proteins
were then analyzed by SDS-PAGE using a 17% polyacrylamide gel and
subjected to fluorography (22).
Signal Peptidase Processing Assay--
One milliliter of M9
media containing 0.5% fructose and 50 µg/ml each amino acid except
methionine was inoculated with 20 µl of an overnight MC1061 cell
culture containing the mutant plasmid of interest. Growth was allowed
to proceed for 3 h, after which expression of the plasmid-encoded
protein was induced with 0.2% arabinose. After a 1-h induction, the
cells were metabolically labeled for 1 min with 50 µCi of
Tran35S-label. Where indicated, CCCP was added at a final
concentration of 50 µM, and after 45 s,
Tran35S-label was added as before. The cells (500 µl)
were transferred to prechilled microcentrifuge tubes containing 500 µl of 20% trichloroacetic acid. After a 30-min precipitation, the
samples were analyzed by immunoprecipitation, SDS-PAGE, and
fluorography as described above.
pH Studies--
One-milliliter samples of M9 medium were
inoculated with 20 µl of an overnight culture of TolC
cells harboring the plasmid of interest. After allowing growth for
3 h, the cells were resuspended in 1 ml of fresh M9 medium having
a pH value of either 6.5 or 7.5. Ten microliters of 20% arabinose was
added to induce synthesis of the plasmid-encoded proteins, and growth
was allowed for 30 min more. The cultures were then labeled with 50 µCi of Tran35S-label for 1 min. Where indicated, 50 µM CCCP (final concentration) was added 45 s prior
to labeling. The cells (500 µl) were aliquoted into tubes containing
an equal volume prechilled 20% trichloroacetic acid, and
immunoprecipitation was performed as before.
Quantitation of Insertion Data--
X-ray films were scanned
using an AppleOne Scanner. The insertion efficiencies were then
determined by quantitation of the appropriate bands on the film using
NIH Image, a public domain program that can be found
on-line.2 For the signal
peptidase cleavage assays, the percent of insertion across the membrane
was determined by calculating the percent of the processed band
appearing on the gels. For full-length constructs, and leader sequence
deletion mutants, two methionines were lost upon signal peptidase
cleavage, so we used Equation 1 to determine membrane insertion.
|
(Eq. 1)
|
For membrane anchor deletions (
48-51,
48-53, and
48-55), two out of six methionines were lost, so we used Equation 2.
|
(Eq. 2)
|
For the protease mapping assays, calculation of insertion
efficiencies of procoat-lep was done in a similar manner as above with
the following exceptions. The percentage of the protease-resistant fragment (prf) (slightly lower molecular weight than the mature band)
was calculated and used as the percentage of membrane insertion. The
percentage of cells that were converted to spheroplasts was determined
for non-CCCP-treated cells with OmpA and ribulokinase controls and used
to calculate the percent of protein that inserted (Equation 3).
|
(Eq. 3)
|
Determination of Summed Hydrophobicity--
The summed
hydrophobicity (H) of the transmembrane regions of procoat
that were manipulated was determined as described (23) using the GES
scale (24). We calculated H for the wild-type and mutant
procoat-lep proteins by adding the hydrophobicity of the uninterrupted
stretch of uncharged amino acids within the predicted transmembrane region.
 |
RESULTS |
The Proton Motive Force Becomes Increasingly Important for
Translocation of Negatively Charged Residues When the Hydrophobicity of
Procoat Is Reduced--
The M13 coat protein is synthesized in a
precursor form, termed procoat, with a 23-residue signal peptide.
Procoat inserts into the membrane as a loop bordered by two hydrophobic
domains (25). One hydrophobic domain is the signal peptide and the
other is a membrane anchor in the mature coat protein. In our studies, the procoat protein contains a leader peptidase fragment of 103 residues fused to the carboxyl-terminal region of procoat, as previously published (13, 17). This protein is termed procoat-lep (Fig.
1A, lep is depicted by a
zig-zag line). The wild-type periplasmic domain of
procoat-lep (see Fig. 1A) contains the following charged residues: glutamic acid (position 25), aspartic acid (position 27),
aspartic acid (position 28), lysine (position 31), and glutamic acid
(position 43).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 1.
Procoat-lep and mutants used to study the
role of hydrophobic forces in membrane insertion. A,
membrane topology of wild-type procoat-lep. The hydrophobic domains are
represented by rectangles; the lep epitope by a
zig-zag line. B, procoat-lep mutants with
sequential deletions in both the signal sequence ( 9-10, 9-12,
9-14) and the membrane anchor ( 48-51, 48-53, 48-55).
The total hydrophobicity of the native and mutant transmembrane
segments was determined (23). The energy of transfer (kcal/mol) from an
aqueous to a nonpolar environment for each amino acid side chains was
summed and used as a measure of hydrophobicity lost. For simplicity, no
charges are shown in the periplasmic loop in procoat-lep in
B.
|
|
Previously, it had been shown that hydrophobic forces play a
considerable role in membrane insertion of the wild-type procoat (25,
26). The pmf plays a secondary role for translocation of the acidic
loop in the wild-type procoat and only stimulates the membrane
insertion across the membrane (13). To test whether the pmf can play a
primary role in membrane insertion if the hydrophobicity of the
signal/membrane anchor is decreased, we first employed oligonucleotide-directed mutagenesis to change the lysine at position 31 and glutamic acid at position 43 to alanines. We termed the resulting construct PClep. All previous studies on the membrane insertion of procoat had utilized constructs that contained the wild-type residues at these positions in order to study the net charge
of the loop (13). We next constructed a series of deletion mutants that
are deficient in hydrophobic amino acids within either the leader
sequence or the membrane anchor sequence, as shown in Fig.
1B. We deleted two, four, and six amino acids in the
moderately hydrophobic signal sequence, and four, six, and eight
residues in the extremely hydrophobic membrane anchor. The total
hydrophobicity for the transmembrane segment from which amino acids
were removed was determined (23). The hydrophobicity scale used was the
transfer free energy (kcal/mol) of each amino acid side chain from
water to a non-polar environment (24).
We used a protease accessibility assay to analyze the insertion of the
procoat mutants into the membrane. We labeled 1 ml of exponentially
growing E. coli cells with 100 µCi of
Tran35S-label and then converted the cells into
spheroplasts using the lysozyme/EDTA method previously described (27).
These cells, or cells lysed with Triton X-100, were digested for 1 h with proteinase K. Aliquots were immunoprecipitated with antiserum to
leader peptidase and analyzed by SDS-PAGE and fluorography. Digestion
of inserted procoat-lep with the protease results in a
protease-resistant fragment (prf) of slightly lower molecular weight
than the mature band that is visible after immunoprecipitating with
antibody to the carboxyl-terminal leader peptidase epitope, as
published previously (13, 17). We quantitated the relative amount of
the precursor form of procoat-lep that remained in the cytoplasm (see
upper band, depicted by p in Fig.
2A, in protease-treated lanes)
and compared this to the amount that had inserted across the membrane and was digested with protease (see the protease-resistant lower molecular weight fragment, prf). In addition, the
protease-treated samples were compared with an aliquot of spheroplasts
which were not subjected to digestion. We also performed a control in
which we lysed an aliquot of spheroplasts with Triton X-100 to confirm that the periplasmically situated domains were not protease-resistant. Pulse-chase experiments and studies using IT41 (28), a
temperature-sensitive signal peptidase strain, revealed which bands
correspond to the mature and precursor form of the protein (data not
shown). The upper band in Fig. 2A
(non-protease-treated lanes) represents the precursor protein
(p) in the cytoplasm, whereas the lower band
(non-protease-treated lanes) corresponds to PClep that had inserted
across the membrane and had been cleaved to the mature protein by
signal peptidase. In the experiments where deletions were made in the
signal sequence, we could not use the processing of procoat to coat by
signal peptidase as an indication of insertion. This was due to the
reduced efficiency of signal peptidase cleavage caused by the shortened
signal peptide.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 2.
Protease mapping to determine the insertion
efficiencies of negatively charged procoat-lep mutants.
A, insertion across the membrane of ( 3) mutants with the
pmf. MC1061 cells bearing plasmids encoding procoat-lep mutants were
grown in M9 medium containing 50 µg/ml each amino acid except
methionine and 0.5% fructose to mid-log phase. After an induction with
0.2% arabinose for 1 h, the cells were metabolically labeled with
100 µCi of Tran35S-label for 1 min. The cells were then
converted to spheroplasts and analyzed by protease mapping, as outlined
under "Experimental Procedures." After proteolysis, samples were
immunoprecipitated with antisera to leader peptidase, run on 17%
SDS-PAGE, and subjected to fluorography. p, precursor;
prf, protease-resistant fragment (represents inserted
material). B, membrane insertion of ( 3) mutants in the
absence of the pmf. MC1061 cells expressing the mutants containing
three negatively charged amino acids were treated exactly as in
A, except for the following. CCCP (final concentration of 50 µM) was added to collapse the pmf for 45 s prior to
labeling with Tran35S-label. p, precursor;
f, fragment (a possible proteolytic fragment that does not
insert across the membrane). C, controls used to determine
the degree of spheroplasting and lysis. Samples were immunoprecipitated
with antisera to OmpA (an outer membrane protein that is potential
dependent for translocation) and ribulokinase (Rib) (a
cytoplasmic protein).
|
|
For negatively charged procoat-lep constructs, as the hydrophobicity of
each transmembrane segment is incrementally decreased, insertion across
the membrane also decreases in stepwise increments (see Table
I for percent efficiencies and Fig.
2A for protease mapping data). PClep(
3), the full-length
construct, inserts 94% efficiently into the membrane with the pmf
present (
CCCP). As can be seen, PC-lep (
3) is
efficiently processed by signal peptidase to the mature protein
(band below the precursor form). The mature form of PClep
(
3) was cleaved to a slightly lower molecular weight form
(prf) by proteinase K. For the series of constructs
9-10 (
3),
9-12(
3), and
9-14(
3), which contain increasingly
larger deletions in the leader sequence, the amount of prf decreases as
the size of the deletion increases (compare lanes 5, 8, and 11 in Fig. 2A). The series of three mutants
possessing membrane anchor deletions,
48-51(
3),
48-53(
3),
and
48-55(
3) shows the same trend, with the largest deletion
almost collapsing insertion at 15%. The major lower bands in the
non-protease-treated lanes 1, 4, 7, 13, 16, and
19 are the result of processing by signal peptidase to the
mature protein as the periplasmic loop of procoat crosses the membrane.
The lowest band seen in lanes 7 and 16 is most likely the result of cleavage by an unknown protease. Testing these constructs in the temperature-sensitive signal peptidase strain,
showed that the lowest band (f) in these lanes was not the
result of signal peptidase cleavage. Fig. 2C shows
representative controls for these experiments. We immunoprecipitated an
equal portion of sample with antiserum raised against OmpA, a protein that is exported in a pmf-dependent manner to the periplasm
(8). In the presence of the pmf, this control shows the degree to which the cells were converted to spheroplasts when comparing the
non-protease-treated cells with those that had been digested by
proteinase K (compare lanes 1 and 2). We also
immunoprecipitated with antiserum to ribulokinase, a cytoplasmic
protein. Intact ribulokinase in the protease-treated lanes (see
lane 2) indicates that minimal lysis occurred when forming
spheroplasts. We had a difficult time obtaining intact spheroplasts for
48-55(
3), so the insertion efficiency, when corrected for lysis,
is higher than that predicted from the gel (lanes 19 and
20).
View this table:
[in this window]
[in a new window]
|
Table I
Mutants containing negative charges in the periplasmic loop and their
insertion efficiencies with and without the
pmf
|
|
We next examined the effects of collapsing the pmf by adding 50 µM CCCP, a protonophore, 45 s before labeling the
cells with Tran35S-label. Comparing lanes 1 and
2 in Fig. 2A with lanes 1 and
2 in Fig. 2B shows that the insertion of the
full-length procoat-lep under these conditions decreases from 95 to
about 69%, confirming that this protein is partially dependent on the
pmf for insertion (10, 13), see also Table I. Interestingly, except for
the
9-10 and
9-12 deletion mutants, membrane insertion of the
mutants was abolished without a pmf (CCCP-treated cells), as shown by the small amounts of prf produced in the protease-treated lanes. The
lower molecular weight band (depicted by a f) in lanes
4-5, 7-8, 10-11, 13-14, 16-17, and 19-20 is most
likely the result of proteolysis by an unknown protease within the
cytoplasm that occurs downstream of the signal peptidase-processing
site. This in vivo proteolytic fragment of procoat does not
contain a domain exposed across the membrane since its molecular weight
does not shift upon treatment with proteinase K. Even a two-amino acid deletion in the leader sequence (
9-10(
3)) has a measurable effect on procoat assembling across the membrane, at 20% (the prf in lane 5 can be discerned directly below the proteolytic
fragment f described above). A six amino acid deletion
within the signal sequence (
9-14(
3)) completely abolishes
insertion (lanes 10 and 11). Immunoprecipitating
with OmpA antiserum served to confirm that the pmf was actually
collapsed (see representative data in Fig. 2C). The
precursor form of the protein (proOmpA), which is visible in
lanes 4 and 5, is not digested by protease,
indicating that this outer membrane protein remained in the cytoplasm.
The spheroplasts were mostly intact as shown by immunoprecipitation with antiserum to ribulokinase, a cytoplasmic protein. These results show that the pmf is absolutely required to insert procoat across the
membrane when the hydrophobicity is reduced, indicating that the pmf
plays a primary role for inserting negatively charged residues across
the membrane when the hydrophobic forces of the inserting procoat are compromised.
Fig. 3 shows the results of protease
accessibility assays on mutants of procoat-lep containing no charges in
the periplasmic loop. The glutamic acid residue at position 25 and the
aspartic acid residues at positions 27 and 28 were changed to glutamine and asparagine residues, respectively. In addition, the hydrophobic amino acids in the signal and membrane anchor regions (refer to Fig.
1B) were deleted as in the (
3) series of mutants. Table II shows the insertion efficiencies of
these seven mutants. As the hydrophobicity is decreased, there is a
concomitant decrease in membrane insertion, as evidenced by a decrease
in the amount of prf (compare in Fig. 3 lanes 4 and
5, and 7 and 8, etc.). These results
show hydrophobic forces are important not only to insert a negatively
charged region but also to insert an uncharged hydrophilic domain. Next
we collapsed the pmf with CCCP. Here, in contrast to the (
3) mutants,
the neutral mutants insert across the membrane with similar
efficiencies as when the pmf is present. These results, when compared
with the results for the (
3) mutants, confirm that the pmf only
contributes to insertion of procoat when acidic amino acids are
translocated.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 3.
Pmf-independent translocation of uncharged
loops when the hydrophobicity of procoat is reduced. A,
insertion of neutral mutants of procoat-lep in the presence of the pmf.
MC1061 cells bearing the plasmid of interest were treated exactly as in
the legend to Fig. 2A. p, precursor;
prf, inserted fragment. B, membrane insertion of
neutral constructs in the absence of the pmf. MC1061 cells harboring
the plasmid of interest were treated exactly as in Fig. 2B.
C, controls as in Fig. 2.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Mutants containing no charges in the periplasmic loop and their
insertion efficiencies with and without the
pmf
|
|
Positive Charges in the Periplasmic Loop Impede Translocation in
the Presence of the pmf--
We next investigated whether the
translocation of positively charged residues would be hindered by the
pmf, as postulated by an electrophoretic mechanism. For these
experiments, we used the assay that relies on signal peptidase
processing to ascertain whether membrane insertion has occurred. We
labeled exponentially growing cells with Tran35S-label and
immunoprecipitated procoat-lep with leader peptidase antiserum. We
analyzed the processing efficiencies by signal peptidase cleavage of
full-length procoat proteins and of mutants possessing two different
deletions in the membrane anchor sequence, all containing positive
charges inserted into the periplasmic loop (see Table III for positions of basic amino acid
substitutions and insertion efficiencies). After labeling 1 ml of
exponentially growing cells with 50 µCi of Tran35S-label
for 1 min, we acid-precipitated the proteins with an equal volume of
20% trichloroacetic acid. The proteins were immunoprecipitated with
antibody to leader peptidase and analyzed on 17% SDS-PAGE gels.
View this table:
[in this window]
[in a new window]
|
Table III
Mutants containing positive charges in the periplasmic loop and their
insertion efficiencies with or without the
pmf
|
|
Fig. 4A shows the signal
peptidase processing results for mutants possessing a positively
charged lysine residue near the center of the periplasmic loop at
position 32. The top band in each lane represents the
uninserted precursor protein (p), and the lower
band corresponds to the mature form (m) which results from signal peptidase cleavage. This mature protein band was identified using a temperature-sensitive signal peptidase strain (28). The results
(Fig. 4A) show that as the hydrophobicity of the membrane anchor was decreased, processing of the procoat protein was decreased. The positively charged procoat protein with an intact membrane anchor
(PClep(+1)) was 61% processed in a 1-min pulse label, compared with 55 (
48-51(+1)) and 23% (
48-53 (+1)) processing with 4 and 6 residues deleted in the membrane anchor, respectively (compare lanes 1, 3, and 5). Strikingly, when the pmf is
collapsed, the insertion of procoat with a positively charged residue
becomes better, as indicated by increased processing of procoat (see
lanes 2, 4, and 6). This enhanced translocation
in the absence of the pmf increases as the hydrophobicity declines;
there is a 20% increase in translocation when CCCP is added for
PClep(+1) but a 37% increase for
48-53(+1). The efficient
translocation of a positive charge with high hydrophobicity of the
membrane anchor shows that hydrophobic forces can overcome such a
barrier even in the presence of the pmf.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 4.
The pmf inhibits translocation of positively
charged residues. A, translocation of one positive
charge in the center of the loop of procoat mutants. MC1061 cells in M9
medium bearing the plasmid of interest were grown for 3 h, induced
for 1 h with a final concentration of 0.2% arabinose. The cells
were then metabolically labeled with 50 µCi of
Tran35S-label for 1 min. For the pmf study, cells were
treated 45 s prior to labeling with 50 µM CCCP
(final concentration). The samples were then subjected to
immunoprecipitation, SDS-PAGE, and fluorography. B,
translocation of two spaced positive charges within procoat mutants.
MC1061 cells were treated as before. C, translocation of two
basic residues immediately after the signal sequence. D,
translocation of two positive residues in the middle of the periplasmic
loop of procoat mutants.
|
|
It should be noted that, in some cases, the addition of CCCP results in
a decrease in protein synthesis of procoat-lep (see data for
48-51
and
48-53). This most likely is due to CCCP, which is an uncoupler
of oxidative phosphorylation, lowering ATP levels in the cytosol.
Fig. 4B depicts the translocation results of constructs
containing two lysine residues spaced within the loop at positions 25 and 32. These mutants are termed procoat-lep (+2S) with the S indicating that the charges are spaced apart. Again, as
the hydrophobicity of the membrane anchor decreases, the processing of
procoat decreases in the presence of the pmf (
CCCP) (compare lanes 1, 3, and 5). Procoat processing is 58, 34, and 0% for PClep (+2S),
48-51 (+2S), and
48-53(+2S), respectively (see Table III). When the pmf
is collapsed with CCCP, the results are even more dramatic than for a
single positive charge in the periplasmic loop. PClep(+2S)
with two positively charged residues and an intact membrane inserts
17% better in the absence of the pmf. The positively charged mutant
PClep
48-51(+2S) (lanes 3 and 4),
with a deletion of 4 residues in the membrane anchor, inserts across
the membrane a dramatic 35% better when the pmf is collapsed compared
with when the pmf is maintained. The
48-53(+2S) mutant
with a larger deletion within the membrane anchor cannot insert at all
in the presence of the pmf, although it inserts to significantly high levels (44%) in the absence of the pmf (see lanes 5 and
6). This is approaching the insertion efficiency of the
procoat mutant with two positively charged residues within an intact
membrane anchor (PClep(+2S)) in the presence of the pmf. The
processing results of procoat mutants possessing two positively charged
lysine residues directly adjacent to the signal sequence (+2H1
constructs) at positions 25 and 26 are very similar to the findings for
the (+2S) mutants (Fig. 4C). Finally, when we
changed two adjacent residues at positions 32 and 33 in the middle of
the loop to positively charged lysine residues (see
PClep(+2M) constructs in Fig. 4D), we
found that even the full-length as well as deletion mutant proteins
were prohibited from insertion with or without the pmf present. This
suggests that the center of the periplasmic loop cannot tolerate more
than one positively charged residue for translocation. The data for
Fig. 4, taken together, provide direct evidence that the pmf inhibits
translocation of positive charges.
The
pH Is Not Required for the Insertion of Procoat--
We
wanted to determine whether the
pH component of the pmf is necessary
for the translocation effects of charged residues. We examined
insertion of the procoat mutants under conditions where the
transmembrane pH gradient was close to 0 or 1 by changing the pH of the
periplasmic medium to 7.5 or 6.5, respectively. This was done using a
TolC
mutant as described under "Experimental
Procedures." In E. coli, the cytoplasmic pH is maintained
at about 7.5 (29). The studies presented thus far in the paper were
performed in a medium with a pH of 7.0, a pH gradient close to 0.5.
Fig. 5 shows the processing of procoat
with either no charges in the periplasmic loop (
48-53(0)), three
negative charges adjacent to the leader sequence (
48-51(-3)), or
two positive charges spaced apart in the periplasmic loop
(48-53(+2S)). We grew E. coli cells expressing
these procoat mutants to mid-log phase in M9 medium and then
resuspended the cells in minimal medium with a pH value of 7.5. After
induction of the procoat mutants with 0.2% arabinose for 30 min, we
labeled the cells with Tran35S-label for 1 min as in the
studies shown in Fig. 4. To half of the cultures, we added CCCP for
45 s prior to labeling. Fig. 5 shows the signal peptidase
processing results. As seen in Fig. 5A, the neutral and
negatively charged procoat protein inserts across the membrane with a
similar pmf requirement at pH 7.5 as it did at pH 7.0 (see Fig. 2,
lanes 13 and 14 for
48-51(
3), Fig. 3,
lanes 16 and 17 for
48-53(0)). The pmf
requirement of procoat with negatively charged residues at pH 7.5 suggests that the pH gradient is not essential for the insertion of
procoat across the inner membrane. Similarly, the positively charged
procoat protein cannot insert at pH 7.5 as was observed at pH 7.0 (see Fig. 4B, lanes 5 and 6 for
48-53(+2S)) but can when the pmf is dissipated with
CCCP. These data are consistent with the transmembrane electrical
gradient driving negatively charged residues across the membrane and
hindering positively charged residues.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Membrane insertion of positively charged,
neutral, and negatively charged mutants at different pH values.
A, insertion of a positively charged, a neutral, and a
negatively charged construct at pH 7.5 TolC cells
harboring the plasmid of interest were grown for 2 h in M9 medium.
The cells were then pelleted and resuspended in minimal medium with a
pH of 7.5. Expression of procoat was induced for 30 min by the addition
of 0.2% arabinose (final concentration). The cells were then
metabolically labeled for 1 min with 50 µCi of
Tran35S-label. Where indicated, CCCP was added 45 s
before labeling at a final concentration of 50 µM. The
samples were analyzed as in Fig. 4. B, insertion of the
above constructs at pH 6.5. The same protocol was followed except,
after growth in M9 medium at pH 7.0, the cells were resuspended in
minimal medium at pH 6.5. The samples were precipitated by adding an
equal volume of 20% trichloroacetic acid and subjected to
immunoprecipitation, SDS-PAGE, and fluorography as before.
|
|
However, it is still possible that a pH gradient greater than the
normal physiological level can assist in insertion. To test this idea,
we performed an identical study to the one shown in Fig. 4A,
except that we resuspended the cells in a minimal medium with a pH of
6.5. Fig. 5B shows that there may be a small contribution of
this increased pH gradient for the
48-51(
3) (lanes 3 and 4) and
48-53(0) mutant (lanes 1 and
2). Insertion across the membrane of
48-53(+2S) is still blocked with the increased
pH (lane 5). Interestingly, the positively charged mutant
inserts more efficiently at this pH when CCCP is added to collapse the normal pmf (compare Fig. 5, A and B, lane
6). These studies with charged residues suggest that it is
primarily the 
component of pmf that affects that translocation
of charged residues and not the pH component.
 |
DISCUSSION |
The mechanism of membrane insertion of the M13 procoat protein has
been studied in great detail (25, 30). Procoat inserts across the
membrane in a sec-independent manner, since conditions that block the
sec machinery have no effect on its insertion (31). It is thought that
it inserts across the membrane by a spontaneous mechanism since procoat
can assemble into protein-free liposomes (32). Translocation of its
acidic loop to the periplasm is hypothesized to be driven by a
synergistic entry of the two hydrophobic regions into the lipid bilayer
(33). Although the pmf stimulates translocation, it is not absolutely
required for insertion (13). The positively charged residues before the
signal sequence and immediately after the membrane anchor remain in the
cytoplasm. Thus, the topology of the procoat protein agrees nicely with
the positive inside rule, which states that basic domains act as
topological determinants because they are retained in the cytoplasm
(for reviews see Refs. 1, 34, and 35).
Several factors have been hypothesized to contribute to the positive
inside rule. First, anionic phospholipids are determinants of membrane
protein topology. van Klompenburg and colleagues (5) showed that the
membrane orientation of leader peptidase constructs depended on the
anionic phospholipid content within the membrane. A second factor
contributing to the positive inside rule is the internal dipole
potential within the membrane that may be as large as 240 mV, interior
positive (36, 37). This dipole potential would make it difficult for
positive charges to enter the membrane. The anionic and dipole
potential may explain why membrane proteins in the ER and thylakoid
membranes obey the positive inside rule even though these membranes
have a very low transmembrane potential. However, in large part, the
available data in bacteria are consistent with the pmf (positive
outside, negative inside) determining the asymmetry of the topology via
an effect on positively charged residues (4). In this report, we
provide direct evidence that charged amino acids in hydrophilic domains
of membrane proteins are directed to the correct compartment of
E. coli by the pmf.
We show here that the pmf plays a critical role in translocating
negatively charged residues across the membrane when the hydrophobicity
of the signal or membrane anchor is reduced. The pmf only stimulates
translocation at most 30% for the wild-type protein (13) and a mutant
containing only three negatively charged residues in the periplasmic
loop (Fig. 2A). A small deletion of four amino acids in the
membrane anchor or six in the signal sequence renders the negatively
charged periplasmic loop translocation-incompetent in the absence of
the pmf. This exemplifies the necessity for both hydrophobic and proton
motive forces for translocating negative charges across the membrane.
The addition of the protonophore, CCCP, was used in this study at
concentrations that dissipate the pmf to inhibit or block the
translocation of the periplasmic region of procoat across the membrane.
The CCCP treatment blocks the translocation step of the membrane
biogenesis pathway of the M13 coat protein (10, 13). It is unlikely
that CCCP treatment inhibits the folding of procoat in the cytosol or
electrostatic binding of procoat to the surface of the membrane because
treatment of the neutral mutants with CCCP shows no effect on translocation.
Whereas the pmf is important for translocation of negative charges
across the membrane, our results so far with procoat do not reveal that
negative charges play an active role in the translocation of the
hydrophilic domain in the presence of the pmf. This is in striking
contrast to the case of the Pf3 coat protein, which only moves its
N-tail across the membrane in the presence of the pmf and when
negatively charged residues are present (38). In the case of Pf3-leader
peptidase, we found that one of the acidic residues in the
amino-terminal tail plays an active role in protein translocation of
the amino terminus of Pf3-leader peptidase (19). However, this occurred
only when the hydrophobicity of apolar domain one of leader peptidase
was decreased (19). Therefore, these recent results indicate that
different sec-independent proteins, although requiring the pmf to
translocate acidic amino acids when the hydrophobicity is low, may
insert into the membrane by different mechanisms.
When no charges are present in the translocated domain, procoat can
insert across the membrane rather efficiently with large deletions in
the signal or membrane anchor (Fig. 3). For all the neutral mutants
studied, the pmf did not stimulate translocation, suggesting that the
pmf is required only in the presence of negatively charged residues.
Translocation of an uncharged region is more efficient compared with
translocation of a negatively charged region even in the presence of a
pmf (compare Fig. 2 with 3). Thus, even with a pmf, negative charges
can still act as a barrier. Intriguingly, even when most of the
hydrophobic core of the signal is deleted, over 60% of the procoat
protein can translocate when this region lacks charges. This is in
contrast to translocation of three negatively charged residues with
procoat with a signal peptide deletion in the presence of the pmf,
which is only 19%. This confirms the results of Rohrer and Kuhn (39)
in which they show the function of a leader peptide is to translocate a
charged region. It is important to emphasize the significance of the
pmf in membrane insertion for sec-independent proteins containing charged residues. The pmf appears to act directly on the polar domains
of the proteins, since no protein translocation machinery has been
found to be required. In contrast, for sec-dependent proteins, the pmf is required in a late stage of translocation for
efficient export of periplasmic domains after entering the proposed
SecYEG channel (40-42) and may act on the machinery itself (43).
Moreover, it has been found that the pmf is required even for the
sec-dependent secretion of an uncharged protein domain (44). Our work here illustrates the mechanistic differences between
sec-independent proteins and proteins that are known to require this machinery.
Most intriguingly, we have also shown directly for the first time that
the pmf (positive outside, negative inside) inhibits translocation of
positively charged residues across the membrane. Our results provide
additional evidence that the pmf can act as a determinant of the
positive inside rule of bacterial membrane proteins. Moreover, we show
that the inhibitory effect of the pmf on translocating positive charges
is larger when the hydrophobicity of the membrane anchor is decreased
(Fig. 4). This shows that hydrophobic forces assist in driving the
charged residues across the membrane. When the hydrophobic forces are
compromised, the charges become a barrier to insertion, especially in
the presence of a positive outside potential. In previous studies that
examined positive charges in the periplasmic loop of procoat, the
negatively charged amino acid near the membrane anchor was always
present, and thus complicated the results (13). In the present report, we examine the effects of just one or two positive charges in the
absence of the acidic amino acid. Particularly compelling is the
finding that two positively charged mutants with large deletions in the
membrane anchor,
48-53(+2S), and
48-53(+2H1), did
not insert across the membrane until the pmf was dissipated with CCCP.
This is in contrast to the mutant
48-53(+1), which partially
inserts into the membrane in the presence of the pmf. Thus, the effects
of the positive charges are accumulative, lending further evidence that
an electrophoretic mechanism is in operation to help insert
sec-independent membrane proteins. It is unclear why none of the (+2M)
mutants inserted into the membrane, even without a pmf present.
The requirement for specific components of the pmf has been examined
for several secreted proteins (45, 46) but for only one sec-independent
protein, the E. coli melibiose permease (11). Those studies
found that both components of the pmf could be utilized for secretion
or insertion. Here we show for the first time that the 
is
sufficient to hinder the insertion of basic residues and to promote the
translocation of negatively charged amino acids (Fig. 5A).
The finding that positive charges are able to better translocate when
the pH of the periplasm is set at 6.5 and when CCCP is added was
unexpected. This may be due to the fact that anionic phospholipids can
act as determinants of membrane topology as well and prevent
translocation of positively charged residues (5). It is possible that
the positive charges in the periplasmic loop of procoat were able to
move across the membrane more efficiently when CCCP abolishes the pH
gradient. These positive charges might not be attracted as strongly to
the anionic phospholipids (at pH 6.5) because of increased protonation
of these anionic lipids on the cytoplasmic side of the membrane.
The significance of our studies may also extend into the eukaryotic
realm. A subset of inner mitochondrial membrane proteins is sorted from
the matrix by a process analogous to the sec-independent process found
in bacteria. It has been found that certain amino-terminal region and
loops of these proteins require the 
for insertion and that these
domains are often enriched in acidic residues (for review see Ref. 47).
In addition, the topology of mitochondrial inner membrane proteins
conforms to the positive inside rule that is found in E. coli (48). Most importantly, the introduction of positively
charged residues blocks amino-terminal translocation from the matrix
(47, 49), thereby lending evidence that the pmf is one basis for the
positive inside rule in mitochondria.