From the Department of Chemistry, The Ohio State University,
Columbus, Ohio 43210
We have shown previously that the first
transmembrane segment of leader peptidase can function to translocate
the polar amino-terminal Pf3 domain across the membrane into the
periplasm independently of the proton motive force (pmf) (Lee, J. I., Kuhn, A., and Dalbey, R. E. (1992) J. Biol.
Chem. 267, 938-943). We now show that when the first
transmembrane segment lacks a strong hydrophobic character, the pmf is
required for translocation. In addition, we find that the
amino-terminal acidic residue proximal to the transmembrane domain
plays a critical role in pmf-dependent amino-terminal
translocation. Moreover, the pmf is required to hold the amino-terminal
domain in the periplasm to prevent it from slipping such that the amino terminus is no longer exposed to the periplasm. In all cases, translocation occurs under conditions in which the function of the Sec
machinery is impaired. These studies show that the low hydrophobicity
of the first apolar domain (the translocation signal) can be
compensated for by a negative charge in the amino-terminal region, upon
which the pmf acts.
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INTRODUCTION |
The mechanisms by which a protein integrates into the
membrane and assumes its correct topology has been studied with great interest (1, 2). It has been shown that membrane proteins in bacteria
can utilize the Sec machinery to assemble across the plasma membrane of
Escherichia coli. However, there are a growing number of
membrane proteins that apparently do not use the Sec machinery (3).
This sec-independent class includes membrane proteins that are made
without a signal sequence and that are oriented with the amino terminus
on the periplasmic side of the plasma membrane
(NoutCin orientation).
Although the exact mechanism by which these proteins assemble across
the membrane is not known, the factors that influence the membrane
topology have been elucidated. For example, it has been previously
found that the asymmetric distribution of charged residues,
particularly basic residues flanking a membrane-spanning domain on the
cytoplasmic side, acts as a topogenic determinant for translocation
(4-7). Positive charges are retained in the cytoplasm and inhibit
translocation of polar regions (8-11). Negatively charged amino acids,
in low abundance, do not inhibit translocation (11, 12). Several groups
have postulated that the membrane topology may be determined by
alignment of dipoles and charged groups of the protein within the
electrical field of the proton motive force or
pmf1 (10-14). This
electrophoretic force is a result of the transmembrane electrical
gradient, 
(positive outside, negative inside), which may
actively promote translocation of acidic residues (11, 12, 15) and
impede translocation of basic residues (14). In support of this
hypothesis, Kiefer and colleagues (16) found that acidic residues in
the amino-terminal region of the Pf3 coat protein (NoutCin) are translocated across the membrane
by the pmf and can act as topogenic determinants.
Another factor that affects membrane insertion of a protein is the
overall hydrophobicity (or length) of a transmembrane segment (17). It
is thought that apolar residues within hydrophobic domains allow their
partitioning into the membrane in an energetically favorable manner,
driving insertion (18-20). Although there have been several studies
conducted on the role of hydrophobicity in cleavable signal peptides
(21-26) and uncleaved signals (17, 27) which undergo
sec-dependent translocation in bacteria, the role of the
hydrophobicity of transmembrane domains which support amino-terminal
translocation has not been investigated.
What are the structural requirements for amino-terminal
translocation for proteins that lack signal (or leader) peptides? In
bacteria, translocation of amino-terminal sequences requires a
downstream hydrophobic segment (28, 29) and is most efficient when the
amino-terminal region is short (14) and contains few positively charged
residues (14, 29). Typically, the energy source driving translocation
is the pmf which is most likely correlated with the acidic charge
content of the periplasmic amino-terminal domains. Translocation of
amino-terminal segments is believed to be Sec-independent because
translocation is unaffected by treatments that block the normal
function of the Sec machinery (3). In eukaryotes, amino-terminal
translocation is dependent not only on the charge difference (30)
between the transmembrane flanking segments with the more positive side
typically retained in the cytoplasm but also on the folding state of
the amino-terminal domain and the hydrophobicity of the translocation
signal (31). Long hydrophobic sequences favor amino-terminal
translocation, whereas short hydrophobic sequences favor
carboxyl-terminal translocation (32).
We have used leader peptidase (lep) of Escherichia
coli as a model system for studying amino-terminal translocation
(14, 28). Lep spans the membrane twice with its amino terminus on the
periplasmic surface of the membrane and its large carboxyl-terminal domain protruding into the periplasm (28, 33, 34). We have shown
previously that the first hydrophobic domain (H1) of lep can function
to translocate a short, polar amino-terminal 18 amino acid antigenic
peptide from the phage Pf3 coat protein across the plasma membrane of
Escherichia coli (28). We have now examined the energetic
requirements necessary for the insertion of amino-terminal periplasmic
domains and have determined that the hydrophobicity of the
transmembrane domain and the pmf are both required to varying degrees.
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EXPERIMENTAL PROCEDURES |
Strains and Plasmids--
E. coli strain MC1061
(
lacX74, araD139,
(ara,
leu)7697, galU, galk, hsr, hsm, strA) was from
our laboratory. XL1-blue (supE44, hsdR17,
recA1, endA1, gyrA46, thi
relA1, lac
) was acquired from Stratagene.
Pf3-lep and its derivatives were expressed using the pING plasmids
(35), which contains the arabinose promoter and the arabinose
regulatory elements (Ingene, Inc.).
Materials--
Lysozyme, amino acids, PMSF, and CCCP were
purchased from Sigma. DpnI was from New England Biolabs.
Pfu DNA polymerase was from Stratagene. Proteinase K was
from Boehringer Mannheim. Tran35S-label, a mixture of 85%
[35S]methionine and 15% [35S]cysteine,
1000 Ci/mmol, was from ICN. Dideoxynucleotides were from Amersham
Pharmacia Biotech. Ampliwax was from Perkin-Elmer. PAGE-purified
oligonucleotides were from Integrated DNA Technologies and Qiagen midi
kits were from Qiagen.
DNA Manipulations--
Site-directed mutagenesis was
accomplished using the Stratagene QuikChange procedure with a few
alterations. The mutagenesis reaction was physically separated into two
portions with Ampliwax beads. The lower portion contained 2.5 µl of
the 10-fold reaction buffer, the mutagenic oligonucleotides (125 ng
each), deoxynucleotide triphosphates (50 µM each, final
concentration), and double distilled water to a final volume of 25 µl. An Ampliwax bead was placed on top, and a wax layer was created
by heating and chilling the tube. The upper reagent, administered above
the wax layer, contained 2.5 µl of the 10-fold reaction buffer, 50 ng
of double-stranded DNA, 1 µl of Pfu DNA polymerase and
double distilled water to a final volume of 25 µl. Mutagenesis was
then carried out in a Progene thermocycler following the QuikChange
protocol. Following DpnI treatment and transformation,
mutations were screened by sequencing double-stranded DNA (36) using
U. S. Biochemical Corp. Sequenase version 2.0. Mutant constructs
identified by sequencing were then transformed into MC1061 using the
calcium chloride method (37).
Protease Mapping Studies--
MC1061 cells (1 ml) bearing the
pING plasmid encoding Pf3-lep proteins were grown to the midlog phase
in M9 minimal media (38) containing ampicillin (100 µg/ml) at pH 7.0 with 0.5% fructose and 50 µg/ml of each amino acid except
methionine. The cells were induced with arabinose (0.2%, final
concentration) for 1 h to express the Pf3-lep mutant proteins.
Cells were labeled with 100 µCi of
trans-[35S]methionine for 1 min, chilled on
ice, then collected by centrifugation (16,000 × g,
4 °C, 40 s). After resuspending in 0.25 ml of Tris acetate, pH
8.2, 0.5 M sucrose, and 5 mM EDTA, the cells
were treated with lysozyme (80 µg/ml, final concentration) and 0.25 ml of ice-cold water. After incubating for 5 min, 30 µl of 1 M MgSO4 were added to stabilize the
spheroplasts, and the cells were collected by centrifugation
(16,000 × g, 4 °C, 40 s). The spheroplasts
were gently resuspended in 50 mM Tris acetate, 0.25 M sucrose, 10 mM MgSO4 and
incubated with or without proteinase K (1.5 mg/ml) for 1 h on ice.
Another aliquot of spheroplasts was treated with 2% Triton X-100 and
incubated with proteinase K (1.5 mg/ml). After quenching the protease
with PMSF (5 mM, final concentration) for 5 min, the
samples were acid-precipitated and immunoprecipitated with antibody
against lep, ribulokinase, a cytoplasmic marker, and outer membrane
protein A (OmpA), as described (11). Samples were then analyzed by
SDS-PAGE with a 15% polyacrylamide gel and subjected to fluorography
(39).
Quantitation of the Translocation Data--
Fluorographs were
scanned using an AppleOne Scanner. The bands were then quantitated by
using the public domain program NIH Image, developed at the National
Institutes of Health.2 The
percent of amino-terminal translocation was determined by taking into
account the number of methionines lost during proteolysis by proteinase
K. For most of the constructs there was one methionine lost out of
eight (see Equations 1, 3, and 4), whereas for
4-9,
4-22, and
the constructs that contain both the amino-terminal domain and the
membrane-spanning domain of Pf3 coat there was one methionine
lost out of seven (see Equations 2-4).
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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Determination of Summed Hydrophobicity--
The summed
hydrophobicity (H) of the transmembrane region flanking the
amino-terminal domain was determined as described (17) using the GES
scale (40). We calculated H for the native and mutant
Pf3-lep proteins by adding the hydrophobicity of the uninterrupted stretch of uncharged amino acids within the transmembrane region.
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RESULTS |
The Role of Amino-terminal Aspartic Acids in Amino-terminal
Translocation--
The amino terminus of Pf3-lep has previously been
shown to translocate in a pmf- and sec-independent manner (28). This is in contrast to amino-terminal translocation of the sec-independent Pf3
coat protein (see Fig. 1A for
topology), which requires the pmf (41) and the aspartyl residue at
position 18 of the amino-terminal domain (16) also present in Pf3-lep.
Therefore, we have investigated the role of acidic residues in
amino-terminal translocation using Pf3-leader peptidase (Pf3-lep) with
an F79R mutation as our model protein (Fig. 1A). This
construct contains the 18 amino acid Pf3 region, from the amino
terminus of Pf3 coat, fused to the fourth amino acid of leader
peptidase (lep) with a threonine linking the two domains (Fig.
1B). Pf3-lep is oriented in the plasma membrane with the
amino-terminal Pf3 domain in the periplasm, a single hydrophobic
membrane-spanning domain (H1), and the carboxyl-terminal lep domain in
the cytoplasm (Fig. 1A). The introduction of an arginine at
position 79 of lep inhibits insertion of the second transmembrane
domain and translocation of the carboxyl terminus (14, 27). The
resulting protein contains only one translocated region which allows us
to monitor translocation of the amino terminus of Pf3-lep across the
plasma membrane via protease mapping techniques.

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Fig. 1.
Mutants used to analyze the importance of
acidic residues for amino-terminal translocation. A,
membrane topology of Pf3-lep and Pf3 coat in the plasma membrane of
E. coli. Apolar segments are represented by cylinders, and
polar segments are depicted by lines. B, mutants
of Pf3-lep and their membrane translocation properties.
Numbers to the right of lep indicate the position
of the acidic residue within the Pf3 coat amino-terminal domain in
which an aspartic acid was mutated to an alanine residue.
Amino-terminal translocation was determined in the presence
( CCCP) or absence (+CCCP) of a pmf.
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We conducted site-directed mutagenesis on the Pf3 domain of Pf3-lep to
determine whether a similar requirement for the aspartyl residues
existed for efficient translocation of Pf3-lep. We replaced the
aspartyl residues at positions 7 and 18 of the Pf3 domain with
alanines, either as a single mutation or as a double mutation (Fig.
1B). Amino-terminal translocation was monitored by protease mapping in the absence or presence of the protonophore CCCP, which destroys the pmf.
Expression of Pf3-lep in exponentially growing cells was induced with
0.2% arabinose for 1 h at 37 °C. Cells producing Pf3-lep or
aspartyl residue mutants were then pulse-labeled with 100 µCi of
35S-trans-labeled methionine for 1 min and then
converted to spheroplasts (42). To examine translocation in the absence
of a pmf, CCCP was added for 45 s prior to labeling. Protease
mapping was accomplished by dividing spheroplasts into three equal
fractions. The first fraction was incubated on ice for 1 h and
then trichloroacetic acid-precipitated. The second fraction was
incubated on ice with 1.5 mg/ml proteinase K for 1 h, quenched
with PMSF (final concentration, 5 mM), followed by
trichloroacetic acid precipitation. The third fraction was treated for
1 h with both 1.5 mg/ml proteinase K and 2% Triton X-100 followed
by treatment with PMSF and trichloroacetic acid precipitation. After
each sample was divided into two, one-half was immunoprecipitated with
anti-leader peptidase antibody and the other half was
immunoprecipitated with anti-OmpA and anti-ribulokinase antibodies. The
samples were then applied to a 15% SDS-PAGE and the results
analyzed by fluorography.
As shown in Fig. 2A,
amino-terminal translocation was observed in Pf3-lep (lanes
1-3), 7A Pf3-lep (lanes 4-6), 18A Pf3-lep (lanes 7-9), and 7,18A Pf3-lep (lanes 10-12),
in the presence (
CCCP) of a pmf. This is detected by the appearance
of a smaller protease-resistant fragment seen in lanes treated with
proteinase K (see arrow). Since the proteinase-treated
fragments differ by only 18 amino acids, there is a slight shift in
molecular weight. These results show that the acidic residues are not
required for translocation of the amino terminus of Pf3-lep, in
contrast to Pf3-coat protein (16). When the translocation study is
carried out in the absence (+CCCP) of a pmf, amino-terminal
translocation is still very efficient as seen in Fig. 2B
(lanes 1-3, 4-6, 7-9, and 10-12). The total
amount of translocated domain in the presence or absence of a pmf was
quantitated by scanning the fluorograms and using the public domain
program NIH Image to determine the percent translocated. As shown in
Fig. 1B, 98% of the proteins translocated their
amino-terminal domains efficiently both in the presence and in the
absence of a pmf. Successful conversion to spheroplasts was monitored
in all experiments by digestion of OmpA, which is exported across the
inner membrane and is completely accessible to proteinase K. The
integrity of the spheroplasts was determined by monitoring the
stability of cytoplasmic ribulokinase in the presence of proteinase K
(Fig. 2). Furthermore, the depletion of the proton gradient was
demonstrated by the inhibition of pro-OmpA translocation (+CCCP), as
the accumulated pro-OmpA is protease-resistant in these studies (Fig.
2B).

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Fig. 2.
Protease mapping to monitor amino-terminal
translocation of the Pf3-lep mutants in the presence or absence of a
pmf. A, exponentially growing cells (1 ml) of MC1061 with
plasmids expressing the parent Pf3-lep or Pf3-lep mutants were
pulse-labeled with 100 µCi of
trans-[35S]methionine for 1 min and then
converted into spheroplasts as described under "Experimental
Procedures." B, for those samples treated with CCCP, 5 µl of 10 mM CCCP was added 45 s prior to adding
[35S]methionine and analyzed in the exact manner as
before. Aliquots of the spheroplasts (100 µl) were incubated on ice
with or without proteinase K (final concentration 1.5 mg/ml) on ice for
1 h. A lysis control was included by adding proteinase K (1.5 mg/ml final concentration) and Triton X-100 (2% final concentration).
Proteinase K was inactivated with 5 mM PMSF for 5 min. The
samples were acid-precipitated and then immunoprecipitated with
antisera to leader peptidase (lep), OmpA, and ribulokinase
(Rib).
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Although both Pf3-lep and the Pf3 coat protein contain the same
amino-terminal 18 amino acid residues, they do not have the same
requirement for aspartic acid residues for efficient amino-terminal translocation. This indicates that there is some other important factor
in the lep domain of Pf3-lep that promotes translocation.
The PMF Can Promote Translocation When Apolar Domain 1 Has Low
Hydrophobicity--
To determine the role of the hydrophobicity of an
NoutCin transmembrane segment in amino-terminal
translocation, we constructed several deletion mutations within the
transmembrane domain of Pf3-lep. The constructs are shown in Fig.
3 in the order of most hydrophobic to
least hydrophobic transmembrane domains. The total hydrophobicity was
determined for the uninterrupted stretches of hydrophobic residues
(40). The hydrophobicity scale used in these studies is the transfer
free energy (kcal/mol) of the amino acid side chains from water to a
non-aqueous environment. Since the transmembrane domain is more
hydrophobic at the amino end than the carboxyl end, deletions at this
location are more detrimental to amino-terminal translocation. Protease
mapping was then performed as before, in the presence of the pmf (Fig. 4A). Amino-terminal
translocation was quantitated and OmpA and ribulokinase controls were
also carried out as before (data not shown). Although Pf3-lep with an
intact transmembrane domain translocates efficiently (99%), an effect
can already be seen on the translocation of the smallest deletions,
17-22 (lanes 1-3) and
4-9 (lanes 4-6),
with 93 and 91% translocating, respectively. For
13-22 (lanes
7-9) and
4-12 (lanes 10-12), translocation is
roughly half as efficient as it is for the full-length protein (66 and 54%, respectively). Finally, translocation is completely abolished for
the larger deletions
9-22 (lanes 13-15),
4-17
(lanes 16-18), and
4-22 (lanes 19-21),
showing that the hydrophobicity of a downstream transmembrane domain is
essential for amino-terminal translocation.

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Fig. 3.
Summary of deletions within the H1 of
Pf3-lep. Amino-terminal translocation was monitored in cells with
or without a pmf. The summed hydrophobicity H is also
indicated.
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Fig. 4.
Amino-terminal translocation of deletion
mutants in which the hydrophobicity of the first transmembrane segment
is reduced. E. coli strain MC1061 containing the plasmid
encoding Pf3-lep 17-22, 4-9, 13-22, 4-12, 9-22, 4-17,
and 4-22 was analyzed for amino-terminal translocation either with
(A) or without (B) the pmf, as described in Fig.
2.
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In contrast, when translocation was carried out in the absence of a pmf
(CCCP treated cells), only the most hydrophobic mutant,
17-22
(lanes 1-3), was partially translocated (54%, Fig.
4B), whereas the other deletion mutants were completely
blocked. Therefore, amino-terminal translocation only occurs with the
full-length Pf3-lep and
17-22 in the absence of a pmf. This
indicates that the pmf can promote membrane insertion of proteins with
apolar domains with low hydrophobicity.
Acidic Residues Are Required for Amino-terminal Translocation When
the Hydrophobicity of the Transmembrane Domain Is
Decreased--
Although we have shown that negatively charged amino
acids are not required for translocation of the amino terminus of
Pf3-lep with a full-length transmembrane domain (Fig. 2), we wanted to test whether translocation of the amino terminus of Pf3-lep with a
truncated H1 requires the amino-terminal aspartyl residues. This is of
particular interest since
13-22 requires the presence of a pmf for
translocation, as shown in Fig. 4. We used site-directed mutagenesis to
create mutations within the Pf3 domain of
13-22, by substituting the
aspartic acid residues at positions 7 and 18 of the amino-terminal
domain with alanines as before (Fig. 5A). We then conducted
protease mapping experiments using cells that were pulse-labeled with
trans-[35S]methionine in the presence and in
the absence of the pmf to determine the competency for amino-terminal
translocation. Whereas the amino-terminal domains of
13-22 and 7A
13-22 translocate with equal efficiency in the presence of a pmf, at
66 and 58%, respectively, the amino-terminal domain does not
translocate in the 18A
13-22 mutant and the double mutant 7,18A
13-22 (Fig. 5B). We should note that the low molecular
weight bands observed in lanes 5, 8, and 11 are
uncharacterized proteolytic fragments. In the absence of the pmf,
translocation of all four of these proteins is abolished (data not
shown). Therefore, we found that the acidic residue proximal to the
transmembrane domain is absolutely required for
pmf-dependent translocation of the amino-terminal domain of
13-22 (Pf3-lep with a truncated transmembrane domain), in agreement
with the results found for Pf3 coat (16). This suggests that the pmf is
acting on the aspartyl residue at position 18 of the amino-terminal Pf3
domain of Pf3-lep to drive translocation when the hydrophobicity of the
membrane-spanning domain is low.

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Fig. 5.
The acidic residue at position 18 of the
amino-terminal domain is required for amino-terminal translocation of
13-22. A, construction of acidic amino acid mutants in
the amino-terminal domain. B, protease-mapping of 13-22
mutants. Cells expressing 13-22 native, 7A,18A, and 7 and 18A were
grown, labeled, and analyzed as described in Fig. 2.
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Role of PMF in Sustaining Amino-terminal Translocation--
We
wanted to determine if the pmf was also required to sustain
amino-terminal translocation of Pf3-lep in which the hydrophobicity of
apolar domain 1 had been decreased. In this study, we characterized Pf3-lep with a full-length transmembrane segment,
4-9, which is
dependent on the pmf for translocation, and
13-22, which had an even
stronger defect in translocation as shown in Fig. 4. We pulse labeled a
2-ml culture for 1 min using 200 µCi of
trans-[35S]methionine followed by a 5-min
chase of cold methionine (500 µg/ml). One-third of the sample was
then chilled, and the remaining cells were treated with 5 µl of 10 mM CCCP for 1 min. At this time, one-half of the remaining
sample was then removed and chilled as above, whereas the remaining
cell culture was treated with 2-mercaptoethanol to inactivate the CCCP
and allow the pmf to regenerate (43). These samples were then washed to
remove the 2-mercaptoethanol and incubated at 37 °C for 10 min to
re-establish the pmf (see figure legend). All the samples were then
converted into spheroplasts and protease-mapped as described
previously. We found that Pf3-lep with a full-length apolar domain 1 was unaffected by this treatment (Fig.
6A, lanes 1-9). To our
surprise, we found that both
4-9 and
13-22, which had undergone
amino-terminal translocation (91 and 66%, respectively) after a 5-min
chase (Fig. 6, B and C, lanes 1-3;
denoted as Pulse
CCCP), were not as accessible to protease when the pmf was abolished following pulse labeling (Fig.
6, B and C, lanes 4-6; denoted as
Pulse +CCCP). We found that the extent to which
the amino terminus had "slipped" out of the periplasmic space was
correlated with the total amount of hydrophobicity in the apolar domain
1. Although full-length Pf3-lep does not "slip" out from the
periplasmic space (hydrophobicity of
46 kcal/mol) when the pmf was
abolished, only 62% of
4-9 with a total hydrophobicity of
28
kcal/mol (Fig. 3) and none of
13-22 with a total hydrophobicity of
25 kcal/mol remain translocated. We are convinced that this effect
was due to the abolishment of the pmf because when we allow the pmf to
regenerate in the presence of 2-mercaptoethanol for 10 min,
translocation of the amino-terminal domains is recovered to the same
extent as before and are digested by protease (81 and 50%,
respectively) Fig. 6, B and C, lanes 7-9
(denoted as P/+CCCP 10' Recover). The results
indicate that when the transmembrane domain is not adequately
hydrophobic, as in the cases of
4-9 and
13-22, the pmf is
required not only for initiation of amino-terminal translocation but
also to sustain translocation of the Pf3 region. We hypothesize that
the pmf holds the translocated amino-terminal domain with the proximal
acidic residue in the periplasm and therefore prevents this domain from "slipping" out from the periplasmic space.

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Fig. 6.
The PMF is required to sustain the
amino-terminal region in the periplasmic space when the first apolar
domain lacks strong hydrophobic character. MC1061 cells harboring
plasmids that encode Pf3-lep (A), 4-9 (B), or
13-22 (C) were grown, induced, and labeled as described
previously in Fig. 2. The procedures are outlined under "Results"
and the methods for the pmf recovery followed Wolfe and Wickner (43)
with the following alterations. Cells pulse-labeled, chased, and
CCCP-treated were washed in M9 media twice and incubated in M9 media
containing 10 mM 2-mercaptoethanol, 500 µg/ml cold
methionine, and 1 mg/ml chloramphenicol for 2 min on ice. The cells
were then washed to remove the 2-mercaptoethanol and incubated in M9
media for 10 min at 37 °C with shaking. The cells were then
harvested and converted to spheroplasts and protease-mapped as
before.
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PMF-dependent Translocation of the Amino Terminus of
Pf3-lep When H1 of Lep Is Replaced with the Transmembrane Segment of
the Pf3 Coat Protein--
Although the Pf3 coat protein requires a
pmf, Pf3-lep does not. One possible reason for this difference is that
the transmembrane segment of Pf3 coat is less hydrophobic than that of
lep. In view of our results with respect to hydrophobicity, we wanted
to determine the outcome of replacing the H1 of lep, with a summed
hydrophobicity of
46 kcal/mol, with that of the Pf3 coat protein,
which has a hydrophobicity of
39 kcal/mol. Oligonucleotide-directed
mutagenesis was used to create this mutant. The
4-22 Pf3-lep mutant,
in which the first transmembrane domain of lep was deleted, was used as a template for mutagenesis. The insertion of the Pf3 coat transmembrane domain was completed in two rounds of mutagenesis in which nine amino
acids were first inserted, followed by the insertion of the remaining
nine amino acids. The completed mutant was sequenced and the
transmembrane segment was found to be genetically identical to that of
the Pf3 coat protein. This mutant protein, Pf3H1-lep, contains the
amino-terminal domain and the transmembrane domain of the Pf3 coat
protein fused to the 23rd amino acid of lep. Pf3H1-lep was
protease-mapped in cells that were analyzed for translocation in the
presence and absence of a pmf, as described previously (Fig.
7). Pf3H1-lep translocates efficiently in
the presence of a pmf (lanes 1-3) (82%) and is impeded
without a pmf (lanes 4-6) (61%). However, it has been
shown that translocation of Pf3 coat is abolished in the absence of a
pmf (16). Since lep also has two additional hydrophobic regions (H2 and
H3) unlike Pf3 coat, we deleted them to determine if amino-terminal
translocation occurs in a manner more similar to that of Pf3 coat. In
the presence of a pmf, Pf3H1-lep
62-98 translocated with a 99%
efficiency (lanes 7-9), and 33% was translocated in the
absence of a pmf (lanes 10-12). These results show that
pmf-dependent translocation of the amino terminus of
Pf3-lep with the transmembrane segment of the Pf3 coat protein is
enhanced when H2 and H3 are omitted.

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Fig. 7.
Protease accessibility of Pf3-lep mutants
where H1 of lep has been replaced with the apolar domain of the
Pf3-coat protein. Exponentially growing MC1061 cells containing
plasmids expressing Pf3-lep with the apolar domain of the Pf3-coat
protein were analyzed for amino-terminal translocation with either H2
and H3 regions intact or deleted, in the presence or absence of a
pmf.
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The Amino-terminal Domain of Pf3-lep Translocates under Conditions
Where the Sec Machinery Is Impaired--
We tested whether Pf3-lep
becomes dependent on the Sec machinery when the hydrophobicity of the
first hydrophobic domain is decreased. To test this, we conducted
protease mapping experiments on cells expressing
13-22 in which the
function of the Sec machinery was compromised. As a control, we
confirmed that Pf3-lep with a full-length apolar domain 1 inserts
independently of a functioning Sec machinery (14). Cells were grown to
mid-log phase and induced with arabinose to express the plasmid-encoded
proteins for 1 h. The cells were treated with azide for 5 min,
followed by pulse labeling for 1 min, and then analyzed by protease
mapping; azide has been shown to inhibit the SecA ATPase activity (44).
We show that azide treatment does not affect amino-terminal
translocation of either of these proteins (Fig.
8, A and B, lanes
1-3). Furthermore, translocation is not affected in
SecAts or SecYts cells when grown at the
non-permissive temperature, 42 °C. As shown in Fig. 8A,
Pf3-lep translocates its amino terminus to the same extent in cells
with impaired SecA (lanes 4-6) or SecY (lanes 7-9) as in cells with functional SecA and SecY (Fig. 2A,
lanes 1-3). Similar results are found with
13-22 (Fig.
8B, lanes 4-9). Under these experimental
conditions where the function of SecA or SecY is impaired, the
translocation of OmpA is completely blocked as indicated by the fact
that pro-OmpA is protease-resistant. These results suggest that
amino-terminal translocation occurs in a Sec-independent manner when
the transmembrane segment has either high or low hydrophobicity.

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Fig. 8.
Translocation of the amino-terminal domain is
not dependent on functional Sec proteins when H1 hydrophobicity is
reduced. A, amino-terminal translocation of Pf3-lep.
B, amino-terminal translocation of 13-22. For azide
studies, MC1061 cells bearing a plasmid encoding Pf3-lep and 13-22
were grown to the mid-log phase. After a 1-h induction of
plasmid-encoded proteins by arabinose (0.2%, final concentration),
cells were treated with 3 mM azide for 5 min and then
labeled with 100 µCi of
trans-[35S]methionine for 1 min. Aliquots were
analyzed for protease mapping as described in Fig. 2. For
SecAts studies, E. coli CJ105 cells bearing the
plasmid encoding Pf3-lep and 13-22 were grown to the mid-log phase
at 30 °C, shifted to 42 °C, and induced with 0.2% arabinose for
1 h and then labeled with 100 µCi of
trans-[35S]methionine for 1 min. For
SecYts studies, E. coli CJ107 cells bearing the
plasmid encoding Pf3 lep or 13-22 were grown and labeled as
described for the SecA studies. Following pulse labeling, the cells
were converted to spheroplasts and analyzed as described in Fig. 2.
CJ105 and CJ107 are derivatives of HJM114 (F' lac, pro[ lac pro])
and have been described (46).
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DISCUSSION |
In this report we have examined why amino-terminal translocation
of some proteins, such as Pf3 coat (41) and ProW (45), requires the pmf
and leader peptidase (28) does not. Our results show that when the
hydrophobic character of a transmembrane segment is high,
amino-terminal translocation occurs independently of the pmf.
However, translocation is dependent on the pmf which acts on a negative
charge when the hydrophobic character of the transmembrane segment is
low.
As a general rule, we found that amino-terminal translocation was
decreased as the hydrophobicity of the transmembrane domain 1 was
decreased. Even the deletion of four amino acids had a noticeable affect on translocation, where translocation decreased from 99 to 91%.
Almost equally surprising was the finding that amino-terminal translocation could be supported, although less efficiently, by a
mutant that contained a stretch of only nine uncharged amino acids,
13-22. However, translocation was blocked by decreasing the
hydrophobic character further.
Strikingly, we observed an increased pmf requirement for
translocation when the hydrophobicity of apolar domain 1 was decreased (Figs. 3 and 4). The only mutant that was able to translocate in a
pmf-independent manner,
17-22, had a very small deletion in the
first transmembrane domain. This suggests that it is the overall high
hydrophobic content of transmembrane segment 1 of the native leader
peptidase that enables translocation of its amino terminus across the
membrane independently of the pmf. This may explain the pmf requirement
of Pf3 coat (16) since the Pf3 coat has a transmembrane segment that is
less hydrophobic than the first transmembrane segment of lep. Indeed,
we observed pmf-dependent translocation of the amino
terminus of Pf3-lep when H1 of lep was replaced with the transmembrane
segment of the Pf3 coat protein. The pmf dependence was even more
dramatic when the H2 and H3 domains of lep were deleted preventing them
from aiding translocation of the amino-terminal domain (Fig. 7).
Fig. 9 illustrates the results in which
the translocation data of the Pf3-lep mutants are plotted against the
hydrophobicity of the respective H1 regions. The hydrophobicity scale
of Engelman et al. (40) was used to determine the
total hydrophobicity. Translocation of the amino-terminal region can
occur efficiently in the presence of a pmf (
CCCP) when the summed
hydrophobicity of the transmembrane segment exceeds a threshold of
roughly
21 kcal/mol, whereas translocation is inefficient even in the
presence of a pmf below a threshold of roughly
18 kcal/mol. These
results indicating that hydrophobicity of a signal domain is required for translocation of amino-terminal domains are in strong agreement with Lee and Manoil (17), where they studied an
NinCout uncleaved signal of the E. coli serine chemoreceptor (Tsr). In their study, they used a
hybrid protein where alkaline phosphatase (phoA) was fused immediately
after the first transmembrane segment of Tsr. They found that arginine
mutants showing high export of phoA had summed hydrophobicities greater
than
23 kcal/mol (for the largest, uninterrupted stretch of
hydrophobic amino acids), whereas low export (<15%) of phoA mutants
had summed hydrophobicities of less than
23 kcal/mol. We obtained
similar results with our single and double arginine mutants (data not
shown).

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Fig. 9.
Plot of amino-terminal translocation
versus transmembrane hydrophobicity. These results are
derived from the experiments in Fig. 4. The quantitation of
translocation and the summed hydrophobicity of the first transmembrane
region for the various mutants is shown in Fig. 3. The pmf was
dissipated by the addition of CCCP.
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The other intriguing finding (see Fig. 9) is that to achieve
approximately 50% amino-terminal translocation, a summed H1
hydrophobicity of
21 kcal/mol is required in the presence of the pmf,
compared with a summed hydrophobicity of
32 kcal/mol in the absence
of the pmf. This suggests that the pmf contributes energy that is equivalent to
11 kcal/mol of hydrophobicity, i.e. four
leucine residues.
Our results also demonstrate that the amino-terminal aspartyl residue
flanking the transmembrane segment is essential for pmf-dependent translocation of
13-22. This confirms that
aspartic residues can play an active role in amino-terminal
translocation as previously suggested (16). It is striking that the pmf
is not only required for initiation of translocation but also to stabilize the amino-terminal domain of
4-9 and
13-22 in the periplasm to prevent it from slipping away from the periplasmic space
so that it is not accessible to protease (Fig. 6, B and C). In contrast, slipping is not observed for Pf3-lep which
has a transmembrane segment with high hydrophobicity (Fig.
6A). How the pmf holds the amino-terminal domain on the
periplasmic side of the inner membrane is not known at this time.
Although it is possible that the amino-terminal polar domain is merely
slipping into the membrane bilayer, we think that this would be
unlikely because it would be energetically unfavorable for the polar
domain to reside within the apolar membrane bilayer.
One possible role of a pmf is that it is affects translocation, via
direct interaction of the negatively charged residues of the inserting
membrane protein, with its electrical potential component (positive
outside, negative inside). Our data do not directly address this
possibility. However, if such a simple electrophoretic mechanism was
operating in E. coli it does not explain why the acidic
residue at position 18 would be required and not the one at position 7. A second possibility is that the pmf acts via a transmembrane pH
gradient. If this were the case, a protein machinery, which protonates
and deprotonates amino acid residues, would most likely be
involved.
In conclusion, the present data provide strong evidence that the pmf,
acting on an acidic residue, can compensate for the low hydrophobicity
of the translocation signal. This indicates that hydrophobic forces and
proton motive forces work together in the translocation process.