Defective translocation of a signal sequence mutant in a
prlA4
suppressor strain of
Escherichia coli
Hendrik Adams
1
, Pier A. Scotti
2
*, Joen Luirink
2
and Jan Tommassen
1
1
Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, The Netherlands;
2
Department of
Microbiology, Institute of Molecular Biological Sciences, Biocentrum Amsterdam, The Netherlands
In the accompanying paper [Adams, H., Scotti, P.A., de
Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Bio-
chem. 269, 5564–5571.], we showed that the precursor of
outer-membrane protein PhoE of Escherichia coli with a Gly
to Leu substitution at position )10 in the signal sequence
(G-10L) is targeted to the SecYEG translocon via the signal-
recognition particle (SRP) route, instead of via the SecB
pathway. Here, we studied the fate of the mutant precursor
in a prlA4 mutant strain. prlA mutations, located in the secY
gene, have been isolated as suppressors that restore the
export of precursors with defective signal sequences.
Remarkably, the G-10L mutant precursor, which is nor-
mally exported in a wild-type strain, accumulated strongly in
a prlA4 mutant strain. In vitro cross-linking experiments

binds to particularly hydrophobic signal sequences when
they emerge from the ribosome [12,13]. The resulting SRP–
ribosome-nascent-chain (SRP–RNC) complex is then tar-
geted via FtsY to the inner membrane [14,15]. Upon release
of the SRP, the nascent chain inserts into the membrane
near the translocase components SecA, SecY, and SecG,
indicating that the SecB-targeting and SRP-targeting path-
ways converge at a common translocon [16].
The components of the Sec system were originally
identified by two different genetic approaches. One method
implicated the isolation of conditionally lethal mutants with
generalized secretion defects. The second approach eman-
ated from the idea that the signal sequence is recognized by
components of the export apparatus, and that specific
mutations in genes for Sec components would restore the
recognition of mutated signal sequences. Indeed, this
method resulted in the isolation of extragenic suppressor
mutations in prl (protein localization) genes directly
involved in protein translocation (prlA alleles of secY, prlD
alleles of secA and prlG alleles of secE) [17]. However, the
lack of allele specificity of prlA and prlG mutations with
respect to the suppression of signal sequence defects and the
observation that these prl mutants are able to translocate
even proteins without a signal sequence [18–20] argue
against the basic idea of the screening method. Studies with
a prlA4 suppressor strain revealed that the Sec translocon in
this strain facilitates translocation of preproteins with folded
domains [21], showed an increased affinity for SecA [22,23],
and is composed of subunits that are more loosely
associated than in the wild-type strain [24]. Therefore, it

laboratory, we use the SecB-dependent outer-membrane
protein PhoE [3,31] as a model protein to study protein
transport. In previous studies, we showed that a single
amino-acid substitution, G-10L (the residue at position )1
precedes the signal-peptidase cleavage site), in the hydro-
phobic core of the signal sequence of PhoE relieved the pmf
dependency of protein translocation [29] and shunts the
precursor via the SRP pathway to the Sec machinery [32].
Other experiments revealed that the prlA4 mutation in secY
reduced the pmf dependency of protein translocation of
wild-type precursors [21]. The initial goal of the present
study was to investigate whether the prlA4 mutation is able
to suppress the pmf dependency of the translocation of
(G-10L)prePhoE even further. Instead, we found a strong
accumulation of the (G-10L)PhoE precursor in the prlA4
mutant strain, and the step that was blocked in the
biogenesis pathway was identified.
MATERIALS AND METHODS
Reagents and biochemicals
Restriction enzymes were purchased from either Boehringer
Mannheim or Pharmacia. MEGAshortscript T7 transcrip-
tion kit was from Ambion, and [
35
S]methionine and
Tran
35
S-label were from Amersham International. The
cross-linker disuccinimidyl-suberate (DSS) and Super Signal
West Pico Chemiluminescent Substrate were from Pierce.
Bacterial strains and plasmids

CAG12071 zhd-3082::Tn10 [59]
CE1510 NT1004 zhd-3082::Tn10 This study
CE1511 CE1224 zhd-3082::Tn10 This study
CE1512 CE1224 prlA4 zhd-3082::Tn10 This study
POP1730 ompR::Tn10 DmalB K. Bauer
AF111 MC4100 lamB14D [60]
ROA7 AF111 prlA7 T.J. Silhavy
ROA11 AF111 prlA11 T.J. Silhavy
ROA202 AF111 prlA202 T.J. Silhavy
CE1518 AF111 ompR::Tn10 This study
CE1519 ROA7 ompR::Tn10 This study
CE1520 ROA11 ompR::Tn10 This study
CE1521 ROA202 ompR::Tn10 This study
CU165 MC4100 secY40 zhd33::tet [44]
Plasmids
pLep-WT Amp
r
, lepB behind ara promoter [40]
pJP29 Cam
r
, wild-type phoE [33]
pNN6 pJP29 derivative encoding (G-10R)prePhoE [29]
pNN8 pJP29 derivative encoding (G-10L)prePhoE [29]
pNN100 Amp
r
, phoE gene behind tac promoter [61]
pHA106 pNN100 derivative encoding (G-10R)prePhoE This study
pHA108 pNN100 derivative encoding (G-10L)prePhoE This study
pC4Meth94PhoE Amp
r

-thiogalactopyranoside for 1 h. Cells were pulse-labeled
for 45 s with Tran
35
S-label followed either by a chase period
with an excess of nonradioactive methionine/cysteine or by
chilling on ice. Subsequently, proteins were precipitated
with 5% (w/v) trichloroacetic acid, followed by immuno-
precipitation with a polyclonal PhoE-specific antiserum [35].
The precipitated proteins were separated by SDS/PAGE
[36] and visualized by autoradiography. Radiolabeled
proteins were quantified using the Imagequant software
(Molecular Dynamics) after scanning of the autoradiogram.
In vitro
transcription, translation, targeting
and cross-linking analysis
To generate truncated mRNA, plasmids encoding truncated
nascent chains of FtsQ or (G-10L)prePhoE (Table 1) were
linearized and transcribed as described [12]. The resulting
mRNAs, encoding (G-10L)94PhoE and 101FtsQ, were
translated in vitro in a lysate of strain MC4100 as described
[12,37] to produce RNC complexes. To allow SRP–RNC
complex formation, 350 n
M
reconstituted SRP was added
to the translation reaction [16]. After 5 min of incubation at
25 °C, samples were chilled on ice, and the resulting SRP–
RNC complexes were purified from the translation mixture
by centrifugation through a high-salt sucrose cushion [38]
and resuspended in RN buffer (100 m
M

integral membrane from soluble and peripheral cross-linked
complexes, samples were treated with 0.18
M
Na
2
CO
3
(pH 11.3) for 15 min on ice. The membrane fractions
containing integral-membrane proteins were pelleted by
ultracentrifugation (10 min, 110 000 g) and resuspended in
RN buffer. Supernatant and pellet fractions were precipi-
tated with 10% (w/v) trichloroacetic acid, washed with cold
acetone, and resuspended in RN buffer. Samples were
immunoprecipitated as described [39] or mixed directly with
2 · SDS/PAGE gel loading buffer before electrophoresis.
Samples were analyzed on 12% or 4–15% gradient SDS/
polyacrylamide gels. Radiolabeled proteins were visual-
ized by phosphorimaging using a PhosphorImager 473
(Molecular Dynamics).
In vivo
membrane-targeting assay
Membrane targeting of leader peptidase was studied in vivo
essentially as described [40]. Briefly, cells containing pLep-
WT, encoding leader peptidase, were induced with 0.2%
arabinose and labeled in the mid-exponential phase with
Tran
35
S-label. After spheroplasting, the cells were treated
with proteinase K to degrade translocated proteins, fol-
lowed by immunoprecipitation [35] with polyclonal anti-

). Subse-
quently, 2 m
M
phenylmethanesulfonyl fluoride was added
to the cell suspension, and incubation was continued for
5 min on ice. Proteins were precipitated with 5% (w/v)
trichloroacetic acid and analyzed by SDS/PAGE and
autoradiography.
Western immunoblot analysis
Total cellular proteins were solubilized in sample buffer for
10 min at 100 °C, followed by separation by SDS/PAGE
on 15% polyacrylamide gels. After transfer of proteins to
nitrocellulose filters (0.45 lm; Schleicher and Schuell) using
a Mini Trans-Blot Cell (Bio-Rad Laboratories), the blots
were incubated with antibodies directed against SecB [41]
and developed by chemiluminescence according to the
manufacturer’s (Pierce) recommendations.
RESULTS
Accumulation of (G-10L)prePhoE in a
prlA4
mutant strain
We have previously shown that the prlA4 mutation in secY
reduced the pmf dependency of protein translocation [21].
Similarly, a single amino-acid substitution, replacing the
helix-breaking glycine at position )10 in the hydrophobic
core of the PhoE signal sequence by leucine (G-10L),
relieved the pmf dependency of PhoE protein translocation
5574 H. Adams et al.(Eur. J. Biochem. 269) Ó FEBS 2002
[29]. To investigate whether the reported effects are additive,
we studied the in vivo translocation kinetics of wild-type and

led G-10R mutant PhoE was processed directly after the
pulse in the wild-type strain. In the prlA7 and prlA202
suppressor strains, processing was improved and the
amount of mature PhoE increased to 50% and 60%,
respectively, of the total amount of PhoE synthesized during
the pulse. The prlA11 suppressor increased the amount of
PhoE only by 2% compared with wild-type cells. Although
the prlA7 and prlA202 alleles tested improved the pro-
cessing of (G-10R)prePhoE, precursor accumulation of
(G-10L)prePhoE was not observed in these prlA suppres-
sors. In conclusion, these data suggest that (G-10L)prePhoE
accumulation is specific for the prlA4 allele.
Targeting of G-10L nascent PhoE to the PrlA4 Sec
translocon
Whereas wild-type PhoE is targeted to the Sec translocon by
SecB [3,31], targeting of (G-10L)prePhoE is mediated by
SRP [32]. Therefore, the accumulation of (G-10L)prePhoE
in the prlA4 mutant may result from a defect in SRP-
mediated targeting to the mutant translocon. To study this
possibility, we examined the targeting of (G-10L)prePhoE
nascent chains to SecY in vitro in cross-linking experiments.
After translation, RNCs of (G-10L)94PhoE polypeptides
were saturated with reconstituted SRP. The SRP–RNC
complexes were purified and incubated with IMVs, derived
from either a wild-type or a prlA4 mutant strain to allow
targeting. After cross-linking with the bifunctional cross-
linking reagent DSS, peripheral and soluble cross-linked
complexes were separated from integral-membrane cross-
linked complexes by Na
2

-thiogalactoside, aliquots of each culture were pulse-labeled with Tran
35
S-label for 45 s, followed by the addition of an equal volume of ice-cold
10% trichloroacetic acid. Radiolabeled proteins were subsequently analyzed as described under (A). G, R and L indicate PhoE proteins with a Gly,
Arg and Leu at position )10 in the signal sequence () 10), respectively.
Ó FEBS 2002 Defective translocation in a prlA4 mutant (Eur. J. Biochem. 269) 5575
and SecA (Fig. 2B, lanes 1 and 3). In addition, cross-linking
adducts of  220 kDa and  40 kDa were also immuno-
precipitated from the Na
2
CO
3
pellet with SecA antiserum.
We assume that the  220-kDa product corresponds to
cross-linked complexes between (G-10L)94PhoE and the
dimeric form of SecA. The  40-kDa product in the
Na
2
CO
3
pellet probably contains proteolytic fragments of
the SecA cross-linked products, which is in agreement with
earlier reports [42]. The fuzzy  46-kDa product (Fig. 2A,
lanes 4 and 5) was immunoprecipitated with anti-SecY
serum (Fig. 2B, lanes 2 and 4), demonstrating that the
(G-10L)94PhoE nascent chains are targeted to the Sec
translocon in the prlA4 IMVs as well as in wild-type IMVs.
The small difference in the electrophoretic mobilities of the
PrlA4-(G-10L)94PhoE adduct and SecY-(G-10L)94PhoE
adduct (Fig. 2A, lanes 4 and 5) probably results from the

(G-10L)prePhoE is inefficiently translocated
in the
prlA4
mutant
Whereas the targeting of the (G-10L)prePhoE and other
SRP substrates to the translocon is apparently unaffected in
the prlA4 mutant, their subsequent insertion into the mutant
translocon might be impaired. To test this possibility,
protease-accessibility experiments were conducted after
pulse-labeling of cells expressing the G-10L mutant protein
(Fig. 3). Indeed, the precursor of G-10L PhoE that
accumulated in the prlA4 mutant was not sensitive to
proteinase K after spheroplasting of the cells (Fig. 3, lane
2), whereas mature PhoE, which is translocated, was
degraded. These results show that the precursor of
(G-10L)PhoE, although correctly targeted to the translocon,
Fig. 2. Targeting of SRP–RNCs to the prlA4 mutant Sec translocon.
[
35
S]Methionine-labeled (G-10L)94PhoE or 101FtsQ was incubated
with 350 n
M
reconstituted SRP. The SRP–RNCs were subsequently
purified and incubated with NT1060 (prlA
+
) and NT1004 (prlA4)
IMVs. The cross-linker DSS was used to analyze SRP–RNC interac-
tions. After quenching, peripherally bound and soluble proteins were
separated from the inner membranes by carbonate extraction. Samples
were either directly (A) or after immunoprecipitation with the indica-

may be involved in protein export and membrane protein
insertion. To test whether the secY40 mutation affects the
translocation of wild-type and (G-10L)prePhoE differently,
pulse–chase experiments were conducted. Indeed, even at
the permissive temperature, the (G-10L)prePhoE was only
slowly processed in the secY40 mutant, whereas wild-type
prePhoE was completely processed within 30 s after the
pulse (Fig. 5). These results are consistent with the hypo-
thesis that wild-type and G-10L mutant prePhoE are
targeted to different domains of the SecYEG translocon.
DISCUSSION
Signal sequences often contain an a-helix breaker in the
hydrophobic core [45]. We have previously shown that
substitution of the helix breaker in the signal sequence of
Fig. 4. In vivo membrane insertion of leader peptidase in wild-type and
prlA4 mutant cells. (A) Cells of strain NT1060 (prlA
+
)oritsprlA4
derivative NT1004, carrying plasmid pLep-WT, were grown in syn-
thetic minimal medium to mid-exponential phase at 30 °C. Expression
of leader peptidase was induced for 5 min by the addition of 0.2%
arabinose. Subsequently, cells were pulse-labeled for 15 s with Tran
35
S-
label and chased with unlabeled methionine/cysteine for the indicated
periods. Insertion of leader peptidase was assessed by analyzing its
protease accessibility after spheroplasting of the cells. The proteins
were analyzed after immunoprecipitation with anti-Lep antibodies by
SDS/PAGE and autoradiography. (B) As a control for membrane
intactness, the spheroplasts were treated with proteinase K (PK),

expressedinaprlA4 mutant strain and that its translocation
across the inner membrane is impaired.
The results described here for (G-10L)prePhoE are
reminiscent of those reported previously for staphylokinase
(Sak), a secreted protein from the Gram-positive bacterium
Staphylococcus aureus, which was efficiently processed and
exported to the periplasm in wild-type E. coli cells, but
accumulated in its precursor form when expressed in a prlA4
mutant of E. coli [46]. Sequence examination suggests the
presence of an unusually long a-helix in the core region of
the Sak signal sequence as is the case in the (G-10L)prePhoE
signal sequence. The export defect of Sak in the prlA4
mutant was suppressed by a small four-amino-acid deletion
or by amino-acid substitutions introducing a strong helix
breaker, such as glycine, into the a-helical core region of the
signal sequence [46]. Similarly, streptokinase, an extracellu-
lar protein of streptococcal strains, is also blocked from
being secreted in E. coli prlA4 mutant cells [47], presumably
because of the long a-helix in its signal sequence.
The prlA4 allele actually contains two missense mutations
in the secY gene, resulting in the amino-acid substitutions
F286Y and I408N in transmembrane segments (TMS) 7
and 10, respectively [48]. The mutation in TMS 10 is
responsible for the suppressor phenotype and enables the
translocation of preproteins with a defective or completely
missing signal sequence [20,25,49]. Furthermore, this muta-
tion was reported to result in a looser association between
the subunits of the SecYEG translocon [24] and in increased
affinity of SecA for the SecY protein [23]. The mutation in
TMS 7 was probably acquired as a secondary mutation

substitution in cytoplasmic domain 5 of SecY) was shown to
be defective in inner-membrane protein insertion, whereas
protein export was unaffected by the mutation [44]. Our data
show that the translocation of the SRP substrate (G-
10L)prePhoE is affected by the secY40 mutation as well,
whereas the SecB substrate wild-type PhoE is not, indica-
ting that these highly related precursors are differently
processed at the SecYEG translocon. Furthermore, the (G-
10L)prePhoE accumulation in prlA4 cells is caused by
inefficient translocation, whereas wild-type prePhoE is
correctly translocated. However, the membrane insertion
of another SRP substrate, leader peptidase, was not affected
by the prlA4 mutation. To explain our results, we propose
that SRP and SecB substrates are targeted to different
domains of the translocon. The cytoplasmic domain 5, where
the secY40 mutation is located, is involved in the docking of
SRP substrates. Also after docking at the translocon, SRP
substrates and SecB substrates are differently processed by
the translocon. In the case of SRP substrates, the translocon
opens laterally to allow the insertion of integral-membrane
proteins. However, when the hydrophobic a-helix is too
short to span the inner membrane, the SRP substrate may be
transferred to the translocation pathway, which is normally
used by SecB substrates. This transfer appears to be defective
in the prlA4 mutant.
ACKNOWLEDGEMENTS
We would like to thank Malene Urbanus for her efforts with the
cross-linking experiments. Furthermore, we would like to thank
Elaine Eppens and Margot Koster for helpful discussions and
interest in the work, William Wickner, Annemieke van Dalen and

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