269, 5564–5571.], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position10 in the signal sequence G-10L is targeted to
Trang 1Defective translocation of a signal sequence mutant in a prlA4
Hendrik Adams1, Pier A Scotti2*, Joen Luirink2and Jan Tommassen1
1
Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, The Netherlands;2Department 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
revealed that the precursor is correctly targeted to the prlA4 mutant translocon However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells Furthermore, the mutant precursor was found to accumu-late when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in pro-tein translocation Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon
Keywords: inner membrane; prlA4; protein insertion; protein translocation; Sec translocon
Most proteins destined for the periplasm or the outer
membrane of Escherichia coli are transported across the
inner membrane by the membrane-embedded Sec system, a
complex consisting of the SecYEG translocon, the
hetero-trimer SecDFyajC and the peripheral ATPase SecA [1,2]
Targeting to the translocon is usually mediated by SecB,
which interacts with the mature portion of presecretory
proteins [3,4] The SecB–preprotein complex is then targeted
to SecA, which in turn binds with high affinity to SecYEG
[5,6] Upon initiation of translocation, SecB is released [7]
and the preprotein is translocated through the translocon by
an insertion–deinsertion cycle of SecA [8] At the
periplas-mic side of the membrane, leader peptidase removes the
signal sequence from the precursor, and the mature protein
is released into the periplasm [9] Targeting of
inner-membrane proteins is primarily controlled by the
signal-recognition particle (SRP) pathway [2,10,11] The E coli
SRP consists of a single protein, P48, and a 4.5S RNA, and
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 has been proposed that prlA mutations cause a general relaxation of the export apparatus [21,24] rather than a specific change that results in bypassing a proofreading mechanism of the Sec machinery [25]
The energy for precursor translocation is supplied by the hydrolysis of ATP by SecA and by the proton-motive force
Correspondence to J Tommassen, Department of Molecular
Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht,
The Netherlands Fax: + 31 30 2513655, Tel.: + 31 30 2532999,
E-mail: J.P.M.Tommassen@bio.uu.nl
Abbreviations: SRP, signal-recognition particle; RNC,
ribosome-nascent chain; pmf, proton-motive force; DSS,
disuccinimidyl-suberate; IMV, inverted inner-membrane vesicle; Sak, Staphylokinase;
TF, trigger factor; TMS, transmembrane segment.
*Present address: IECB-E´cole polytechnique ENSCPB,
Talence cedex, France.
(Received 29 May 2002, revised 10 September 2002,
accepted 16 September 2002)
Trang 2(pmf) [26] Energy from ATP binding and hydrolysis is
probably used to confer conformational changes in the
SecA molecule, which lead to a cycle of insertion and
deinsertion of SecA into the membrane [8,27] and
move-ment of the precursor across the membrane [28] The
mechanism by which the pmf stimulates the translocation
process is less clear However, recent experiments indicate
that the insertion of the signal sequence in a transmembrane
orientation is stimulated by the pmf [29,30] In our
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
M A T E R I A L S A N D M E T H O D S
Reagents and biochemicals Restriction enzymes were purchased from either Boehringer Mannheim or Pharmacia MEGAshortscript T7 transcrip-tion kit was from Ambion, and [35S]methionine and Tran35S-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
The E coli strains and plasmids used in this study are listed in Table 1 To obtain a prlA4 derivative of strain CE1224, a Tn10marker was introduced close to the prlA4 allele in strain NT1004 by P1 transduction using strain CAG12071 as the donor and selection for tetracycline-resistant transductants Subsequently, the resulting strain, CE1510, was used as the donor in a P1 transduction experiment with CE1224 as the recipient To confirm the prlA4 phenotype, tetracycline-resistant transductants were transformed with plasmid pNN6 and tested in pulse-labeling experiments for suppres-sion of the secretion defect of (G-10R)prePhoE, which contains a charged residue in the hydrophobic core of the signal sequence Tetracycline-resistant prlA+ and prlA4
Table 1 Bacterial strains and plasmids used in this study Camrand Amp r indicate resistance to chloramphenicol and ampicillin, respectively.
Strains
CE1224 F – , thr leu D(proA-proB-phoE-gpt) his thi argElacY galK xyl rpsL supEompR [57]
Plasmids
pHA106 pNN100 derivative encoding (G-10R)prePhoE This study
pHA108 pNN100 derivative encoding (G-10L)prePhoE This study
pC4Meth101FtsQ-WTAmpr, encodes truncated 101FtsQ [12]
pC4Meth(G-10L)94PhoE pC4Meth94PhoE derivative encoding (G-10L) mutant PhoE [32]
Trang 3transductants were designated CE1511 and CE1512,
respect-ively CE1518-1521 strains were obtained by P1 transduction
using strains AF111, ROA7, ROA11 and ROA202,
respect-ively, as recipients and strain POP1730 as the donor
For the construction of plasmids pHA106 and pHA108,
encoding the G-10R and G-10L mutant PhoE, respectively,
under control of the tac promoter, a PstI/BamHI fragment
from pNN100 containing the signal-sequence-encoding part
of phoE was replaced by the corresponding fragment of
pNN6 and pNN8, respectively
Pulse-labeling experiments
Strains carrying plasmid pJP29 or derivatives were grown
under phosphate limitation at 30C to induce PhoE
expression [33] Strains, carrying plasmids with phoE under
tac promoter control, were grown for 4 h at 30C in
synthetic minimal medium [34] supplemented with
100 lgÆmL)1ampicillin and 0.4% glucose PhoE expression
was induced under these conditions with 1 mMisopropyl
b-D-thiogalactopyranoside for 1 h Cells were pulse-labeled
for 45 s with Tran35S-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 nM reconstituted SRP was added
to the translation reaction [16] After 5 min of incubation at
25C, 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 mMpotassium acetate,
5 mM magnesium acetate, 2.5 mM Hepes/KOH, pH 7.9)
Inverted inner-membrane vesicles (IMVs) were isolated as
described [37] Targeting reactions were performed as
described previously [16] by incubating purified SRP–
RNC complexes together with 1 mM FtsY, 50 lM GTP,
50 lM ATP, and IMVs (1.25 mgÆmL)1 protein)
Cross-linking was induced with 1 mMDSS for 10 min at 25C
and quenched at 0C by adding 0.1 vol quenching buffer
(1M glycine/100 mM NaHCO3, pH 8.5) To separate
integral membrane from soluble and peripheral cross-linked
complexes, samples were treated with 0.18M Na2CO3
(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 Tran35S-label After spheroplasting, the cells were treated with proteinase K to degrade translocated proteins, fol-lowed by immunoprecipitation [35] with polyclonal anti-bodies directed against leader peptidase and analysis by SDS/PAGE and autoradiography
Proteinase K-accessibility experiments Cells of prlA4 mutant strain NT1004, carrying plasmid pNN8, were grown under phosphate limitation to induce PhoE expression [33] before pulse-labeling with Tran35 S-label for 60 s Directly after the pulse, an excess of nonradioactive methionine and cysteine was added, and the cells were collected by centrifugation (2 min, 16 000 g) For spheroplasting, cells were resuspended in ice-cold buffer
A [40% (w/v) sucrose, 1.5 mM EDTA, 33 mM Tris/HCl (pH 8.0)] and incubated with lysozyme (final concentration
5 lgÆmL)1) After 10 min incubation on ice, incubation was continued at 37C for 10 min followed by addition of
10 mMMgCl2 Aliquots of the spheroplast suspension were incubated on ice for 30 min in the presence or absence of proteinase K (final concentration 50 lgÆmL)1) Subse-quently, 2 mMphenylmethanesulfonyl 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 100C, 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
R E S U L T S
Accumulation of (G-10L)prePhoE in aprlA4 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
Trang 4[29] To investigate whether the reported effects are additive,
we studied the in vivo translocation kinetics of wild-type and
mutant PhoE in prlA4 mutant strain CE1512, using
processing as a criterion for translocation The prlA4
phenotype of the strain was confirmed by studying the
processing kinetics of (G-10R)prePhoE The translocation
of this mutant precursor, which contains a charged residue
in the hydrophobic core of the signal sequence, was severely
hampered in the wild-type strain and considerably improved
in the prlA4 mutant (Fig 1A) Directly after the pulse, the
mature form of the G-10R mutant was barely detectable in
the wild-type strain, whereas the majority of the protein was
already processed in the prlA4 mutant Processing was
completed in the prlA4 cells after a 2-min chase period,
whereas in the wild-type cells, at this time point, still about
50% of the synthesized (G-10R)prePhoE was unprocessed
Interestingly, whereas the (G-10L) mutant precursor was
efficiently processed in wild-type cells, it accumulated
abundantly in the prlA4 suppressor strain, indicating that
its translocation across the inner membrane was affected by
the prlA4 mutation Thus, whereas the prlA4 mutation has
been isolated as a strong suppressor of signal sequence
defects, it seemed to have an adverse effect on the
translocation of the G-10L PhoE precursor
To test whether the (G-10L)prePhoE accumulation is
specific for the prlA4 allele, we determined the effect of
several other prlA suppressor mutations with the
amino-acid substitutions being located in distinct topological
domains of SecY [25] Again, suppression of the
transloca-tion defect of (G-10R)prePhoE was used as a control for the
suppressor phenotype (Fig 1B) Quantification of the
results revealed that 39% of the total amount of
radiolabe-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 linking with the bifunctional cross-linking reagent DSS, peripheral and soluble cross-linked complexes were separated from integral-membrane cross-linked complexes by Na2CO3extraction, and the complexes were analyzed by SDS/PAGE (Fig 2A) In the Na2CO3 pellet, at least two (G-10L)94PhoE cross-linked complexes, one at 110 kDa and one at 46 kDa, could be detected with both wild-type and prlA4 IMVs (Fig 2A, lanes 4 and 5) In both cases, the 110-kDa complex could be immuno-precipitated with antiserum directed against SecA, indica-ting that it is a complex of the radiolabeled (G-10L)94PhoE
Fig 1 Processing kinetics of prePhoE and mutant derivatives in wild-type and prlA mutant strains (A) Cells of prlA+strain CE1511 (prlA+) or prlA4 mutant CE1512, carrying plasmids encoding wild-type PhoE (WT) or mutant derivatives with G-10R or G-10L substititutions in the signal sequence, were grown under phosphate limitation to express PhoE The cells were pulse-labeled at 30 C for 45 s with T ran 35 S-label and chased with unlabeled methionine/cysteine for the indicated periods The proteins were analyzed, after immunoprecipitation with antibodies against PhoE,
by SDS/PAGE and autoradiography The precursor and mature forms of PhoE are indicated by p and m, respectively (B) Cells of strains CE1518 (prlA + ), CE1519 (prlA7), CE1520 (prlA11) and CE1521 (prlA202), carrying plasmids encoding wild-type PhoE (WT) or mutant derivatives under control of the tac promoter, were grown in synthetic minimal medium at 30 C until the mid-exponential phase After 1 h induction with isopropyl b- D -thiogalactoside, aliquots of each culture were pulse-labeled with Tran35S-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.
Trang 5and SecA (Fig 2B, lanes 1 and 3) In addition, cross-linking
adducts of 220 kDa and 40 kDa were also
immuno-precipitated from the Na2CO3pellet 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
Na2CO3pellet 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 amino-acid substitutions in the mutant PrlA4 protein
In the Na2CO3 supernatant, at least three major cross-linking adducts, of apparent molecular mass 110, 65 and 55 kDa, could be detected, both when wild-type and when prlA4 IMVs were present (Fig 2A, lanes 8 and 9) Immunoprecipitation revealed that these adducts represent cross-linking to SecA (data not shown), trigger factor (TF) and P48 (Fig 2C), respectively, demonstrating that (G-10L)94PhoE interacts with P48 and T F in both cases As described in the accompanying paper [32], several additional low-molecular-mass cross-linking adducts (< 30 kDa) were revealed (Fig 2A, lanes 8 and 9), but the identity of these complexes is unknown To investigate whether the cross-linking adducts are similar to those of other substrates
of the SRP pathway, FtsQ was used as a model substrate This class II membrane protein, with a short N-terminal cytoplasmic tail [43], was synthesized as a slightly longer nascent chain (101 residues) than (G-10L)94PhoE to expose properly its signal-anchor domain Indeed, 101FtsQ inter-acted properly with both SecY and SecA (Fig 2A, lanes 13 and 14) as demonstrated by immunoprecipitations with anti-SecY and anti-SecA serum, respectively (Fig 2B, lanes 5–8) In conclusion, the results show that the targeting of the (G-10L)94PhoE nascent chains and other SRP substrates to the SecY protein in the translocon is not defective in the prlA4mutant
(G-10L)prePhoE is inefficiently translocated
in theprlA4 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-ted antisera (B and C) subjecindica-ted to SDS/PAGE, and cross-linked complexes were visualized with a PhosphorImager The positions of molecular mass marker proteins (MW) are indicated on the right Arrowheads indicate relevant cross-linked complexes.
Trang 6is inefficiently translocated across the inner membrane in a
prlA4mutant
To investigate whether membrane insertion of SRP
substrates is also defective in a prlA4 mutant, we examined
the insertion kinetics of leader peptidase [40] Directly after
pulse-labeling, leader peptidase already appeared to be
accessible to proteinase K in spheroplasts of both the
wild-type and the prlA4 mutant strain (Fig 4A) The intactness
of the spheroplasts was confirmed by the inaccessibility to
proteinase K of SecB, which was only degraded after
solubilization of the spheroplasts with Triton X-100
(Fig 4B) These results indicate that insertion of the SRP
substrate leader peptidase into the Sec translocon is not
affected in the prlA4 mutant
Processing of (G-10L)prePhoE is impaired in secY40
mutant cells
The results presented so far indicate that the prlA4 mutation
has distinct effects on the translocation kinetics of a
SecB-targeted and an SRP-SecB-targeted precursor, i.e wild-type and
G-10L mutant PhoE, respectively A possible explanation is
that these substrates are targeted to different domains of
SecYEG A cold-sensitive secY40 mutant has previously
been described that is impaired in inner-membrane protein
insertion, whereas protein export is unaffected [44] In
addition, this result suggested that different regions of SecY
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
D I S C U S S I O N
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+) or its prlA4 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 unS-labeled 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), without or with solubilization by Triton X-100 (TX-100) and analyzed
by immunoblotting using antiserum directed against SecB.
Fig 3 Proteinase K accessibility of (G-10L)prePhoE in prlA4 mutant
cells NT1004 (prlA4) cells, expressing (G-10L)prePhoE from plasmid
pNN8, were pulse-labeled for 60 s with Tran 35 S-label, followed by the
addition of an excess of methionine/cysteine After spheroplasting, the
cells were incubated in the absence or presence of proteinase K (PK).
Subsequently, the undigested proteins were precipitated with
trichloroacetic acid and analyzed by SDS/PAGE and
autoradio-graphy The precursor and mature forms of PhoE are indicated by p
and m, respectively.
Fig 5 Processing of (G-10L)prePhoE is impaired in secY40 cells Cells
of strain CU165 (secY40) carrying plasmid pJP29 encoding wild-type PhoE (WT) or pNN8 encoding (G-10L)prePhoE, were grown under phosphate limitation to express PhoE The cells were pulse-labeled
at 37 C for 45 s with T ran 35 S-label and chased with unlabeled methionine/cysteine for the indicated periods Radiolabeled proteins were analyzed by SDS/PAGE and autoradiography The precursor and mature forms of PhoE are indicated by p and m, respectively.
Trang 7PhoE by a helix-promoting residue (e.g G-10L) relieved the
pmf dependency of the translocation of this precursor [29]
Furthermore, we showed that such substitutions resulted in
the re-routing of this SecB substrate to the SRP-targeting
pathway [32] In the present study, we show that
(G-10L)prePhoE accumulates in its precursor form when
expressed in a prlA4 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 associamuta-tion 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
which restored the stability while conserving the flexibility of
the system Further, it was shown that the mutation in
TMS 7 (F286Y) is responsible for the observed export
defect of Sak [46,48,50], suggesting that this defect is not
related to the suppressor phenotype Consistently, we did
not observe a processing defect when (G-10L)prePhoE was
expressed in other prlA suppressor strains, i.e prlA7 (A277E
in TMS 7 and L407R in TMS 10), prlA11 (V407R in
TMS 10 and V411G in TMS 10), and prlA202 (I287S in
TMS 7) Therefore, we speculate that the export defect of
(G-10L)prePhoE, like that of Sak, is caused by the
secondary mutation in TMS 7 of SecY
Recent studies have indicated that the SecYEG complex
facilitates the insertion of inner-membrane proteins in
addition to catalyzing protein translocation [51,52]
Mem-brane protein insertion and protein translocation are two
distinct processes that are likely to impose different
func-tional requirements on the translocase The mechanism by
which the Sec complex mediates both of these functions is still
unclear Available evidence indicates that the translocon
forms an aqueous channel that permits the translocation of
polypeptides into the periplasm [53,54] However, it can also
open laterally to allow the exit of transmembrane regions
into the lipid bilayer [55] Possibly, the passage of exported
proteins and the insertion of integral membrane proteins are
facilitated by distinct regions of the translocon Indeed,
studies in eukaryotes indicated that signal sequences of
secreted proteins and signal-anchor domains of membrane proteins are positioned differently in the Sec61p translocon [56] Recently, a secY40 mutant of E coli (carrying an A363S 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
A C K N O W L E D G E M E N T S
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 Arnold Driessen for providing antisera, and Katharina Bauer, Ann Flower, Chris Harris and Tom Silhavy for their gifts of strains This work was supported by EU grant HPRN-CT-2000-00075 from the European Community.
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