Furthermore, in vitro cross-linking experiments revealed that the G-10L mutant protein is routed to the SecYEG translocon via the SRP pathway,the targeting pathway that is exploited by i
Trang 1The presence of a helix breaker in the hydrophobic core
of signal sequences of secretory proteins prevents recognition
Hendrik Adams1, Pier A Scotti2*, Hans de Cock1, 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
Signal sequences often contain a-helix-destabilizing amino
acids within the hydrophobic core In the precursor of the
Escherichia coliouter-membrane protein PhoE,the glycine
residue at position)10 (Gly)10) is thought to be responsible
for the break in the a-helix Previously,we showed that
substitution of Gly)10by a-helix-promoting residues (Ala,
Cys or Leu) reduced the proton-motive force dependency of
the translocation of the precursor,but the actual role of the
helix breaker remained obscure Here,we considered
the possibility that extension of the a-helical structure in
the signal sequence resulting from the Gly)10substitutions
affects the targeting pathway of the precursor Indeed,the
mutations resulted in reduced dependency on SecB for
tar-geting in vivo In vitro cross-linking experiments revealed that
the G-10L and G-10C mutant PhoE precursors had a dra-matically increased affinity for P48,one of the constituents of the signal-recognition particle (SRP) Furthermore, in vitro cross-linking experiments revealed that the G-10L mutant protein is routed to the SecYEG translocon via the SRP pathway,the targeting pathway that is exploited by integral inner-membrane proteins Together,these data indicate that the helix breaker in cleavable signal sequences prevents recognition by SRP and is thereby,together with the hydrophobicity of the signal sequence,a determinant of the targeting pathway
Keywords: outer-membrane protein; Sec translocon; SecB; signal-recognition particle; translocation
Most cell envelope proteins of Escherichia coli are
translo-cated across or inserted into the cytoplasmic membrane via
the membrane-embedded Sec translocon Targeting of
precursor proteins to the translocon can be mediated by
components of the Sec pathway or by the signal-recognition
particle (SRP) pathway [1,2] The Sec pathway utilizes a
cytosolic chaperone,SecB,which interacts with the mature
portion of presecretory proteins [3,4] The SecB-preprotein
complex is then targeted to SecA, which in turn interacts
with components of the Sec translocon [5,6] At the onset of
translocation,SecB is released [7] and the preprotein is
translocated by an insertion–deinsertion cycle of SecA into
the SecYEG translocon [8] Energy for the translocation
process is provided by ATP hydrolysis by SecA [8,9] and by
the proton-motive force (pmf) [9] At the periplasmic side of
the membrane,leader peptidase removes the signal sequence
from the precursor,and the mature protein is released into the periplasm [10] The bacterial SRP-targeting route is homologous with,but less complex than,the eukaryotic SRP system [11,12] The E coli SRP consists of a single protein,P48,and a 4.5S RNA,and binds cotranslationally
to hydrophobic sequences [13,14] The ribosome-nascent chain (RNC) complex subsequently binds to FtsY and is targeted to the Sec translocon in the inner membrane [15,16] Whereas the SecB route is predominantly used by a subset of periplasmic and most,if not all,outer-membrane proteins,inner-membrane proteins are particularly depend-ent on a functional SRP pathway [17]
We are using outer-membrane protein PhoE as a model
to study protein export PhoE is targeted via its signal sequence in a SecB-dependent way to the Sec translocon [3] Whereas the signal sequence is necessary and,in most cases, sufficient for translocation across the cytoplasmic mem-brane,its exact role in the export mechanism is far from understood Despite the common function of signal sequences,i.e to direct the translocation of the attached polypeptide chain,there is little sequence homology among them [18] Nevertheless,a common structural organization can be recognized (Fig 1) Signal sequences are character-ized by a positively charged N-terminal region (N domain), followed by a 10–15 residues long hydrophobic core (H domain) and a polar C-terminus (C domain) containing the signal-peptidase cleavage site [19] The importance of a-helix formation in the signal sequence is well documented [20–24] However,NMR studies on the conformation of signal sequences in a membrane mimetic environment showed that the stable a-helix is disrupted towards the C-terminus
of the hydrophobic core [25–27] Furthermore,a statistical
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; pmf,proton-motive
force; RNC,ribosome-nascent chain; BS3
,bis(sulfosuccinimidyl)-suberate; DSS,disuccinimidyl-,bis(sulfosuccinimidyl)-suberate; IMV,inverted
inner-membrane vesicles; TF,trigger factor.
*Present address: IECB-E´cole polytechnique ENSCPB,Talence cedex,
France.
(Received 29 May 2002,revised 10 September 2002,
accepted 16 September 2002)
Trang 2analysis of signal sequences revealed the common
occurrence of a-helix-destabilizing amino acids in the
hydrophobic core [28] In a previous study,the role of the
a-helix-breaking glycine residue at position)10 (Gly)10) of
the signal sequence of PhoE was examined [29] It was
shown that substitution of this residue by a-helix-promoting
residues (Ala,Cys or Leu) reduced the pmf dependency of
the translocation across the inner membrane,but the actual
role of the helix breaker remained obscure It should be
noted that such substitutions extend the a-helix not just by a
single residue,but,probably,over the entire H domain
(Fig 1) Whereas the a-helix in the wild-type signal
sequence is too short to span the inner membrane,the
resulting mutant signal sequences would more closely
resemble the membrane-spanning domains of
inner-mem-brane proteins and might therefore be turned into substrates
for the SRP In this paper,we considered the possibility that
the extended a-helix resulting from the Gly)10substitutions
affects the targeting pathway of the precursor
E X P E R I M E N T A L P R O C E D U R E 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 Tran
35S-label were from Amersham International Bis(sulfo-succinimidyl)-suberate (BS3) and disuccinimidyl-suberate (DSS) were from Pierce,and oligonucleotides were pur-chased from Isogen Bioscience (Maarsen,the Netherlands) Bacterial strains
The E coli K-12 strains used in this study are listed in Table 1 Strains CE1514 and CE1515 were obtained by P1 transduction using strain CE1224 as the recipient and strains IQ85 and strain MM152,respectively,as donor strains To obtain strain CE1513,strain MM88 was used as
Fig 1 Physical characteristics of the PhoE signal sequence The signal sequence consists of the positively charged N domain,the hydrophobic H domain and the C-terminal C domain The a-helix in the H domain is predicted to extend up to the Gly at position )10 in the signal sequence Introduction of an a-helix-stabilizing residue (Ala,Cys or Leu) at position )10 results in extension of the a-helical core region as indicated The leader peptidase cleavage site is depicted with an arrow.
Table 1 Bacterial strains and plasmids used in this study Ts,temperature sensitive Camrand Amp r ,resistance to chloramphenicol and ampicillin, respectively.
Strains
CE1224 F–, thr leu D(proA-proB-phoE-gpt) his thi argElacY galK xyl rpsL supEompR [49]
MM88 F – , DlacU169 araD139 thiA rpsL relA leu::Tn10 secAtsA51 B Oudega (pers comm.) NT1060 F–, DlacU169 araD139 rpsL thi relA ptsF25 deoC1 lamBD60 T.J Silhavy (pers comm.)
, lacDx74 araD139 (araABOIC-leu) D7679 galU galK rpsL ffs::kan/F¢ lac-pro, lacIqPtac::ffs
[52]
Plasmids
pC4Meth(G-10C)94PhoE pC4Meth94PhoE derivative encoding (G-10C) mutant 94PhoE This study
pC4Meth(G-10L)94PhoE pC4Meth94PhoE derivative encoding (G-10L) mutant 94PhoE This study
Trang 3the donor and CE1224 as the recipient in a P1 transduction
experiment
Plasmid construction
Plasmid pJP29 and derivatives carrying mutations in the
PhoE signal-sequence-encoding region and other plasmids
are listed in Table 1 Plasmid pC4Meth94PhoE was used to
generate truncated phoE mRNA,encoding a 94-residue
PhoE polypeptide exposing the signal sequence just outside
the ribosome [13] Plasmid pC4Meth101FtsQ-WT was used
to generate truncated FtsQ mRNA,encoding a 101-residue
FtsQ polypeptide exposing the signal-anchor domain just
outside the ribosome To compensate for the loss of
methionines from the deleted domains of the proteins,
both constructs contain a C-terminal tetra-methionine tag
sequence for labeling To introduce the Cys and Leu
mutations for the Gly)10residue into pC4Meth94PhoE,the
EcoRI/BamHI fragment of the plasmid was replaced by
PCR fragments created using the PhoE forward primer
(5¢-GCCGGAATTCTAATATGAAAAAGAGCACTCT
GGC-3¢) and the 94PhoE reverse primer (5¢-CGCGGA
TCCTTTTTGCTGTCAGTATCAC-3¢),pNN7 and pNN8
as the templates,respectively,and Pfu polymerase The
resulting plasmids were designated pC4Meth(G-10C)94
PhoE and pC4Meth(G-10L)94PhoE,respectively
In vivo pulse–chase experiments
Cells of strain CE1224 or its derivatives each containing a
plasmid expressing (mutant) phoE from its own promoter,
were grown under phosphate limitation at 30C as
described previously [30] Cells of the 4.5S RNA conditional
strain FF283 were grown to D660¼ 1.0 in Hepes-buffered
synthetic medium,supplemented with 660 lM K2HPO4
For the depletion of 4.5S RNA,isopropyl b-D
-thiogalacto-pyranoside was omitted from the growth medium To
induce the expression of (mutant) phoE from its own
promoter,cells were collected by centrifugation and washed
with Hepes-buffered synthetic medium with no phosphate
added The cell pellets were resuspended in the latter
medium at the original absorbance,followed by incubation
at 37C for 30 min For pulse-labeling,cells were incubated
for 45 s with Tran35S-label followed by a chase period with
an excess of nonradioactive methionine/cysteine After
precipitation with 5% (w/v) trichloroacetic
acid,radio-labeled proteins were separated either directly or after
immunoprecipitation with a polyclonal PhoE-specific
anti-serum [31] by SDS/PAGE [32] and visualized by
autoradio-graphy
In vitro transcription, translation, targeting
and cross-linking analysis
To generate truncated mRNA,plasmids (Table 1) encoding
truncated nascent chains were linearized and transcribed as
described previously [13] The resulting mRNAs were
translated in vitro in a lysate of strain MC4100 as described
[13,33] The mixture containing RNCs was chilled on ice
and treated with 1 mM BS3 at 25C for 10 min before
addition of 0.1 vol quench buffer (1M glycine/0.1M
NaHCO3,pH 8.5) After incubation for 20 min at 0C,
cross-linked products were immunoprecipitated [34],and
the precipitates were analyzed by SDS/PAGE (12% gels) Radiolabeled proteins were visualized with a Phosphor-Imager 473 (Molecular Dynamics) and quantified using the Imagequant software (Molecular Dynamics) To test the targeting of wild-type prePhoE RNCs,truncated mRNAs were translated in the presence of inverted inner-membrane vesicles (IMVs) [33] from strain MC4100 After cross-linking with 1 mMDSS for 10 min at 25C,the cross-link reaction was stopped with quench buffer Peripheral and soluble cross-linked complexes were separated from integral-membrane cross-linked complexes by Na2CO3 extraction as described [35] Samples were analyzed either directly or after immunoprecipitation on 12% polyacryla-mide gels and visualized as described above
To probe the molecular environment of membrane-associated RNCs,SRP was reconstituted in vitro from purified 4.5S RNA and purified hexa-His-tagged P48 as described [35] To allow SRP–RNC complex formation (G-10L)94PhoE and 101FtsQ were synthesized in vitro and incubated at 25C with 350 nM reconstituted SRP,and SRP–RNC complexes were purified from the translation mixture by centrifugation through a high-salt sucrose cushion [36] The SRP–RNC complexes were incubated with IMVs from strain NT1060 under conditions as described previously [35] After cross-linking with 2 mM DSS at 25C for 10 min,0.1 vol quench buffer was added and incubation was continued on ice for 15 min Subse-quently,peripheral and soluble cross-linked complexes were separated from integral-membrane cross-linked complexes
by Na2CO3 extraction as described [35] Samples were analyzed either directly or after immunoprecipitation on 12% or 15% gels and visualized as described above
R E S U L T S
SecB dependency of the targeting of mutant prePhoE
By the substitution of an a-helix-promoting residue (Leu, Ala or Cys) for the helix-breaking Gly)10 of the signal sequence of PhoE,the a-helix is expected to be extended considerably (Fig 1) As these mutant signal sequences resemble more closely the membrane-spanning domains of integral-membrane proteins,the mutations might affect the targeting route of the precursors to the SecYEG translocon This possibility was first tested in vivo in pulse–chase experiments The processing kinetics of the wild-type and mutant PhoE proteins were compared in a secB null mutant strain Previously,it was demonstrated that introduction of
an a-helix-stabilizing residue (Ala,Cys or Leu) instead of the Gly)10 did not result in dramatic differences in the processing kinetics of prePhoE in wild-type cells [29] As the export of wild-type PhoE is SecB dependent [3],its precursor strongly accumulated in a secB mutant (Fig 2A) Interestingly,the mutant precursors showed considerably improved processing kinetics compared with wild-type prePhoE in the secB mutant (Fig 2A) After a 5-min chase period,hardly any mutant prePhoE was detected anymore, whereas the vast majority of the wild-type precursor was still not processed Together with the previously reported reduced pmf dependency for translocation of the mutant precursors [29],our results suggest that the SecB depend-ency of prePhoE targeting correlates with its DlH+ dependency for in vitro translocation
Trang 4Of all the precursors tested,the mutant precursor with the
strongest a-helix-promoting residue (Leu) at position)10
appeared to be most efficiently processed in the secB mutant
strain This mutant precursor was used to verify if
translocation is still dependent on the membrane-embedded
SecYEG complex and on SecA For this purpose,pulse–
chase experiments were performed in secA51 and secY24
mutant strains at their nonpermissive temperature In both
strains,processing of the (G-10L)prePhoE protein,like that
of the wild-type precursor,was strongly impaired in
comparison with the processing in the wild-type strain
(Fig 2B) Apparently,substitution of the glycine residue at
position)10 by an a-helix-promoting residue does not alter
the dependency of the precursor on SecA and SecY,
whereas its SecB dependency is reduced
Affinity of mutant prePhoE nascent chains for P48
As the SecB dependency of the translocation of the mutant
prePhoE proteins was clearly decreased,we next considered
the possibility that they had become substrates for the
SRP pathway To determine whether components of the
SRP pathway are indeed involved in the targeting of (G-10L)prePhoE to the translocon, in vitro cross-linking studies were performed Previously,Valent et al [13] analyzed the interaction of nascent prePhoE protein with soluble proteins in an E coli lysate Nascent PhoE 94-mer extended with a tetra-methionine tag-sequence (94PhoE) was synthesized in an E coli lysate and treated with the water-soluble cross-linker BS3 Whereas,in these experi-ments,nascent chains of integral inner-membrane proteins could be cross-linked to the P48 component of SRP,this was not the case for nascent 94PhoE [13] To investigate whether substitution of the Gly)10residue by an a-helix-stabilizing residue resulted in a higher affinity for P48, (G-10L)94PhoE and 94PhoE were synthesized and tested for cross-linking to P48 present in the E coli lysate Whereas hardly any cross-linked 94PhoE could be immu-noprecipitated with anti-P48 antibodies,strong cross-link-ing of (G-10L)94PhoE to P48 was observed (Fig 3A) To determine whether the improved cross-linking of (G-10L) 94PhoE to P48 was due solely to the increased hydropho-bicity of this mutant signal sequence,similar cross-linking experiments were also performed for the (G-10C)94PhoE mutant PhoE protein Even though cysteine has an even lower hydrophobicity than glycine on the consensus hydrophobicity scale of Eisenberg et al [37],the (G-10C) 94PhoE protein was also cross-linked to P48 (Fig 3), although not as efficiently as (G-10L)94PhoE In all cases, antiserum against trigger factor (TF) efficiently precipitated cross-linked complexes (Fig 3A,B), confirming the earlier observation that TF,a cytosolic chaperone,binds to E coli nascent polypeptides [13] Quantification of the data indicated that the cross-linking efficiency of the mutant nascent chains was somewhat reduced (Fig 3B) In conclu-sion,our results show an increased affinity of the G-10C and G-10L prePhoE for the P48 component of SRP
G-10L nascent PhoE interacts with Sec translocon components
As (G-10L)94PhoE nascent chains apparently have a high affinity for P48 in vitro,we subsequently examined whether these nascent chains are targeted to SecY via SRP by performing cross-linking experiments in vitro in the presence
of IMVs To obtain a high cross-linking efficiency,recon-stituted E coli SRP was added after translation of nascent (G-10L)94PhoE polypeptides to saturate the RNCs with SRP The SRP–RNC complexes were purified over a high-salt sucrose cushion and incubated with IMVs to allow targeting After cross-linking with the membrane-permeable linking reagent DSS,peripheral and soluble cross-linked complexes were separated from integral-membrane cross-linked complexes by Na2CO3extraction and analyzed
by SDS/PAGE (Fig 4) In the Na2CO3pellet,at least two major (G-10L)94PhoE cross-linked complexes could be detected,one at 110 kDa and one at 46 kDa (Fig 4A, lane 3) The 110-kDa complex could be immunoprecipitated with antiserum directed against SecA,indicating that it is a complex of the radiolabeled (G-10L)94PhoE and SecA (Fig 4B,lane 1) In addition,cross-linking adducts of
220 kDa and 40 kDa were also immunoprecipitated from the Na2CO3pellet with anti-SecA serum We assume that the 220-kDa product corresponds to cross-linked complexes between (G-10L)94PhoE and the dimeric form
Fig 2 In vivo processing kinetics of prePhoE and mutant prePhoE
proteins in sec mutants (A) Cells of secB mutant strain CE1515
carrying plasmid pJP29 encoding wild-type PhoE (WT) or derivatives
were grown under phosphate limitation to express PhoE The cells
were pulse-labeled,followed by a chase for the indicated periods.
PhoE proteins were immunoprecipitated,separated by SDS/PAGE
followed by autoradiography G-10A (G-10A)prePhoE; G-10C
(G-10C)prePhoE; G-10L (G-10L)prePhoE (B) SecAts51 and
sec-Yts24 derivatives of CE1224 or their isogenic wild-type parental
strain (wt) carrying plasmids pJP29 or pNN8,encoding prePhoE or
(G-10L)prePhoE,respectively,were grown under phosphate limitation
for 3 h at the permissive temperature (30 C),subsequently for 2 h at
the restrictive temperature (42 C),and pulse-labeled at 42 C for 45 s
with Tran35S-label and chased with an excess of unlabeled methionine/
cysteine Aliquots were removed at the indicated periods and analyzed
as described for panel (A) The precursor and mature forms of the
PhoE proteins are indicated by p and m,respectively.
Trang 5of SecA The 40-kDa product in the Na2CO3 pellet
probably contains proteolytic fragments of the SecA dimer
and monomer cross-linking products,which is in agreement
with earlier reports [38] The fuzzy 46-kDa product
(Fig 4A,lane 3) was immunoprecipitated with anti-SecY
serum (Fig 4B,lane 2),showing that the (G-10L)94PhoE
nascent chains are targeted to the SecYEG translocon
In the Na2CO3supernatant,at least three major
cross-linking adducts,of apparent molecular mass 110, 65
and 55 kDa,could be detected (Fig 4A,lane 5) In
addition,several cross-linking adducts of low molecular
mass (< 30 kDa) were detected Immunoprecipitation
revealed that the high-molecular-mass adducts represent
cross-linking to SecA (data not shown),TF and P48 (Fig 4B,lane 5 and 6),respectively The identity of the low-molecular-mass adducts is unknown As the signal sequence
of 94PhoE has no affinity for P48 (Fig 3),and the SecB-binding sites in the mature domain are not exposed from the ribosome in RNCs of 94PhoE,these RNCs cannot be targeted to the translocon Consistently,no cross-linking adducts similar to those obtained with (G-10L)94PhoE were obtained,when 94PhoE and (G-10L)94PhoE nascent chains were incubated with IMVs after cross-linking with DSS (Fig 4C,lanes 1–4) To investigate whether the cross-linking adducts of (G-10L)94PhoE that were obtained are similar to the cross-linking adducts with a known substrate
of the SRP pathway,FtsQ was used as a model This class II membrane protein,with a short N-terminal cytoplasmic tail [39],was synthesized as a slightly longer nascent chain (101 residues) than (G-10L)94PhoE to expose properly its signal-anchor domain Indeed,101FtsQ interacted properly with SecY and SecA (Fig 4A,lane 8; Fig 4B,lane 3 and 4) Furthermore,the same cross-linking efficiency was obtained for P48 (Fig 4B,lane 8) as was observed for the (G-10L) prePhoE (Fig 4B,lane 6),but TF was hardly cross-linked if
at all (Fig 4B,compare lane 5 and 7) In conclusion,these results show that (G-10L)94PhoE nascent chains are correctly targeted to the SecY protein in the translocon via the SRP pathway
SRP dependency of (G-10L)prePhoEin vivo
As the experiments described above show that (G-10L) prePhoE is targeted in vitro to the Sec translocon via the SRP pathway,it was of interest to determine whether it is dependent on this pathway in vivo To test this possibility, wild-type and the (G-10L)prePhoE were expressed in FF283 cells which were depleted of 4.5S RNA After radioactive labeling of the cells,the PhoE forms were immunoprecipitated and analyzed by SDS/PAGE (Fig 5) Depletion of 4.5S RNA did not result in the accumulation
of precursors of either wild-type prePhoE or (G-10L)pre-PhoE Apparently (G-10L)prePhoE translocation is not dependent on the SRP pathway in vivo
D I S C U S S I O N
NMR studies of the signal peptides of LamB [25],OmpA [26] and PhoE [27] showed that the a-helical conformation is disrupted toward the C-terminus of the hydrophobic core near a helix-breaking residue,such as Gly)10in the case of prePhoE Furthermore,a statistical analysis of signal sequences revealed the common occurrence of helix-break-ing residues within the hydrophobic core [28],suggesthelix-break-ing that the disruption of the a-helix is a common feature of signal sequences In a previous study,it was shown that the DlH+dependency of prePhoE translocation was dramati-cally reduced when a helix-promoting residue,such as leucine or cysteine,was substituted for the helix-breaking Gly)10of the signal sequence [29] Such a substitution is expected to result in considerable elongation of the a-helix
in the signal sequence Consistent with a considerable conformational change,these substitutions resulted in a higher electrophoretic mobility of the mutant precursors compared with that of wild-type prePhoE [29] (see also Fig 2A),suggesting a more compact conformation of the
Fig 3 Cross-linking of soluble E coli proteins to PhoE nascent chains
and mutant derivatives (A) [ 35 S]methionine-labeled nascent 94PhoE or
mutant derivatives were synthesized in an E coli lysate and treated
with the homo-bifunctional chemical cross-linker BS 3 After
quench-ing,both P48- and TF-cross-linked complexes were
immunoprecipi-tated with antisera directed against P48 and TF,analyzed on SDS/
PAGE and visualized with a PhosphorImager (B) Quantification of
data presented in panel (A),after correction for translation efficiency.
The highest amounts of immunoprecipitated cross-linked nascent
chains were obtained for (G-10L)prePhoE in the case of P48
linked complexes and for WT prePhoE in the case of the TF
cross-linked complexes These amounts were set to 100%,and the relative
cross-linking efficiencies of the other prePhoE forms to TF and P48 are
shown.
Trang 6signal sequence In addition,CD measurements on synthetic
signal peptides showed a considerable increase in the
a-helical content by the G-10L substitution [40] Because
of the extension of the a-helix,the mutant signal sequences
more closely resemble the signal-anchor sequences of
integral-membrane proteins than does the wild-type signal
sequence Therefore,we considered the possibility that the
Gly)10 mutations affected the targeting pathway The
results from the in vivo pulse–chase experiments showed
that targeting of the mutant PhoE precursors is less
dependent on SecB,indicating that they are targeted to
the Sec translocon via another targeting pathway In vitro
cross-linking with the water-soluble cross-linker BS3
revealed that the G-10C and G-10L 94PhoE nascent chains
had an increased affinity for the P48 component of SRP compared with wild-type 94PhoE nascent chains Further-more,cross-link experiments with nascent chains in the presence of IMVs showed SRP-mediated targeting of (G-10L)94PhoE to the Sec translocon However, in vivo pulse–chase experiments revealed normal translocation kinetics of (G-10L)prePhoE in a 4.5S RNA-depletion strain This result is understandable,as the SecB-binding sites,which are located in the mature domain of the PhoE precursor [3],are not affected in the G-10L mutant precursor Thus,in the absence of SRP,SecB can target the mutant prePhoE to the SecYEG translocon Consis-tently,the processing of the mutant precursors was not completely SecB independent in a strain expressing SRP (Fig 2A) It has been reported previously that the SRP-targeting pathway is easily overloaded by overexpression of SRP substrates [17] Therefore,at the high expression levels used in these experiments,a proportion of the mutant prePhoE molecules may still rely on the SecB pathway, because of overloading of the SRP pathway The re-routing
of (G-10L)prePhoE to the Sec translocon via the SRP instead of the SecB pathway could be explained by the increased hydrophobicity of the hydrophobic core of the mutant signal sequence,because hydrophobicity was previ-ously reported to be an important variable in the interaction with SRP [14,41,42] However, the hydrophobicity of cysteine is even slightly lower than that of glycine [37] Therefore,the cross-linking of (G-10C)prePhoE to P48 indicates that another variable,in addition to hydropho-bicity,contributes to the interaction of signal sequences with
Fig 4 Targeting of SRP–RNCs to the Sec
translocon [35S]Methionine-labeled
(G-10L)94PhoE or 101FtsQ was incubated
with 350 n M reconstituted SRP SRP–RNCs
were purified and targeted to IMVs as
des-cribed in Experimental procedures The
cross-linker DSS was used to analyze SRP–RNC
interactions After quenching,peripherally
bound and soluble proteins were separated
from the inner membranes by carbonate
extraction Samples were either (A) directly or
(B) after immunoprecipitation (IP) with the
indicated antisera,subjected 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 Relevant cross-linked
complexes are indicated with arrowheads (C)
RNCs of wild-type and (G-10L)prePhoE were
synthesized in the presence of IMVs and
sub-sequently incubated with DSS After
quench-ing,cross-linked products were examined as
described above.
Fig 5 SRP dependency of (G-10L)prePhoE translocation in vivo.
Wild-type prePhoE and (G-10L)prePhoE were expressed in cells of
strain FF283 either depleted or not depleted of 4.5S RNA The cells
were pulse-labeled,followed by a chase for the indicated periods PhoE
proteins were immunoprecipitated,separated by SDS/PAGE and
detected by autoradiography.
Trang 7P48 We propose that this additional variable is a-helix
propensity Apparently,the a-helix propensity of cysteine
compensates for its low hydrophobicity,resulting in a better
interaction of the (G-10C)94PhoE protein with P48
The mechanism by which secretory proteins are routed
into the SRP-targeting or the SecB-targeting pathways in
E coli is not fully understood Although E coli SRP has
been shown to interact with cleavable signal sequences
in vitro [41,43–46], it is generally assumed that it binds
efficiently,under physiological conditions,only to
signal-anchor sequences,which contain a longer stretch of
consecutive hydrophobic amino acids Recent studies have
indicated that the hydrophobicity of the targeting signal is
the parameter discriminating between SRP-dependent and
SRP-independent pathways [14] On the other hand, in vitro
cross-linking studies have revealed that the binding of TF to
a sequence within the first 125 amino-acid residues of
pro-OmpA (but beyond the signal peptide) excluded the
association of the precursor to SRP [47] This observation
led to the proposal that secretory precursors are targeted to
the SecB pathway when they emerge from the ribosome by
means of their preferential recognition by TF However,we
found that a single amino-acid substitution (G-10L or
G-10C) in the signal sequence of PhoE results in a high
affinity for P48,even though TF is still bound to the G-10L
PhoE precursor Therefore,TF binding apparently does not
prevent the binding to P48,although we cannot exclude the
possibility that different (G-10L)prePhoE or
(G-10C)pre-PhoE molecules bind to either TF or P48,but not to both at
the same time In the case of the 101FtsQ substrate,TF was
not cross-linked efficiently whereas P48 was,in accordance
with previous observations [35] In general,our results are in
agreement with the reported binding of TF to secretory
precursors [47],but the basis for routing of secretory
proteins to the SecB pathway appears not to be the exclusion
of SRP by TF More likely,the helix breaker present in the
wild-type prePhoE signal sequence prevents interaction with
SRP,whereas the hydrophobic core of the mutant signal
sequences adopts a longer a-helical structure,which is
recognized by SRP as a substrate It is interesting to note
that the natural signal sequences of at least some secreted
proteins of Gram-positive bacteria,which do not possess a
SecB pathway and might therefore be entirely dependent on
the SRP pathway for protein secretion,also contain an
extended a-helix and have functional characteristics similar
to those of the G-10L mutant PhoE [48] In conclusion,our
results indicate that the helix breaker in cleavable signal
sequences prevents recognition by SRP,and it appears that
besides hydrophobicity the a-helix propensity of the
hydro-phobic core of the signal sequence helps to determine the
targeting pathway
A C K N O W L E D G E M E N T S
We would like to thank Elaine Eppens and Margot Koster for helpful
discussions and interest in the work,and Nico Nouwen for construction
of strain CE1513 Our thanks also go to William Wickner and Arnold
Driessen for providing antibodies against SecY and SecA,respectively.
Further,we thank Bauke Oudega for providing strain MM88,and
Tom Silhavy for his gift of strain NT1060 Finally,we thank Malene
Urbanus for her efforts with the cross-linking experiments This work
was supported by EU grant HPRN-CT-2000-00075 from the European
Community.
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