To study the importance of signal peptide hydrophobicity for protein export in both organisms, the a-amylase AmyQ was provided with leucine-rich high hydrophobicity or alanine-rich low h
Trang 1protein export by Bacillus subtilis
Geeske Zanen1, Edith N G Houben2, Rob Meima2,*, Harold Tjalsma3,†, Jan D H Jongbloed3,‡, Helga Westers1,3, Bauke Oudega2, Joen Luirink2, Jan Maarten van Dijl1,§ and Wim J Quax1
1 Department of Pharmaceutical Biology, University of Groningen, the Netherlands
2 Department of Molecular Microbiology, Vrije Universiteit, Amsterdam, the Netherlands
3 Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, the Netherlands
Bacillus subtilisand Escherichia coli are used as
proto-type models for studies on protein translocation and
secretion in Gram-positive and Gram-negative
bac-teria, respectively The absence or presence of a
hydro-phobic export signal, called signal peptide, determines
whether newly synthesized proteins are retained in the cytoplasm or exported to other cellular compartments Signal peptides and their recognition by cytoplasmic chaperones play a key role in membrane insertion of membrane proteins and in targeting of secretory
Keywords
SRP; signal peptide; protein targeting;
protein translocation; trigger factor
Correspondence
W J Quax, Department of Pharmaceutical
Biology, University of Groningen, Antonius
Deusinglaan 1, 9713 AV Groningen,
the Netherlands
Fax: +31 50 3633000
Tel: +31 50 3632558
E-mail: W.J.Quax@farm.rug.nl
Present addresses
*DSM Food Specialties, Postbus 1, 2600
MA Delft, the Netherlands; †Department of
Clinical Chemistry, University Medical
Centre Nijmegen, PO Box 9101, 6500 HB
Nijmegen, the Netherlands; ‡Department of
Clinical Genetics, University Medical Center
of Groningen, P.O box 30001, 9700 RB,
Groningen, the Netherlands; §Laboratory of
Molecular Bacteriology, Department of
Medical Microbiology, University Medical
Center of Groningen and University of
Groningen, Hanzeplein 1, PO Box 30001,
9700 RB Groningen, the Netherlands
(Received 29 March 2005, revised 03 May
2005, accepted 18 May 2005)
doi:10.1111/j.1742-4658.2005.04777.x
Signal peptides that direct protein export in Bacillus subtilis are overall more hydrophobic than signal peptides in Escherichia coli To study the importance of signal peptide hydrophobicity for protein export in both organisms, the a-amylase AmyQ was provided with leucine-rich (high hydrophobicity) or alanine-rich (low hydrophobicity) signal peptides AmyQ export was most efficiently directed by the authentic signal peptide, both in E coli and B subtilis The leucine-rich signal peptide directed AmyQ export less efficiently in both organisms, as judged from pulse-chase labelling experiments Remarkably, the alanine-rich signal peptide was functional in protein translocation only in E coli Cross-linking of in vitro synthesized ribosome nascent chain complexes (RNCs) to cytoplasmic pro-teins showed that signal peptide hydrophobicity is a critical determinant for signal peptide binding to the Ffh component of the signal recognition particle (SRP) or to trigger factor, not only in E coli, but also in B subtilis The results show that B subtilis SRP can discriminate between signal peptides with relatively high hydrophobicities Interestingly, the B subtilis protein export machinery seems to be poorly adapted to handle alanine-rich signal peptides with a low hydrophobicity Thus, signal peptide hydro-phobicity appears to be more critical for the efficiency of early stages in protein export in B subtilis than in E coli
Abbreviations
DSS, disuccinimidyl suberate; RNCs, ribosome nascent chain complexes; SRP, signal recognition particle; TF, trigger factor.
Trang 2proteins to the translocation machinery in the
mem-brane, the so-called Sec machinery in particular [1–4]
Signal peptides are usually sophisticated N-terminal
extensions, containing multipurpose functional
infor-mation A signal peptide can be divided into three
distinct domains; the N-, H-, and C-domains [5,6] The
N-domain interacts with the translocation machinery
and the negatively charged phospholipids in the lipid
bilayer of the membrane [7,8] The H-domain can adopt
an a-helical conformation in the membrane due to a
stretch of hydrophobic residues [9] To allow the
forma-tion of a hairpin-like structure that can insert into the
membrane, helix-breaking glycine or proline residues are
often present in the middle of the hydrophobic stretch
Unlooping of this hairpin might result in the insertion
of the complete signal peptide into the membrane [8]
Analyses of the H-domain show that the hydrophobic
core is the dominant structure in determining signal
peptide function [10–12] The C-domain contains the
cleavage site for specific signal peptidases that remove
signal peptides from the mature part of the exported
protein during or shortly after translocation [13,14]
Although the overall structure of signal peptides is
quite similar, small variations can result in export via
different targeting pathways [15–17] Signal peptides
directing proteins into the signal recognition particle
(SRP)-dependent pathway have a significantly more
hydrophobic H-domain than those mediating
SRP-independent targeting, at least in E coli [18,19]
Reduction of the net positive charge or the
hydro-phobicity of certain signal peptides decreases the
effectiveness of SRP recognition However, in E coli a
high degree of H-domain hydrophobicity can
compen-sate for the loss of basic residues in the N-domain and
restore SRP binding [20] Signal peptides containing an
(S⁄ T)RRXFLK motif in E coli or an RRXFF motif
in B subtilis (F is a hydrophobic residue, X can be
any residue) are candidates to be translocated via the
twin arginine translocation (Tat) pathway [15,21] In
general, Tat-targeting signal peptides have H-domains
which are less hydrophobic than signal peptides that
target proteins to the Sec machinery [22] Upon
emer-gence from the ribosome, the signal peptide of a
nascent secretory protein can be recognized by several
cytoplasmic chaperones and⁄ or targeting factors, such
as Ffh or trigger factor (TF) [23] In contrast to Ffh,
which is required for cotranslational protein export in
E coli, the cytoplasmic chaperone SecB has mainly
been implicated in post-translational protein targeting
For E coli it has been shown that by increasing the
hydrophobicity of signal peptides, exported proteins
can be re-routed from SecB into the SRP pathway
[19,24,25] Altogether, this means that different
specifi-city determinants are involved in early stages of pro-tein export from the cytoplasm
Most research on the interactions between signal pep-tides and cytoplasmic chaperones has so far been per-formed in E coli However, as shown by Collier, signal peptides can behave differently in different hosts [26] Notably, B subtilis lacks a SecB homologue, the chaper-one that is involved in post-translational targeting of the secretory proteins in E coli [2] Moreover, signal pep-tides of Gram-positive organisms are usually longer and more hydrophobic than those of Gram-negative organ-isms [2,27,28] Until now, it is not known whether this difference in hydrophobicity and length of signal pep-tides represents a functional difference in these species
In the present studies, we have addressed the effects
of major variations in signal peptide hydrophobicity
on translocation, processing, and signal peptide inter-action with cytoplasmic chaperones using a combined
in vivo and in vitro approach in both E coli and
B subtilis The results show interesting differences for the translocation of an a-amylase of B amyloliquefac-iens (AmyQ) with altered signal peptides in these organisms Whereas E coli translocates AmyQ with a less hydrophobic alanine-rich signal peptide, even in a secB mutant, B subtilis accumulates the respective pre-cursor intracellularly Cross-linking studies show that
TF of B subtilis interacts with the authentic signal peptide of AmyQ, whereas Ffh and TF of E coli com-pete to interact with this signal peptide Remarkably, a more hydrophobic leucine-rich signal peptide resulted
in reduced AmyQ translocation efficiencies, both in
B subtilis and E coli Taken together, these findings suggest that the hydrophobicity of signal peptides is more critical for early stages in protein translocation
in B subtilis than in E coli
Results
Changing the hydrophobicity of the AmyQ signal peptide
To study the effects of signal peptide hydrophobicity on the export of the a-amylase AmyQ of B amylolique-faciensby E coli or B subtilis, plasmids were construc-ted encoding AmyQ precursors with signal peptides of distinct hydrophobicity Specifically, an Ala-rich signal
TSAVN) and a Leu-rich signal peptide (MIQKRKR TVSLLLLLLCLLLLLQPITKTSAVN) were designed Hereafter, these mutant signal peptides are referred to
as Ala or Leu signal peptides These signal peptides have grand average of hydropathicity (Gravy) values that are significantly lower (0.341 for the Ala signal
Trang 3peptide) or higher (0.903 for the Leu signal peptide)
than that of the authentic AmyQ signal peptide (MIQK
value 0.591)
In vivo translocation and processing of a-amylase
in E coli and B subtilis
To study the effects of the different signal peptides on
in vivo translocation of AmyQ, E coli TG90 and
B subtilis 168 were transformed with the E coli–
B subtilis shuttle vectors pKTHM10, pKTHM101 or
pKTHM102 These vectors encode the authentic
pre-AmyQ, pre-AmyQ with the Ala signal peptide, and
pre-AmyQ with the Leu signal peptide, respectively
Cells were grown overnight and samples were prepared
for western blotting experiments and immunodetection
with specific antibodies against AmyQ As shown in
Fig 1A, mature AmyQ was detectable in cellular
sam-ples of E coli, irrespective of the signal peptide used
Pre-AmyQ was only detectable in significant amounts
when the Ala signal peptide was used, and it was
barely detectable when the Leu signal peptide was
used When expressed in B subtilis, mature AmyQ
was secreted into the growth medium when synthesized
with the authentic or Leu signal peptide In contrast,
no mature AmyQ was secreted when this protein was
synthesized with the Ala signal peptide (Fig 1B) To
verify whether the AmyQ secreted by B subtilis was
active, an activity assay was performed that is based
on the degradation of starch in agar plates As reflec-ted by the formation of halos upon staining with iodine vapour, active AmyQ was secreted when this protein was provided with the authentic or Leu signal peptide, but not when the Ala signal peptide was pre-sent (Fig 1C)
To examine the effects of signal peptide hydropho-bicity on the kinetics of pre-AmyQ processing, pulse-chase labelling experiments were performed with
B subtilis 168 or E coli TG90 cells producing AmyQ with the authentic, Ala, or Leu signal peptides After pulse labelling of newly synthesized proteins with [35S]methionine for 1 min, excess nonradioactive methionine (chase) was added (t¼ 0) After different periods of chase, samples were taken from which AmyQ was precipitated with specific antibodies As shown in Fig 2A, the authentic pre-AmyQ was almost completely processed after 5 min of chase when pro-duced in E coli In contrast, processing of AmyQ pre-cursors with the Leu or Ala signal peptides was significantly less efficient After 5 min chase, 46% or 53% of the AmyQ molecules synthesized with the Leu
or Ala signal peptides, respectively, were still in the precursor form (note that pre-AmyQ with the Ala sig-nal peptide has a lower mobility on SDS⁄ PAGE than pre-AmyQ with the authentic or Leu signal peptides)
In contrast, 45% of the authentic pre-AmyQ molecules was processed to the mature form within 1 min of chase Processing of AmyQ with the Ala signal peptide was so slow, that even after a chase of 30 min precur-sor molecules were still detectable (data not shown) The observation that, in E coli, AmyQ precursors with the Leu signal peptide were processed less efficiently was unexpected, since Doud and coworkers have previ-ously shown that signal peptides with increased hydro-phobicity improved the export efficiency for PhoA in this organism [29] Also in B subtilis, the processing of AmyQ with the Leu signal peptide occurred at a lower rate than that of AmyQ with the authentic signal pep-tide (Fig 2B) After 2 min of chase 53% of the AmyQ with the Leu signal peptide was processed to the mature form, whereas 71% of the AmyQ with the authentic signal peptide was processed within this time
of chase About 68% of the AmyQ molecules synthes-ized with the Leu signal peptide were mature after
5 min of chase A completely different result was obtained for AmyQ synthesized with the Ala signal peptide While AmyQ precursors with this signal pep-tide were processed in E coli, no processing of these precursors could be observed in B subtilis (Fig 2B) and even after a chase of 60 min no mature AmyQ was detected (data not shown) Notably, AmyQ mole-cules with the authentic signal peptide were processed
A
B
C
Fig 1 AmyQ production and secretion (A) AmyQ production in
cells of E coli as determined by western blotting using the proteins
from total cell extracts separated by SDS ⁄ PAGE (B) AmyQ
secre-tion into the growth medium of B subtilis as determined by
west-ern blotting using the proteins from culture supwest-ernatants separated
by SDS ⁄ PAGE The images in A and B relate to equal numbers of
E coli or B subtilis cells, respectively (C) Plate assay for AmyQ
secretion by B subtilis The signal peptides fused to AmyQ are
indicated p, Pre-AmyQ; m, mature AmyQ.
Trang 4more efficiently in B subtilis than in E coli, and the
same was true for AmyQ molecules with the Leu
sig-nal peptide
The fact that no processing of AmyQ with the Ala
signal peptide could be detected in B subtilis raised
the question whether this precursor was translocated
across the membrane To determine the topology of
(pre)AmyQ at steady state, protoplasts of B subtilis
cells were incubated with trypsin In parallel,
proto-plasts were incubated without trypsin or with trypsin
plus Triton X-100 As shown in Fig 3, cells producing
the authentic AmyQ or AmyQ with the Leu signal
peptide contained both precursor and mature forms of
AmyQ Notably, the accumulation of pre-AmyQ in
wild-type cells of B subtilis 168 is commonly observed,
despite the fact that this precursor is shown to be
processed efficiently in pulse-chase labelling
experi-ments [21,30,31] In contrast to AmyQ with the
authentic or Leu signal peptides, all AmyQ synthesized
with the Ala signal peptide was present in the
precur-sor form As previously shown, all AmyQ molecules
synthesized with the authentic signal peptide were
accessible to trypsin upon protoplasting of the cells
[31] In contrast, the situation was slightly different for
AmyQ synthesized with the Leu signal peptide: while
all mature molecules were accessible to trypsin upon
protoplasting, a significant fraction of the pre-AmyQ
molecules remained inaccessible to trypsin The latter
pre-AmyQ molecules were only degraded by trypsin in
the presence of Triton X-100, indicating that they were
protected against trypsin activity by the cytoplasmic
membrane Strikingly, none of the AmyQ molecules
synthesized with the Ala signal peptide was accessible
to trypsin upon protoplasting These precursor
mole-cules were, however, degraded by trypsin when the
protoplasts were lysed with Triton X-100 As controls for these fractionation experiments, the lipoprotein PrsA, which is localized at the membrane–cell wall interface, and the cytoplasmic protein GroEL were used Figure 3 shows that, irrespective of the cells used, the accessibility of PrsA and GroEL to trypsin was consistent with the subcellular location of these proteins While all PrsA was accessible to trypsin upon protoplasting, GroEL was only degraded by trypsin when the protoplasts were lysed with Triton X-100 Notably, microscopic inspection of the cells suggested that none of the strains investigated contained AmyQ inclusion bodies in the cytoplasm (data not shown) Consistent with the fact that AmyQ molecules synthes-ized with the authentic, Leu or Ala signal peptides were processed in E coli, subcellular localization experiments in this organism revealed that all corres-ponding precursor and mature AmyQ molecules were accessible to trypsin upon spheroplasting (data not shown) Taken together, these observations show that AmyQ molecules with the Leu signal peptide are trans-located across the cytoplasmic membranes of B subtilis and E coli, but with a slightly lower efficiency than AmyQ molecules with the authentic signal peptide In contrast, AmyQ molecules with the Ala signal peptide are translocated across the cytoplasmic membrane in
E coli, but not in B subtilis
Although the processing of the AmyQ precursor containing the Leu signal peptide was slower than that of wild type AmyQ in E coli and in B subtilis, processing of the AmyQ precursor containing the Ala signal peptide was only observed in E coli Since
E colicontains the cytoplasmic chaperone SecB, which
is absent from B subtilis, the influence of SecB on the processing of AmyQ containing the Ala signal peptide
A
B
Fig 2 Processing of pre-AmyQ Processing
of AmyQ precursors with different signal peptides in E coli (A) or B subtilis (B) was analysed by pulse-chase labelling at 37 C Cells were labelled with [ 35 S]methionine for
1 min prior to chase with excess nonradio-active methionine Samples were withdrawn
at the times indicated The presence of pre-cursor or mature forms of AmyQ in cells plus growth medium was visualized by immunoprecipitation, SDS ⁄ PAGE and fluo-rography The percentage of processed (mature) AmyQ relative to the total amount
of AmyQ (precursor + mature) in each lane
is indicated (%) The signal peptides fused
to AmyQ are indicated p, Precursor;
m, mature AmyQ.
Trang 5was investigated Pulse-chase labelling experiments
with E coli MC4100 and the corresponding secB
mutant strain were performed at 30C, because the
growth of both strains at 37C was severely impaired
when transformed with the plasmid for AmyQ-Ala
expression (note that this was not the case in E coli
TG90) The results obtained with E coli MC4100
showed a less efficient processing of AmyQ precursor
containing the Ala signal peptide at 30C, as
com-pared to the processing of this precursor in E coli
TG90 at 37C (compare Fig 2A and Fig 4A) As
shown in Fig 4A, the processing rate of AmyQ with
the Ala signal peptide was mildly reduced in secB
mutant cells as compared to cells of E coli MC4100
Compared to the SecB-dependent OmpA protein, the effect of the absence of SecB on the processing of AmyQ with the Ala signal peptide was less evident (Fig 4)
In vitro cross-linking of a-amylase nascent chains
in E coli and B subtilis The influence of signal peptide hydrophobicity on its interactions with E coli and B subtilis cytoplasmic proteins was investigated by chemical cross-linking of
in vitro translated nascent chains In this approach, truncated mRNAs were translated in an E coli trans-lation lysate in the presence of [35S]methionine to
Fig 3 Localization of AmyQ in B subtilis To analyse the subcellular localization of AmyQ molecules synthesized with different signal pep-tides, cells of B subtilis were grown overnight at 37 C in TY medium, diluted 50-fold in fresh TY medium and incubated at 37 C for 3 h prior to protoplasting Protoplasts were incubated for 30 min without further additions, in the presence of trypsin (T; 1 mgÆmL)1), or trypsin + Triton X-100 (1%) Samples were used for SDS ⁄ PAGE and western blotting Specific antibodies were used to detect AmyQ, PrsA, or GroEL The positions of (pre)AmyQ, PrsA, and GroEL (c), and degradation products of PrsA (d*) are indicated The signal peptides fused to AmyQ are indicated A cartoon of the protoplasting and protease protection experiment is shown to illustrate the effects of trypsin (T) and Triton X-100.
A
B
Fig 4 Processing of AmyQ with the Ala
signal peptide in E coli secB Processing of
pre-AmyQ containing the Ala signal peptide
(A) and pro-OmpA (B) in E coli MC4100
secB or the parental strain (wt) were
ana-lysed by pulse-chase labeling at 30 C and
subsequent immunoprecipitation,
SDS ⁄ PAGE, and fluorography Cells were
labelled with [35S]methionine for 1 min prior
to chase with excess nonradioactive
methio-nine Samples were withdrawn at the times
indicated The percentage of processed
(mature) AmyQ relative to the total amount
of AmyQ (precursor + mature) in each lane
is indicated (%) p, Precursor; m, mature.
Trang 6generate radioactively labelled ribosome-nascent chain
complexes (RNCs) A C-terminal 4· methionine tag
was introduced into the nascent chains to increase the
labelling efficiency The nascent chain corresponding
to the authentic preprotein comprised 101 amino
acids, while the nascent chain corresponding to the
Leu and Ala preproteins comprised 105 amino acids
Thus, the lengths of these nascent chains allows
opti-mal cytoplasmic exposure of the signal peptides,
tak-ing into consideration that approximately 30 amino
acids will be located within the ribosome
(schemati-cally represented in Fig 5A) The RNCs were purified
over a high-salt sucrose cushion to remove all loosely
associated E coli components originating from the
translation lyate Subsequently, they were either
incu-bated with crude E coli MC4100, B subtilis 168, or
B subtilis DTF cell lysates The latter strain lacks the
TF, which is known to interact with peptides
emer-ging from the ribosome [23] The DTF strain was
used for these experiments, because no anti-body against the B subtilis TF is currently available
As a negative control, the purified RNCs were incu-bated with incubation buffer only Interactions between RNCs and cytoplasmic components of E coli
or B subtilis were fixed by adding the homobifunc-tional lysine-lysine cross-linking reagent disuccinimidyl suberate (DSS)
Incubation of AmyQ nascent chains containing the authentic signal peptide with E coli lysate in the presence of DSS generated cross-linking adducts
of 25 kDa, 60 kDa, 68 kDa, and 80 kDa (Fig 5B, lane 3) The 25 kDa adduct could be immu-noprecipitated using antiserum raised against the E coli ribosomal protein L23 (Fig 5B, lane 6) In fact, cross-linking to L23 was not only shown for RNCs with the authentic signal peptide, but also for RNCs with the Leu and Ala signal peptides (Fig 5B, lanes 2–5, lanes 10–13, lanes 18–21) As shown by immunoprecipitation
A
B
Fig 5 Cross-linking of AmyQ nascent
chai-ns to soluble E coli and B subtilis compo-nents The 101AmyQ wt, 105AmyQ Leu and 105AmyQ Ala RNCs were synthesized
in an E coli MC4100 translation lysate (A) Schematic representation of the translation reactions The different signal peptides used and lysine residues (K) that may participate
in cross-linking reactions are indicated (B) After translation, the RNCs were purified over a high-salt sucrose cushion, incubated with crude E coli MC4100, B subtilis 168,
B subtilis DTF cell lysates or incubation buffer and treated with DSS Cross-linking was quenched by adding TCA ⁄ acetone Immunoprecipitations were subsequently carried out as indicated in Experimental procedures IP, Immuno-precipitation; E, crude cell lysate of E coli MC4100; B, crude cell lysate of B subtilis 168; BD, crude cell lysate of B subtilis DTF; NC, nascent chain;
?, unknown cross-linking adducts;
*, cross-linking adducts with E coli L23;
d , cross-linking adducts with E coli Ffh;
s , cross-linking adducts with B subtilis Ffh;
n, cross-linking adducts with E coli TF;
h , cross-linking adducts with B subtilis TF.
Trang 7with Ffh- and TF-specific antibodies, the 60 kDa
cross-linking adduct contained E coli Ffh, while both
the 68 kDa and 80 kDa adducts contained the
E coli TF (Fig 5B, lanes 7, 8) Adducts of TF
fre-quently appear as a doublet [18,32], but it is not known
why the ratio between the immunoprecipitated
AmyQ-TF adducts differs from the ratio in the nonprecipitated
sample (Fig 5B, lanes 3 and 8) Unfortunately, E coli
SRP could not be removed completely from the
ribo-somes by high-salt treatment (Fig 5B, lane 2 closed
cir-cle) Consequently, cross-links to E coli Ffh were also
detected upon incubation of RNCs containing the
authentic signal peptide with B subtilis 168 lysate in the
presence of DSS Cross-linking of these RNCs to B
sub-tilisFfh could not be demonstrated by
immunoprecipi-tations using antibodies specific for B subtilis Ffh (data
not shown) On the other hand, two dominant
cross-linking adducts of 69 kDa were detected upon
incuba-tion of authentic pre-AmyQ RNCs with B subtilis 168
lysate (Fig 5B, lane 4 open squares) Such adducts were
not observed after incubating these nascent chains with
B subtilisDTF lysate in the presence of DSS (Fig 5B,
lane 5), which implies that the 69 kDa adducts
represent cross-links to TF of B subtilis Interestingly,
incubation with the B subtilis DTF lysate resulted in
40-kDa cross-linking adducts that were not observed
upon incubation with the B subtilis 168 lysate (Fig 5B,
lane 5 question mark) Unfortunately, the B subtilis
protein(s) in these 40-kDa adducts could not be
identified
Nascent chains of AmyQ with the Leu signal peptide
generated 60-kDa and 70-kDa cross-linking
adducts upon incubation with E coli lysate in the
pres-ence of DSS (Fig 5B, lane 10 closed circles), which
both represented cross-linking to E coli Ffh (data not
shown) Cross-linking of these RNCs to E coli Ffh was
even detected upon incubation with B subtilis 168
ly-sate in the presence of DSS (Fig 5B, lane 14) This
cross-linked E coli Ffh was derived from the
transla-tion lysate, despite the high-salt purificatransla-tion
Never-theless, as shown by immunoprecipitation with specific
antibodies, a 60-kDa cross-linking adduct containing
B subtilisFfh was formed upon incubation with B
sub-tilis 168 lysate in the presence of DSS (Fig 5B, lane
15) Note that the antibodies against Ffh of B subtilis
do not cross-react with Ffh of E coli (Fig 5B, lane 16)
The 48-kDa cross-linking adduct obtained upon
incu-bation of RNCs containing the Leu signal peptide with
the B subtilis 168 lysate was not identified (Fig 5B,
lane 12 question mark) Interestingly, no evidence for
specific cross-links between RNCs with the Leu signal
peptide and the B subtilis or E coli TFs was obtained
(Fig 5B, lanes 11–13 and data not shown)
Finally, nascent chains of AmyQ containing the Ala signal peptide generated strong cross-links to E coli and B subtilis TF (Fig 5B, lane 19–21 open and closed squares), while neither cross-links to E coli Ffh nor B subtilis Ffh were observed (data not shown) Incubation of these nascent chains with the B subtilis DTF lysate again generated unidentified 40-kDa cross-linking adducts (Fig 5B, lane 21 question mark)
In conclusion, these findings show that RNCs contain-ing the authentic signal peptide can be cross-linked with L23, Ffh, and TF of E coli and with TF of
B subtilis RNCs containing the highly hydrophobic Leu signal peptide can be cross-linked with L23 of
E coli, Ffh of E coli and B subtilis, but not detecta-bly with TF of these organisms In contrast, RNCs containing the mildly hydrophobic Ala signal peptide are efficiently cross-linked with L23 of E coli, TF of
E coli and B subtilis, but not detectably with Ffh of these organisms
Discussion
Several studies indicate that signal peptide hydrophob-icity is an important determinant for SRP-mediated protein targeting to the E coli inner membrane [18,33] Cross-linking of nascent PhoA-derivatives revealed an almost linear correlation between hydrophobicity and SRP cross-linking [18] In addition, hydrophobic alter-ations in the signal peptides of SecB-dependent pro-teins, re-routed these proteins into the SRP pathway [19,24,25] Precursor proteins from Gram-positive bac-teria contain signal peptides that are usually longer and more hydrophobic than the signal peptides of pre-cursor proteins from Gram-negative bacteria [2,27,28]
It was therefore hypothesized that the higher hydro-phobicity of signal peptides in Gram-positive bacteria, lacking SecB, has evolved as an adaptation to the SRP-dependent translocation pathway [2]
Changes in hydrophobicity of the signal peptide of a-amylase AmyQ seem to have different effects on the translocation of pre-AmyQ in E coli or B subtilis For E coli cells, changing the alanine or leucine content and, consequently, the hydrophobicity of the signal peptide did not lead to major translocation defects However, processing was less efficient for AmyQ precursors containing the Leu or Ala signal peptides when compared to AmyQ with the authentic signal peptide Importantly, significant amounts of mature AmyQ were released into the periplasm irres-pective of the signal peptide used In B subtilis, mature AmyQ directed to and across the membrane with help of the authentic or Leu signal peptides, was efficiently secreted resulting in active AmyQ in the
Trang 8growth medium In contrast, AmyQ containing the
Ala signal peptide was not translocated at all This
implies that B subtilis is not able to translocate
pre-cursor proteins with Ala-rich signal peptides of low
overall hydrophobicity This could be due to specific
not previously reported effects of Ala residues in a
B subtilis signal peptide For example, the relatively
small size of the Ala side chain might be of relevance
with respect to the recognition of the Ala signal
peptide by the secretion machinery of B subtilis
Nevertheless, certain B subtilis signal peptides of
which the in vivo activity has been demonstrated
con-tain a relatively large amount of alanine For example
the YxkA signal peptide contains 12 Ala residues
[2,4], but has a grand average of hydrophaticity of
0915 This suggests that the low hydrophobicity
rather than the high Ala content is responsible for
the observed malfunction of the Ala signal peptide
Clearly, this malfunction cannot be explained by the
absence of a SecB homologue in B subtilis, because
SecB contributes only to a minor extent to the export
of AmyQ with the Ala signal peptide in E coli
The processing of AmyQ with the authentic or Leu
signal peptides was faster in B subtilis than in E coli
This is likely due to the overall characteristics of the
AmyQ signal peptide Precursors have normally
shor-ter signal peptides in E coli than in B subtilis [2,4],
and thus the signal peptides used in this study are
probably suboptimal for E coli Nevertheless, when
produced in E coli, most AmyQ molecules with the
authentic signal peptide are processed within 5 min of
chase Finally, in both species the processing rate for
AmyQ precursors containing the Leu signal peptide
was lower compared to those containing the authentic
signal peptide A possible explanation for this
observa-tion could be that the Leu-rich H-domain of high
hydrophobicity, perhaps in combination with the four
positively charged residues already present in the
N-domain, results in a tighter binding of a
signalpep-tide to SRP This might slow down the release of the
precursor protein from SRP, which would result in
slower translocation and processing by signal
pepti-dase Another possibility could be that the Sec
trans-locon has a lower affinity for more hydrophobic
AmyQ-derived signal peptides However, it has been
shown that an increased leucine content of a signal
peptide increases the cross-linking to Sec of E coli
[34] Taken together, our observations indicate that, in
particular, a low signal peptide hydrophobicity
com-pletely impairs precursor translocation in B subtilis,
but not in E coli
Together with the DnaK system, the
ribosome-asso-ciated chaperone TF promotes the folding of newly
synthesized proteins in the cytosol of E coli [35,36]
E coli TF interacts with virtually all nascent polypep-tides, whereas Ffh interacts specifically with hydropho-bic signal peptides [23] The present studies show for the first time that the hydrophobicity of a signal pep-tide has a critical impact on its binding to TF or Ffh in
B subtilis In addition, our studies provide first support for binding of the B subtilis TF to nascent chains While the authentic AmyQ signal peptide binds both to
TF and (E coli) Ffh, the less hydrophobic Ala signal peptide only binds to TF, and the more hydrophobic Leu signal peptide binds mainly to Ffh This obser-vation suggests that TF of B subtilis plays also an important role in the early stages of signal peptide recognition In E coli, ribosomal protein L23 is located near the exit site of the ribosomal tunnel that runs from the peptidyl transferase centre to the surface of the large ribosomal subunit [37] Interestingly, all AmyQ nascent chains tested were found to bind L23 present in the E coli lysates used for in vitro translation Remark-ably, nascent chains containing the authentic or Leu signal peptides were cross-linked to E coli Ffh, even in the presence of a B subtilis lysate This implies that
E coli Ffh could not be removed completely from the RNCs by high salt treatment and that B subtlis Ffh was unable to compete efficiently with E coli Ffh This
is probably the reason why binding of B subtilis Ffh to RNCs with the authentic AmyQ signal peptide could not be visualized in our cross-linking experiments even though it seems most likely that this binding does occur
in vivo As previously pointed out by Walter and Blobel [38], such inefficient binding can be exacerbated by the fact that the H-domains of signal peptides lack lysine residues, which are required for cross-linking with the lysine-specific reagent DSS Nevertheless, binding of
B subtilis Ffh to RNCs with the Leu signal peptide could be demonstrated This indicates that Ffh of
B subtilis has a higher affinity for hydrophobic signal peptides, such as the Leu signal peptide, and that Ffh
of B subtilis can effectively compete with Ffh of E coli for the binding of this signal peptide Alternatively, RNCs with exposed Leu signal peptides may not be saturated with E coli Ffh, which would allow for more efficient binding of B subtilis Ffh This latter possibil-ity would imply that B subtilis Ffh does not bind effi-ciently to the RNC with the authentic AmyQ signal peptide, which seems rather unlikely At present it is not clear why AmyQ with the Ala signal peptide is translocated in E coli, but not in B subtilis Import-antly, this precursor is still translocated in a secB mutant of E coli, indicating that factors other than SecB are required for this process in E coli This suggests that the absence of a SecB homologue in
Trang 9B subtilis is not responsible for the lack of export of
AmyQ containing the Ala signal peptide
In conclusion, our present observations imply that
signal peptide hydrophobicity is critical for early stage
signal peptide recognition by SRP and TF, not only in
E coli, but also in B subtilis This view is supported
by the fact that the sequences of TF and L23 that are
required for ribosome docking of TF [39] are
con-served in the corresponding proteins of B subtilis
Even though the signal peptide of AmyQ is already
longer and more hydrophobic than the average E coli
signal peptide, its binding to Ffh of B subtilis can be
enhanced by further increasing its hydrophobicity
Thus, the B subtilis SRP system is able to disciminate
between signal peptides with relatively high
hydropho-bicities Conversely, the B subtilis machinery for
pro-tein export appears poorly adapted to handle signal
peptides with a low hydrophobicity These findings are
likely to be of biological relevance since the average
hydrophobicity of B subtilis signal peptides is
signifi-cantly higher than that of E coli signal peptides
Experimental procedures
Plasmids, bacterial strains and media The plasmids and bacterial strains used are listed in Table 1 TY medium contained Bacto tryptone (1%), Bacto yeast extract (0.5%), and NaCl (1%) S7-MAM medium was essentially prepared as S7 medium [40] with the differ-ence that the MAM amino acid mixture from Becton Dick-inson (Franklin Lakes, NJ, USA) was used instead of the amino acid mixture normally used to supplement S7 medium [41] If required, media for E coli were supple-mented with ampicillin (100 lgÆmL)1); or chloramphenicol (10 lgÆmL)1), and media for B subtilis with chlorampheni-col (5 lgÆmL)1); or kanamycin (10 or 20 lgÆmL)1)
DNA techniques Procedures for PCR, DNA purification, restriction, liga-tion, agarose gel electrophoresis, and transformation of
E coli were carried out as described by Sambrook et al [42] Competent B subtilis cells were transformed as
Table 1 Plasmids and bacterial strains.
Plasmids
pMTL23Q3 pMTL23 carrying the 712 bp EcoRV-SphI fragment of pKTH10, encompassing the
5¢-terminus of the amyQ gene
This paper pQ1 pMTL23Q3 carrying silent mutations in the 5¢-terminus of the amyQ gene, creating
HindIII, SpeI and KpnI sites at the nucleotides that specify the signal peptidase cleavage site
This paper
pKTH10 B subtilis vector; encodes the a-amylase AmyQ of B amyloliquefaciens [55]
pKTHM10 E coli–B subtilis shuttle vector The EcoRV–SphI fragment of pKTH10 is replaced
by the EcoRV–PvuII fragment of pQ10
This paper
pC4Meth94Bla E coli cloning vector used for in vitro transcription-translation [18]
pC4Meth95AmyQ wt pC4Meth94Bla containing the first 95 codons of the wild type amyQ gene This paper pC4Meth95AmyQ Ala pC4Meth94Bla containing the first 95 codons of the amyQ gene from pKTHM101 This paper pC4Meth95AmyQ Leu pC4Meth94Bla containing the first 95 codons of the amyQ gene from pKTHM102 This paper Strains
B subtilis
E coli
MC4100 F–; araD139; D(argF-lac); U169; rspL150; relA1; flbB5301; fruA25; deoC1; ptsF25 [60]
laboratory collection
Trang 10previously described [30] Restriction enzymes and the
Expand long template PCR system were obtained from
Roche Diagnostics GmbH (Mannheim, Germany) T4
DNA ligase was obtained from Epicenter Technologies
(Omaha, NE, USA)
Construction of AmyQ derivatives containing
altered signal peptides
To study the effect of the amino acid composition of the
H-domain of the signal peptide on protein export, a series
of plasmids was constructed encoding AmyQ derivatives
with modified H-domains First, an SphI–EcoRV fragment
of pKTH10 containing the 5¢-terminus of the amyQ gene
was subcloned in the E coli cloning vector pMTL23
result-ing in pMTL23Q3 Usresult-ing pMTL23Q3 as a template, five
silent mutations were introduced into the signal
peptide-encoding region of amyQ by two subsequent rounds of
PCR mutagenesis resulting in plasmids pQ1 and pQ10,
respectively The primers used are listed in Table 2 Thus,
restriction sites were introduced for replacement of the
sig-nal peptide or parts thereof (e.g the H-domain) with
exist-ing or designed amino acid sequences To construct an
E coli–B subtilisshuttle vector, EcoRV-digested pQ10 was
fused to plasmid pKTH10, which was cut with EcoRV–
PvuII This resulted in plasmid pKTHM10, which carries
a full-length amyQ gene that encodes the authentic AmyQ
precursor
Next, two modified H-domains were introduced to
replace the original H-domain of the AmyQ signal peptide
Firstly, complementary oligonucleotides (see Table 2) were
annealed and cloned into plasmid pCR2.1-TOPO
(Invitro-gen Life Technologies, Paisley, UK), using the overhanging
HindIII and SpeI compatible sticky ends Secondly, the fragments were transferred to plasmid pQ10 using the same restriction sites resulting in plasmids pQ101 and pQ102 Finally, the EcoRV–SphI fragments of pQ101 and pQ102 were ligated to EcoRV–SphI digested pKTHM10 This resulted in plasmids pKTHM101 (encoding pre-AmyQ with
a Leu-rich signal sequence) and pKTHM102 (encoding pre-AmyQ with an Ala-rich signal sequence) Though not used
in the present studies, the Cys residue in the centre of the H-region of the authentic AmyQ signal peptide was main-tained in the H-regions of the Ala- and Leu-rich signal pep-tides to facilitate future cross-linking experiments
The ‘grand average of hydrophathicity’ (Gravy) value for the signal peptide was calculated with the protparam tool (http://www.expasy.org/tools/protparam.html) as the sum
of hydrophobicity values of all the amino acids, divided by the number of residues in the sequence [43,44]
SDS/PAGE, western blotting and immunodetection
To visualize proteins of E coli or B subtilis by western blotting, cells were separated from the growth medium by
centrifugation (3 min, 12 900 g, 20C) Cellular samples of
E coli and B subtilis, and growth medium samples of
B subtilis were prepared for SDS⁄ PAGE as described pre-viously [40,45] After separation by SDS⁄ PAGE, proteins were transferred to a Protran nitrocellulose transfer mem-brane (Schleicher and Schuell, Dassel, Germany) Western blotting was performed as described by Kyhse-Andersen [46] AmyQ, GroEL, and PrsA were visualized with specific antibodies and horseradish peroxidase-conjugated goat anti-rabbit IgG or alkaline phosphatase-conjugated goat
Table 2 Overview of primers used in the present study Restriction sites are indicated in bold.
CGGCACTAGTTTTTGTAATCGGCAAGCTTACAAATAACAG
Mutagenesis primer for introduction of KpnI, SpeI, and HindIII sites into the AmyQ signal peptide coding region
AmyQ signal peptide coding region
CACATAAGCACAAGTCTGAAGCTTACTGTCCGCTTTCG
TTTTTGAATCATATGTC
Second round mutagenesis primer (introduction of NdeI and HindIII sites upstream of H-region)
GCGCTGCAGCCGATTACAAAAA
Oligo encompassing AmyQ H-region, complementary with h-ala-rev
CGGCGCACGCAGCCGCGGCCGCCA
Oligo encompassing AmyQ H-region, complementary with h-ala-fwd
AGCCGATTACAAAAA
Oligo encompassing AmyQ H-region, complementary with h-leu-rev
AAAAGGAGAAGCAGCA
Oligo encompassing AmyQ H-region, complementary with h-leu-fwd
amyQ_ATG CGCGAATTCTAATATGATTCAAAAACGAAAGCGGA Amplification primer for construction of truncated
AmyQ variants for synthesis of nascent chains
AmyQ variants for synthesis of nascent chains