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J. Biol. Chem., Vol. 275, Issue 31, 23439-23445, August 4, 2000
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§, and
From the Fakultät für Biologie, Universität
Konstanz, 78457 Konstanz, Germany and the Department of
Microbiology and Molecular Genetics, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, April 3, 2000, and in revised form, May 10, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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One isoform of trehalase, TreF, is present in the
cytoplasm and a second, TreA, in the periplasm. To study the questions
of why one enzyme is exported efficiently and the other is not and whether these proteins can fold in their nonnative cellular
compartment, we fused the signal sequence of periplasmic TreA to
cytoplasmic TreF. Even though this TreF construct was exported
efficiently to the periplasm, it was not active. It was insoluble and
degraded by the periplasmic serine protease DegP. To determine why TreF was misfolded in the periplasm, we isolated and characterized Tre+ revertants of periplasmic TreF. To further
characterize periplasmic TreF, we used a genetic selection to isolate
functional TreA-TreF hybrids, which were analyzed with respect to
solubility and function. These data suggested that a domain located
between residues 255 and 350 of TreF is sufficient to cause folding
problems in the periplasm. In contrast to TreF, periplasmic TreA could
fold into the active conformation in its nonnative cellular
compartment, the cytoplasm, after removal of its signal sequence.
Secretory and cytoplasmic proteins differ not only by the signal
sequence but also in their folding properties. It is thought that the
export competence of secretory proteins is the result of slow folding
prior to export. Cytoplasmic proteins are believed to fold more rapidly
and thus are not substrates of the cellular secretion apparatus. For a
better understanding of the mechanism of translocation, the folding of
cytoplasmic and secretory proteins needs to be characterized. Folding
of polypeptides is determined by the primary amino acid sequence but
also could be influenced by the particular properties of a cellular
compartment. However, little is known about whether and how the
cytoplasm and the periplasm specifically influence protein folding,
i.e. whether there are other elements besides redox state
and the presence/absence of ATP. To study these aspects, we used TreA
and TreF, the two trehalases of Escherichia coli, as model proteins.
Periplasmic TreA is synthesized as a precursor of 565 amino acids. The
signal sequence of 30 amino acids is rather long. Mature TreA has a
molecular mass of 58 kDa (1, 2). The Km of the
purified enzyme is 0.8 mM, the Vmax
is 66 µmol of trehalose hydrolyzed/min/mg of protein, and the pH
optimum is 5.5 (3). The expression of treA is independent of
the presence of trehalose in the growth medium but is stimulated
10-fold at high osmolarity (1, 2). Also, treA is regulated
by RpoS, the stationary phase sigma factor (4).
Cytoplasmic TreF has 549 amino acids and a molecular mass of 64 kDa.
The Km of the purified enzyme is 1.9 mM,
the Vmax is 54 µmol of trehalose
hydrolyzed/min/mg of protein, and the pH optimum is 6.0. Like
treA, the expression of treF is independent of
trehalose, is dependent on RpoS, and is induced at high osmolarity. Both TreA and TreF are monomeric enzymes, and they share an amino acid
sequence identity of 47%. TreA has an extended C terminus of about 30 residues, which is not present in TreF, and TreF has a 61-residue
extension at its N terminus, which is not present in TreA (5).
Trehalose metabolism in E. coli can occur either in the
periplasm via TreA or after transport into the cytoplasm via a
trehalose-specific enzyme II encoded by treB (6). Transport
results in the formation of trehalose 6-phosphate. Trehalose
6-phosphate is cleaved by TreC into glucose 6-phosphate and glucose
(7). When trehalose is synthesized in the cytoplasm in response to high
osmolarity of the growth medium, it can be degraded by cytoplasmic
TreF. Cells expressing treF from the chromosome exhibit very
low trehalase activity (5).
We are interested in the phenomenon that, despite their high
similarity, TreA and TreF are localized in different
cellular compartments, and we wished to determine whether these
compartments have an effect on folding and specific activity. To study
these questions we expressed both enzymes in their nonnative cellular compartment, i.e. treA in the cytoplasm and
treF in the periplasm, and investigated whether they would
fold into the active conformation. Whereas treA was actively
expressed in the cytoplasm, periplasmic TreF was inactive. To determine
why TreF is misfolded in the periplasm, we isolated and characterized
Tre+ revertants of periplasmic TreF. To identify regions in
TreF responsible for misfolding in the periplasm, we selected and
characterized TreA-TreF hybrid proteins.
Bacteria and Plasmids--
E. coli strains used were
derivatives of DHB3 (araD139 Construction of Plasmids--
p
ptreA'-'treF was constructed by subcloning a 2.27-kilobase
EagI fragment from ptre11 into
pBADtreF which was cleaved with EagI. This
plasmid contains the wild-type treA promoter and the 404 codons of the 5'-end of wild-type treA followed by
treF containing a deletion of the first 165 codons.
p
ptreA'-treF expresses a gene fusion of the first 265 codons
of treA and all of treF. It was constructed by
cloning a polymerase chain reaction product of treF flanked
by a KpnI restriction site at the 5'-end and an
EcoRI restriction site at the 3'-end into ptre11
cleaved with KpnI and EcoRI. Primers used were
5'-GGGGTACCatgctcaatcagaaaattcaaaac-3' and 5'-CGGAATTCttatggttcgccgtaca
aacc-3'. p
psstreF was constructed by the subcloning of a 789-base pair
polymerase chain reaction fragment containing the treA
promoter up to the treA signal sequence flanked by a
KpnI restriction site at the 3'-end as a
BamHI-KpnI fragment into ptreA'-treF
cleaved with BamHI and KpnI. Primers used were
5'-GGGGTACCttcttctgcctgcaccgat-3' and 5'-aatgggcatgcaaggagatgg-3'. This
construct contained a treA sequence including the first two
codons of mature treA, followed by codons for Gly and Thr
and the entire treF gene. The additional Gly and Thr
residues were a consequence of the introduced KpnI site
immediately upstream of the translational initiation codon of
treF.
Trehalase Assay--
To determine the Km for
trehalose of the various trehalase constructs, treA treC
mutant strains KU92 or KU95 expressing the trehalase constructs from
plasmids were grown overnight in rich medium. Cultures were washed
twice in minimal medium and were broken in a french pressure cell at
9000 p.s.i. The remaining intact cells were removed by
centrifugation (15,000 rpm, 30 min, 4 °C, SS34 rotor). The DNA of
the cell extract was precipitated with 2% streptomycin sulfate and
pelleted by centrifugation (15,000 rpm, 30 min, 4 °C, SS34 rotor).
Subsequently, the protein concentration of the cell extract was
adjusted to 0.2-0.4 mg/ml. Alternatively, to assay periplasmic
trehalase activity of whole cells, cells were grown overnight in rich
medium. Cultures were washed twice in minimal medium and were
resuspended in trehalase assay buffer.
Trehalase assays were performed in 100 µl of 20 mM
potassium phosphate buffer, pH 6.0. The reaction was started by the
addition of 0.25 to 10 mM trehalose and incubated for 5-15
min at room temperature. The reaction was stopped by boiling the
samples for 5 min. Cleavage of trehalose was assayed by determining
glucose concentration by the glucose test kit from Merck.
Labeling of Cells, Cell Fractionation, and Antibodies against
TreF--
Protein was labeled in cultures of strain KU101 expressing
either treA or sstreF from plasmids
ptre11 and psstreF, respectively, growing
exponentially (A600 0.4) in minimal medium 9 (11), 0.2% glucose, 1 µg/ml thiamine, 50 µg/ml ampicillin,
supplemented with each common amino acid except Cys and Met. 1 ml of
cells was exposed to [35S]methionine (>1000 Ci/mmol) at
50 µCi/ml for 1 min and subsequently cooled on ice for
immunoprecipitation or the labeling period was followed by a chase with
excess cold methionine (50 mM final concentration) for the
time indicated. To assay the effect of the temperature-sensitive lepB9 mutation on secretion of ssTreF, strain IT41 (12) was grown at 28 °C and shifted to 42 °C for 30 min before exposure to
[35S]methionine. Immunoprecipitation and gel
electrophoresis were done as described by Ito et al. (13)
and Laemmli (14). To precipitate TreA, TreF, and DegP, polyclonal
antibodies against TreA, TreF, and DegP were used, respectively.
Rabbit polyclonal antiserum against TreF was obtained by immunization
with purified TreF. The antiserum against TreF cross-reacts only weakly
with TreA. Rabbit polyclonal antisera against TreA, MBP, DegP, or SecA
were kind gifts of W. Boos, J. Beckwith, and P. C. Tai. The cold
osmotic shock procedure was carried out according to Neu and Heppel
(15).
Selection of Tre+ Revertants of sstreF--
Strain
KU101 containing psstreF was grown in LB in the presence of
diaminopurine (600 µg/ml). psstreF was isolated and
retransformed into KU101. Tre+ revertants were selected on
minimal medium A containing 0.2% trehalose (11). After purification on
the selection medium, candidates were tested for elevated trehalase
activity on MacConkey agar plates containing 1% trehalose. Candidates
exhibiting red color on MacConkey plates were further tested by
trehalase assays. The mutations of the Tre+ revertants were
determined by nucleotide sequencing of the entire treF gene.
Selection of TreA/TreF and of Electron Microscopy--
For electron microscopy, strains were
grown overnight at 28 °C in LB medium. Cells were harvested by
centrifugation (4000 × g, 10 min, 4 °C) and fixed
for 1.5 h at room temperature with 2.5% glutaraldehyde in buffer
A (0.1 M sodium phosphate buffer, pH 7.1). The pellet was
washed three times with buffer A and embedded in 2% low melting
agarose. These samples were fixed in buffer A containing 2%
OsO4 (1 h, room temperature), washed five times with buffer
A, dehydrated in 50 and 70% acetone (10 min, room temperature),
contrasted with 2% uranylacetate in 70% acetone (1 h, room
temperature), and subsequently further dehydrated with 90 and 100%
acetone. Embedding was done according to Spurr (16). After
polymerization ultrathin sections were prepared and contrasted with 2%
aqueous uranylacetate and lead citrate (17). The specimen were examined
in a EM10 C2 (Zeiss, Oberkochen, Germany) under 80 kV at a primary
magnification of ×17,600.
The two trehalases of E. coli are highly homologous and
have similar enzymatic properties. The main apparent difference is their cellular localization. Therefore, these enzymes must contain signals allowing either efficient translocation and folding in the
periplasm or exclusion from export and proper folding in the cytoplasm.
To obtain information on how these isoenzymes have adapted to their
environment we expressed both in their nonnative cellular compartment,
periplasmic treA in the cytoplasm, and cytoplasmic treF in the periplasm.
Expression of treA and treF in Their Nonnative Cellular
Compartments--
To test whether periplasmic TreA can fold in the
cytoplasm, we constructed a signal sequenceless TreA derivative. This
plasmid was termed p
To test whether treF could be actively expressed in the
periplasm, we fused the signal sequence of TreA to the N terminus of
TreF. This plasmid was termed psstreF. TreF was efficiently exported to the periplasm. Pulse-chase experiments indicated that more
than 95% of the signal sequences of the TreF precursor population were
processed within 1 min (Fig.
1A). Only under nonpermissive conditions in the temperature-sensitive lepB9 strain IT41
(12) could the ssTreF precursor be detected (Fig. 1B). We
concluded that processing of the signal sequence occurred as a
consequence of translocation. Also, if a cytoplasmic protease would be
responsible for generating the mature form of ssTreF, TreF would fold
into the active conformation in the cytoplasm. Because we did not
detect trehalase activity in whole cell extracts (see below), this
explanation for efficient processing of ssTreF could be excluded.
sstreF was expressed at lower levels compared with
treA, i.e. 37 ± 6% of wild-type
treA (Fig. 1). Because treF was expressed under
treA promoter control and was fused to the TreA signal
sequence, we expected comparable levels of expression. Because the
periplasmic DegP protease is known to degrade a large variety of
misfolded proteins (19), we tested whether DegP is involved in
degradation of periplasmic TreF. We detected an increase in
treF expression in the degP null mutant strain
KU104. This effect of the degP mutation was stronger in
overnight than in log phase cultures (Fig. 1C). It should be
noted that 60 times more cells were used for Western blotting compared
with pulse-chase experiments. Other mutants of cell envelope proteases
such as ompT, ptr, hhoA, and hhoB had no pronounced effects on TreF expression.
Enzymatic Activity of Periplasmic TreF--
Periplasmic TreF had
only background enzymatic activity regardless of whether TreF was
expressed in degP+ or degP mutant
strains (Table I). This finding corresponded to the weak growth of
strain KU101 expressing sstreF on minimal trehalose agar
plates. When growth was dependent on the presence of periplasmic TreA,
single colonies were detected after overnight incubation at 28 °C.
When growth was dependent on periplasmic TreF, strains needed 3 days to
form single colonies.
Trehalase activity was determined in either whole cells or whole cell
extracts of treA::spec
In addition, periplasmic TreF was present in an insoluble form. We were
unable to extract TreF from cold osmotic shock fluids (Fig.
2A). Electron microscopy
suggested the presence of inclusion bodies in the periplasm (Fig.
2B). Similar results were obtained with MBP mutants (20).
From these data we conclude that TreF can be exported to the periplasm
where it is present in a misfolded and inactive form and is a substrate
for DegP protease.
Genetic Selection of Periplasmic TreF with Increased Trehalase
Activity--
To determine why TreF is misfolded in the periplasm, we
isolated and characterized Tre+ revertants of periplasmic
TreF. To obtain mutants of periplasmic TreF with increased enzymatic
activity, psstreF was mutagenized using 2-aminopurine.
Mutagenized plasmids were transformed into treA::spec
The expression of treF82 and treF172 was found to
be identical to periplasmic TreF. It is therefore likely that the
mutations did not change the overall structure of periplasmic TreF but
might have an effect only on folding of the catalytic site.
Characterization of a TreA'-TreF Hybrid--
We reasoned that a
hybrid protein composed of a significant fragment of TreA and TreF
might mediate contact to potential periplasmic chaperones or folding
catalysts and may thus influence folding of the TreF moiety. Fusing the
N-terminal 263 amino acids of the TreA precursor to TreF (Fig.
6) led to translocation of the hybrid protein but did not increase trehalase activity (Table
II) or solubility of the protein compared
with periplasmic TreF (Fig. 2).
To test whether the presence of the 233 amino acids of mature TreA
would interfere with folding of TreF we constructed a signal sequenceless derivative. It was expressed in the cytoplasm and exhibited high trehalase activity (Table II). Thus, the N-terminal 233 amino acids of TreA did not interfere with the folding of TreF in the
cytoplasm and did not stimulate folding of periplasmic TreF.
Genetic Selection for Functional TreA'-'TreF Hybrids--
To
identify regions responsible for misfolding of periplasmic TreF, we
wanted to test whether functional hybrids can be constructed composed
of N-terminal fragments of TreA and C-terminal fragments of TreF. The
genetic selection for active trehalase hybrids was identical to that
used for selection of Tre+ revertants of sstreF.
It was carried out in strain KU92 (treA::spec
When plating cultures of KU92 containing ptreA'-'treF
spontaneous Tre+ revertants were isolated at high
frequency. Plasmids of 12 candidates were retransformed into strain
KU92 and checked for trehalase activity. Three candidates were chosen
for further analysis. Nucleotide sequencing indicated that
treA-F1 encodes a hybrid protein precursor of 535 amino
acids composed of the N-terminal 335 residues of TreA precursor and the
C-terminal 200 residues of TreF. treA-F8 encodes a hybrid
protein precursor of 534 amino acids composed of the N-terminal 239 residues of TreA precursor and the C-terminal 295 residues of TreF.
treA-F10 encodes a hybrid protein precursor of 535 amino
acids composed of the N-terminal 342 residues of TreA precursor and the
C-terminal 193 residues of TreF (Figs. 4 and 6). Processing of each
precursor protein removes the N-terminal signal peptide of 30 residues.
Because the fusion joints of treA-F1 and treA-F10
were nearly identical, we did not further investigate treA-F10.
Enzymatic Activity of Periplasmic TreA'-'TreF Hybrids--
The
hybrid proteins differed in their enzymatic activity. Whereas TreA-F1
exhibited high trehalase activity, TreA-F8 had low trehalase activity.
Like all other constructs described above, the hybrids were substrates
of DegP protease. In degP null mutant derivatives of strain
KU92, protein expression (data not shown) and enzymatic activity
increased (Table II). Also, we detected a difference in solubility.
Whereas about 50% of TreA-F1 could be released by cold osmotic shock,
only about 10% of TreA-F8 was released, indicating that a larger
population of TreA-F8 was misfolded (Fig.
7). Because TreA-F8 had similar
properties to periplasmic TreF but had only about 50% of TreF
sequence, we suspect that the C-terminal half of TreF contains a major
determinant for misfolding of periplasmic TreF. Because TreA-F1
exhibited enzymatic properties similar to TreA (see below) even though
it contains the 200 C-terminal residues of TreF, the region causing
folding problems could be further restricted to residues 255-350 of
TreF.
Because wild-type TreA and TreF differ in their Km
values for trehalose, i.e. 0.3 mM for TreA and
1.5 mM for TreF, we asked whether the trehalase hybrid
TreA-F1 would resemble more closely TreA or TreF. The
Km value for trehalose was determined as 0.3 mM, which was identical to that of periplasmic TreA. Thus,
the high affinity binding site should be located in the TreA part of
TreA-F1. A possible candidate for the high affinity binding site is the
N-terminal trehalase signature (Figs. 4 and 5).
Genetic Selection and Characterization of Functional
We used genetic approaches to study the intramolecular signals and
the effects of the cellular compartments on translocation and folding
of the two trehalases of E. coli. Periplasmic trehalase TreA
folded properly in the periplasm and in the cytoplasm. Also, TreA-TreF
hybrid proteins containing 334 amino acids of TreA and 200 residues of
TreF had enzymatic properties comparable to those of wild-type TreA, no
matter whether these constructs were expressed in the cytoplasm or in
the periplasm. The experimental data obtained from these hybrid
proteins indicated that the main determinant of substrate affinity must
be localized in the N-terminal 334 residues of TreA. Trehalases have
two conserved signatures typical for glycosyl hydrolases. One is
localized at the N terminus and the other at the C terminus (Fig. 4).
Thus, we speculate that the N-terminal trehalase signature is
responsible for the lower Km values of TreA.
In contrast to TreA, cytoplasmic TreF could not fold in its nonnative
cellular compartment. Periplasmic TreF was misfolded and enzymatically
inactive. One TreF mutation, T172I, located in the N-terminal trehalase
signature, led to 6-fold higher periplasmic trehalase activity. This
finding supported the model that the N-terminal Tre box is important
for enzymatic activity. However, this mutation did not abolish the
problems of solubility and protease sensitivity of periplasmic TreF.
It is difficult to secrete native cytoplasmic proteins of E. coli, which is consistent with the idea that cytoplasmic proteins fold too rapidly to be substrates of the secretion apparatus. Therefore, successful translocation of native cytoplasmic E. coli proteins has been reported only for a few cases. Thioredoxin
1 can be translocated to the periplasm when fused to the signal sequence of alkaline phosphatase or of DsbA (21, 22), and A model predicting that the cytoplasm and the periplasm have different
properties influencing the folding of polypeptides may explain why TreF
did not fold properly in the periplasm. Periplasmic E. coli
amylase MalS is another example because signal sequenceless MalS cannot
fold into the active conformation in the cytoplasm (25). The cellular
factors responsible for these effects are unknown. Possible candidates
are different sets of molecular chaperones, for example
ATP-dependent chaperones are present in the cytoplasm but
not in the periplasm. Also, because TreF contains three Cys residues,
which tend to form intermolecular disulfide bonds in purified
cytoplasmic TreF, these Cys residues could be responsible for
misfolding of periplasmic TreF. When testing whether the inability of
periplasmic TreF to fold was dependent on DsbA, a catalyst for
disulfide bond formation, no increase in TreF activity or solubility
was observed in dsbA knock out
strains.1 Also, there are no
Cys residues in the TreF part of the hybrid TreA-F8, which was as
insoluble as full-length ssTreF. However, one region comprising
residues 255-350 of TreF was identified as sufficient to cause folding
problems in the periplasm. These 95 residues, of which only 24 are
nonconserved with respect to TreA, are located between the fusion
joints of treA-F1 and treA-F8. We are expecting
that further work on TreF will allow us to study the determinants of
solubility, folding of the active site, and protease sensitivity by
using genetic methods.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(ara-leu) 7697 lacX74 phoA (Pvull) phoR
malF3 galE galK thi rpsL) (8) or of MC4100 which is
F
lacU169 araD136 rbsR relA rpsL
thi (9). KU92 is DHB3,
treA::SpecR treRBC
ara714 leu::Tn10
TetR; KU93 is KU92 prlA4..KanR; KU95
is MC4100 treA::SpecR
treC-lacZ 
placMu55 KanR
ara71 leu::Tn10
TetR; KU101 is KU92 ara714
leu+; KU104 is KU101
degP::TetR; KU105 is KU104
prlA4..KanR. Plasmids used were pBAD22, a
pBR322-derived plasmid that has the arabinose promoter followed by a
linker containing multiple restriction sites (10); pBADtreF
has the coding region of wild-type treF in pBAD22 (5);
ptre11 has wild-type treA under its own promoter
in pBR322 (2).
sstreA
was generated by cleaving ptre11 with BsgI
followed by T4 DNA polymerase treatment and PstI digestion.
The 2.7-kilobase DNA fragment containing
sstreA was ligated into pBAD22, which was
cleaved with Asp178I, followed by treatment with Klenow
enzyme and PstI digestion. Because of these manipulations,
treA was expressed without its signal sequence and the first
five amino acids of mature TreA (Glu, Glu, Thr, Pro, and Val) were
replaced by Met, Val, and Leu.
sstreA'-'treF was constructed by subcloning
the 3.05-kilobase EagI-ScaI fragment from
pBADtreF into psstreA which was
cleaved with EagI and ScaI.
sstreA'-treF was constructed by
subcloning of a 2.41-kilobase KpnI-PstI fragment
into psstreA cleaved with KpnI and
PstI.
ssTreA/TreF Hybrids--
Strain
KU92 containing ptreA'-'treF was grown overnight in rich
medium. A 25-ml culture was washed twice in minimal medium, and
aliquots corresponding to 2.5 ml of cell culture were plated onto one
minimal medium agar plate containing 0.2% trehalose. After incubation
overnight, Tre+ candidates were purified twice on the
selection medium. To show that complementation of the tre
defect was linked to ptreA'-'treF, plasmid DNA of such
candidates was retransformed into strain KU92. In all cases growth on
minimal trehalose media was detected. This indicated that
plasmid-derived mutations were sufficient to generate growth on
selection plates. These cells were used for further characterization.
The fusion joints were determined by nucleotide sequencing. Selection
of ssTreA/TreF hybrids was carried out as described above for
selection of TreA/TreF hybrids except that strains KU93 and KU95
containing psstreA'-'treF were used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sstreA.
sstreA was expressed in
treA strain KU101. Signal sequenceless
treA was actively expressed in the cytoplasm (Table
I). The kinetic parameters of cytoplasmic
TreA were determined in whole cell extracts of strain KU101. Signal
sequenceless TreA had a Km of 0.16 mM,
whereas periplasmic TreA had a Km of 0.31 mM trehalose. Cytoplasmic wild-type TreF had a
Km of 1.5 mM. In addition, signal
sequenceless TreA remained export competent, because it could be
exported in prlA4 background (data not shown).
prlA mutants are derivatives of secY allowing the export of signal sequenceless secretory proteins (18).
Enzymatic activity of trehalases
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Fig. 1.
Translocation of ssTreF. A,
cells of strain KU101 expressing either wild-type treA or
sstreF from plasmids ptre11 or
psstreF, respectively, were labeled with
[35S]methionine for 60 s (0) or 60 s followed
by chase periods of 0.5, 1, or 5 min with excess cold methionine. Cells
were lysed and immunoprecipitated with antibodies to TreA or TreF. The
positions of TreA, of mature TreF, and of DnaK are indicated by
arrows. B, cells of strain IT49
(lepB9) expressing sstreF from psstreF
were grown at 28 °C and shifted to 42 °C for 30 min before
exposure to [35S]methionine. Cells were labeled for
60 s (0) or 60 s followed by chase periods of 1, 5, or 10 min
with excess cold methionine. Cells were lysed and immunoprecipitated
with antibodies to TreF and DegP. The positions of DnaK, which was
recognized by the antiserum against TreF, and of precursor and mature
forms of TreF and DegP are indicated. C, Western blot of
strains KU101 (degP+) and KU104
(degP::Tn10) expressing
sstreF after overnight growth in LB. These cultures were
concentrated 10-fold. To detect TreF, polyclonal antibodies against
TreF were used. The positions of TreF and DnaK are indicated.
treBC
strains, lacking periplasmic trehalase TreA and the trehalose
transporter TreB. Trehalase activity of whole cells represented only
periplasmic trehalase activity because in treB
mutants trehalose is not transported across the cytoplasmic membrane.
When trehalase activity was determined in whole cell extracts,
periplasmic and cytoplasmic trehalase contributed to enzymatic
activity. There was no difference when comparing trehalase activity of
whole cells and cell extracts of cells expressing sstreF
(Table I). This indicated that no residual TreF activity was present in
the cytoplasm, which could be the result of inefficient export.
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Fig. 2.
ssTreF and TreA'-TreF are not released by
cold osmotic shock. A, Western blot of periplasmic and
pellet fractions of strain KU104
(degP::Tn10) expressing
sstreF and treA'-treF after overnight growth in
LB. To detect TreF, TreA'-TreF, SecA, or MBP polyclonal antibodies
against TreF, SecA, or MBP were used. SecA and MBP were used as
controls for cytoplasmic and periplasmic proteins, respectively. The
positions of TreF, TreA'-TreF, SecA, and MBP are indicated.
S is cold osmotic shock fluid, and P is the
pellet fraction of osmotically shocked cells. B, electron
microscopy of degP+ and
degP strains expressing sstreF. The images
show representative details of the cell envelope of
degP+ strain KU101 (a and
b) and degP strain KU104
(c and d). Ultrathin sections were contrasted
with uranylacetate and lead citrate. Aggregated forms of improperly
folded ssTreF were detected in the periplasm of the degP
mutant (arrowheads). Magnification was equal, and the bar
represents 100 nm.
treBC strain KU101 and plated on minimal trehalose agar plates (Fig. 3). Colonies showing
improved growth were screened for increased trehalase activity on
MacConkey trehalose agar plates. Subsequently, trehalase activity of 25 candidates was assayed in whole cells. Two candidates showed more than
a 5-fold increase in trehalase activity. Retransformation verified that
the isolated mutations were linked to psstreF. Both mutants,
termed treF82 and treF172, exhibited about a
6-fold increase in trehalase activity (Table I). Nucleotide sequencing
indicated that they were independent mutants, because treF82
had a A82T and treF172 had a T172I exchange (Fig.
4). Interestingly, the T172I exchange
detected in treF172 is located in the N-terminal trehalase
signature. An amino acid sequence alignment shows that most trehalases
have hydrophobic residues at position 172, whereas wild-type TreF has a
Tyr residue (Fig. 5). This may explain
why the T172I exchange leads to elevated trehalase activity.
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Fig. 3.
Genetic selection for elevated periplasmic
trehalase activity. Trehalose enters the cell via the outer
membrane protein LamB (26). In the absence of periplasmic trehalase
TreA and the trehalose transporter TreB, cells depend on the presence
of periplasmic TreF, which needs to be enzymatically active to allow
growth on minimal trehalose plates.
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Fig. 4.
Amino acid sequence alignment of mature TreA
and TreF. sstreF82 and sstreF172 show the amino acid exchange of
sstreF82 and sstreF172, respectively. Numbers
indicate the fusion joints of TreA'-'TreF hybrids. The straight line
indicates the last residue of TreA that is present in the hybrid
protein. sig. 1 and sig. 2 indicate the N- and
C-terminal trehalase signatures. Asterisks indicate
identical residues, and dots indicate conserved
residues.
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Fig. 5.
Consensus sequence of the N-terminal
trehalase signature. Aminoacyl residues 166-180 of TreF and of
ssTreF172 are compared with the relevant sequences of other trehalases.
The sequences were extracted from the latest release of SwissProt data
base. Accession numbers are given in parenthesis. TreA and TreB of
S. ce are from Saccharomyces cerevisiae (P32356
and P35172), C. al is Candida albicans (P52494),
K. la is Kluyveromyces lactis (P49381), N. cr is Neurospora crassa (O42783), B. mo is
Bombyx mori (P32358), T. mo is Tenebrio
molitor (P32359), O. cu is Oryctolagus
cuniculus (P19813), and H. sa is Homo
sapiens (O43280).
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Fig. 6.
Schematic presentation of various
trehalases. 
, indicates TreF or TreF fragments, 
indicates
TreA or TreA fragments, and arrows represent the TreA signal
sequence. Box B1 and box 2 indicate the
localization of the two trehalase signatures (see Fig. 4).
Enzymatic activity of trehalase hybrids
treBC), which requires periplasmic trehalase
activity for growth on minimal trehalose medium (Fig. 3). To select for
functional TreA-TreF hybrids, plasmids were constructed containing the
5'-end of treA until codon 373 fused to the 3'-end of
treF starting at codon 166. The region of overlapping
homology was about 750 base pairs. In this construct, termed
treA'-'treF, the reading frames of treA and
treF were nonidentical. This construct did not confer trehalase activity, and no growth on minimal trehalose medium was
observed after expression in strain KU92.
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Fig. 7.
TreA'-'F1 is released by cold osmotic shock,
whereas TreA'-'F8 is not. Western blot of periplasmic and pellet
fractions of strain KU104 (degP::Tn10)
expressing treA'-'treF1 and treA'-'treF8 after
overnight growth in LB. To detect TreA'-'TreF, SecA, or MBP, polyclonal
antibodies against TreA, SecA, or MBP were used. SecA and MBP were used
as controls for cytoplasmic and periplasmic proteins, respectively. The
presence of MBP in the pellet fraction results from intact cells. The
position of TreA'-'TreF and its derivatives, SecA and MBP, are
indicated. S is cold osmotic shock fluid, and P
is the pellet of osmotically shocked cells.
ssTreA'-'TreF Hybrids--
We repeated the selection described
above using signal sequenceless versions of ptreA'-'treF.
These were expressed in prlA4 strain KU93 allowing export of
signal sequenceless secretory proteins. As above, we selected for
periplasmic trehalase activity. Tre+ revertants were
obtained at a 1000-fold lower frequency compared with the original
selection using ptreA'-'treF encoding the TreA signal
sequence. Fortuitously, one isolate had exactly the same fusion joint
as TreA-F1 and was thus termed ssTreA-F1. The fusion joint was such
that a new trehalase gene was generated, lacking the extended N
terminus of TreF and the extended C terminus of TreA. This trehalase
was composed of 503 residues, whereas wild-type TreA has 535 residues
and wild-type TreF has 549 residues. ssTreA-F1 contained three
additional residues (Met, Val, and Leu) at its N terminus, which are a
consequence of subcloning, 300 residues of TreA (37-334), and the 200 C-terminal residues of TreF (350-549). The enzymatic activity of
ssTreA-F1 was about 12-fold higher as detected for the signal
sequence-containing construct TreA-F1 and was mostly localized in the
cytoplasm (Table II). After expression in prlA degP mutant
strain KU105, the activity of ssTreA-F1 was 0.89 ± 0.02 units,
which was only about 40% higher than detected in
prlA+ degP+ strain KU101
(Table II) indicating that ssTreA-F1 was only a poor substrate of
the prlA secretion apparatus. Therefore, we conclude that
the C-terminal 200 residues of TreF were sufficient to block export by
the PrlA secretion apparatus. The Km of ssTreA-F1
was determined in whole cell extracts as 0.16 mM for
trehalose, which was identical to that of ssTreA. This result and
the finding that the activity of ssTreA-F1 was the same as that of
ssTreA (Tables I and II) indicated that the exclusion from export
was not the consequence of improper folding of ssTreA-F1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase was exported as tripartite protein, LamB-LacZ-PhoA (23), or when fused to the signal sequence of OmpA (24). Compared with
thioredoxin and -galactosidase, secretion of TreF was more efficient, which may be because of the exceptionally long and hydrophobic signal sequence of TreA. We are currently investigating whether a more hydrophobic signal sequence is leading to a more efficient export of native cytoplasmic proteins.
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ACKNOWLEDGEMENTS |
|---|
We thank Eberhard Spiess for help with the EM work done in his lab. We thank Jon Beckwith, P. C. Tai, and Winfried Boos for antibodies; Ross Dalbey for bacterial strains; and Ann Flower for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to M. E.) and by fellowships from the Studienstiftung des deutschen Volkes (to M. M. and C. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Cell Biology, Harvard Medical School, Boston, MA 02115.
¶ To whom correspondence should be addressed: School of Biosciences, Cardiff University, Museum Ave., P. O. Box 911, Cardiff CF10 3US, United Kingdom. Tel./Fax: 44-29-2087-4648; E-mail: ehrmann@cf.ac.uk.
Published, JBC Papers in Press, May 17, 2000, DOI 10.1074/jbc.M002793200
1 K. Uhland, unpublished results.
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REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Boos, W., Ehmann, U., Bremer, E., Middendorf, A., and Postma, P. (1987) J. Biol. Chem. 262, 13212-13218 |
| 2. | Gutierrez, J. A., Ardourel, M., Bremer, E., Middendorf, A., Boos, W., and Ehmann, U. (1989) Mol. Gen. Genet. 217, 347-354 |
| 3. | Tourino-dos-Santos, C., Bachinski, N., Paschoalin, V., Paiva, C., Silva, J., and Panek, A. (1994) Braz. J. Med. Biol. Res. 27, 627-636 |
| 4. | Hengge-Aronis, R., Klein, W., Lange, R., Rimmele, M., and Boos, W. (1991) J. Bacteriol. 173, 7918-7924 |
| 5. | Horlacher, R., Uhland, K., Klein, W., Ehrmann, M., and Boos, W. (1996) J. Bacteriol. 178, 6250-6257 |
| 6. | Klein, W., Horlacher, R., and Boos, W. (1995) J. Bacteriol. 177, 4043-4052 |
| 7. | Rimmele, M., and Boos, W. (1994) J. Bacteriol. 176, 5654-5664 |
| 8. | Boyd, D., Manoil, C., and Beckwith, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8525-8529 |
| 9. | Casadaban, M. J. (1976) J. Mol. Biol. 104, 541-555 |
| 10. | Guzman, L.-M., Belin, D., Carson, M., and Beckwith, J. (1995) J. Bacteriol. 177, 4121-4130 |
| 11. | Miller, J. (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
| 12. | Inada, T., Court, D., Ito, K., and Nakamura, Y. (1989) J. Bacteriol. 171, 585-587 |
| 13. | Ito, K., Bassford, P., Jr., and Beckwith, J. (1981) Cell 24, 707-717 |
| 14. | Laemmli, U. K. (1970) Nature 227, 680-685 |
| 15. | Neu, H., and Heppel, L. (1965) J. Biol. Chem. 240, 3685-3692 |
| 16. | Spurr, A. R. (1969) J. Ultrastruct. Res. 26, 31-43 |
| 17. | Reynolds. (1963) J. Cell Biol. 17, 208-212 |
| 18. | Prinz, W. A., Spiess, C., Ehrmann, M., Schierle, C., and Beckwith, J. (1996) EMBO J. 15, 5209-5217 |
| 19. | Spiess, C., Beil, A., and Ehrmann, M. (1999) Cell 97, 339-347 |
| 20. | Betton, J.-M., and Hofnung, M. (1996) J. Biol. Chem. 271, 8046-8052 |
| 21. | Debarbieux, L., and Beckwith, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10751-10756 |
| 22. | Jonda, S., Huber-Wunderlich, M., Glockshuber, R., and Mossner, E. (1999) EMBO J. 18, 3271-3281 |
| 23. | Snyder, W., and Silhavy, T. (1995) J. Bacteriol. 177, 953-963 |
| 24. | Freudl, R., Schwarz, H., Kramps, S., Hindennach, I., and Henning, U. (1988) J. Biol. Chem. 263, 17084-17091 |
| 25. | Spiess, C., Happersberger, H. P., Glocker, M. O., Spiess, E., Rippe, K., and Ehrmann, M. (1997) J. Biol. Chem. 272, 22125-22133 |
| 26. | Klein, W., and Boos, W. (1993) J. Bacteriol. 175, 1682-1686 |
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