Full text of DOI:10.1016/S0923-2508(02)01338-4

Research in Microbiology 153 (2002) 399–404
www.elsevier.com/locate/resmic
Folding and aggregation of export-defective mutants
of the maltose-binding protein
∗
1
Jean-Michel Betton , Denis Phichith, Sabine Hunke
Unité de Repliement et de Modélisation des Protéines, Institut Pasteur, CNRS-URA 2185, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France
Received 15 April 2002; accepted 11 June 2002
Abstract
We previously characterized a defective-folding variant of the periplasmic maltose-binding protein, MalE31. To examine the alternative
folding pathways open to the MalE31 precursor, we have analyzed the cellular fates of this aggregation-prone protein carrying altered signal
sequences. Our results are most easily interpreted by a kinetic competition between exportation, folding, and degradation.  2002 Éditions
scientifiques et médicales Elsevier SAS. All rights reserved.
Keywords: Heat-shock proteins; Maltose; Protein folding; Protein precursors
1
. Introduction
To study the competitive processes between export, fold-
ing and aggregation, we use a defective folding mutant of
the maltose-binding protein MalE31, the periplasmic re-
ceptor for the high affinity transport of maltose. This vari-
ant carries a double substitution G32D, I33P in a turn of
the N-domain [4]. In vitro, unfolding–refolding experiments
showed that a folding intermediate of MalE31 is kinetically
trapped in an off-pathway folding reaction leading to its ag-
gregation [21]. In vivo, the MalE31 precursor was correctly
exported based on the kinetics of signal sequence process-
ing, but the defective folding of the mature protein in the
periplasm led, at high levels of production, to the formation
of inclusion bodies [4]. At low levels of production, the mis-
folded MalE31 polypeptide chain is completely degraded
by DegP, a heat-shock periplasmic protease [14,24]. A ki-
netic competition between folding, aggregation and degra-
dation was proposed to explain the different fates of newly
translocated proteins in the bacterial periplasm [6]. One ma-
jor physiological consequence for the cells overproducing
Exported proteins with an ultimate destination of the
periplasm or outer membrane in Escherichia coli are synthe-
sized as precursor proteins with an amino-terminal signal se-
quence. Interaction with cellular chaperones maintains these
precursor proteins in a translocation-competent conforma-
tion until they are transported through the inner membrane
by the translocation machinery [9]. As the precursor pro-
teins enter the periplasm, the signal sequence is removed by
signal peptidase, an integral membrane protein with the ac-
tive site facing the periplasm. After translocation and signal
sequence cleavage, the newly exported mature proteins are
subsequently sorted, folded and assembled in the periplasm
and outer membrane [20]. In contrast to the folding and tar-
geting of nascent precursors emerging from the ribosome,
little is known about these processes after release from the
translocation machinery in the periplasm. Recently, studies
of outer membrane protein biogenesis have permitted char-
E
E
acterization of two regulatory pathways, the σ regulatory
MalE31 was an increase in the σ -dependent response but
3
2
system and the CpxRA two-component signal transduction
pathway, which are both activated by the presence of mis-
folded proteins in the periplasm [22].
not that of the general heat shock sigma factor, σ [3].
These results indicated that E. coli senses the presence of
misfolded MalE31 in the periplasm and responds to it in a
manner that is distinct from the heat shock response induced
by protein misfolding in the cytoplasm.
*
Correspondence and reprints.
In this study, we have examined the expression of
MalE31 carrying altered signal sequences to determine the
consequences of a decreased export efficiency on both
E-mail address: jmbetton@pasteur.fr (J.-M. Betton).
Present address: Institut für Biologie, Bakterienphysiologie, Humboldt
1
Universität zu Berlin, Chausseestr. 117, 10115 Berlin, Germany.
0923-2508/02/$ – see front matter  2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
PII: S0923-2508(02)01338-4

400
J.-M. Betton / Research in Microbiology 153 (2002) 399–404
MalE31 folding and on its ability to induce a cellular stress
response.
gels followed by Coomassie blue staining. For quantitative
analyses, gels were scanned with an ImageMaster VDS cam-
era (Amersham Biotech). MalE proteins were visualized by
immunodetection using an antibody raised against MalE as
a primary antibody and an alkaline phosphatase-conjugated
secondary antibody, as described previously [21].
2
. Materials and methods
2
.1. Bacterial strains and plasmids
2
.4. β-galactosidase assays
−
E. coli K12 strain pop6499 (F araD139 ꢀlacU169
rpsL150 relA1 flbB5301 ꢀ(srlR-recA)306::Tn10 deoC1
ptsF25 rbsR ꢀmalE444 malT ) was used as a host strain
for plasmids encoding the various MalE proteins [4]. This
E. coli SR1364 or SR1458 strains, carrying the various
c
plasmids, were grown in LB medium supplemented with
◦
0.2% maltose and 0.1 mg/ml ampicillin at 30 C. After 1.5 h
strain carries a nonpolar deletion of the chromosomal malE
gene [10] and harbors the malT allele which confers con-
of induction, cells were harvested and β-galactosidase ac-
tivity in Miller units was determined as described previ-
ously [16]. A minimum of four independent assays were av-
eraged to obtain the indicated activities.
c
stitutive expression on the maltose regulons. Expression of
Plon::lacZ and PdegP::lacZ fusions was monitored in strains
SR1364 and SR1458, respectively [3]. Plasmids p1H and
p31H carrying the wild-type malE and malE31 genes, both
under the control of their natural promoters, were previously
described [3]. Plasmid pꢀ709, which carries a deletion of
the ribosome binding site and the first 256 codons of the
malE gene [5], was used as a negative control.
3. Results
Our previous studies indicated that the MalE31 precursor
was rapidly and efficiently exported to the periplasm. This
conclusion was based on the findings that the kinetics of
MalE31 maturation was similar to that of the wild type,
and that overexpression of MalE31 could induce a stress
response mediated not by the classical heat-shock sigma
2
.2. Site-directed mutagenesis
Signal peptide mutations were constructed by oligonu-
3
2
cleotide mutagenesis with single-stranded p1H and p31H
DNA containing uracil as the template for primer ex-
tension and ligation [13]. The mutagenic oligonucleotides
used for the deletion of the signal peptide (ꢀSS) and
for creating the M-18R (M-18) substitution were, respec-
factor, σ , but by the alternative heat-shock sigma factor
E
σ , specific to extracytoplasmic stress [3,17]. However, if an
alteration in the signal sequence of the precursor decreases
the efficiency of export, a significant proportion of the
precursor could accumulate inside the cytoplasm.
ꢀ
ꢀ
tively: 5 -TACCTTCTTCGATTTTCATAATCTATGG-3 ,
5
ꢀ
ꢀ
-GGAAAACCTCATCGTC-3 . Both mutations were con-
3.1. Export, folding and aggregation of MalE variants
firmed by dideoxynucleotidesequencing, and subcloned into
parental nonmutagenized p1H and p31H plasmids.
In order to examine the kinetic partition between export,
folding and aggregation, MalE and MalE31 carrying either
a complete deletion of their signal sequence (MalEꢀSS
and MalE31ꢀSS) or a single mutation at position −18 in
the signal sequence (MalEM-18 and MalE31M-18) were
constructed. The latter corresponds to the original malE18-1
mutation for which an arginine is substituted for methionine
at residue −18 in the signal sequence [2]. Manson et al. [15]
showed that 4% of the normal periplasmic amount of mature
MalE could be found in the strain carrying the original signal
sequence mutation malE18-1.
The steady-state production level of each variant was
first analyzed by SDS-polyacrylamide gel electrophoresis
to determine whether the signal sequence mutations could
affect the amount of MalE found in whole cell extracts
(Fig. 1A). All variants except one (MalE31 expressed from
p31M-18) were detected at a yield comparable to that of
the wild-type MalE expressed either from p1H or from
p1ꢀSS (Table 1). In the case of MalE31 expressed with the
export-defective signal sequence, the protein was produced
at a low level. Furthermore, its electrophoretic mobility was
identical to that of the elongation factor protein EF-Tu, the
2
.3. Subcellular fractionation of MalE
Cultures were grown in LB medium supplemented with
◦
0
.1 mg/ml ampicillin at 30 C. Cells were harvested at
A600 = 1.5, and fractionated by spheroplast preparation as
previously described [3]. Cell pellets, normalized to the
same A600, were resuspended in 10 mM Tris-HCl buffer
(
(
pH 7.5) containing 0.5 M sucrose. After adding lysozyme
0.2 mg/ml) and EDTA (10 mM), the suspensions were in-
◦
cubated for 15 min at 4 C. Then, the samples were cen-
trifuged for 5 min at 14 000 rpm, and supernatants contain-
ing the periplasmic fractions were withdrawn. The spher-
oplast pellets were washed, freeze-thawed, sonicated and
centrifuged at 14 000 rpm for 15 min. Supernatants were
collected to obtain the cytoplasmic fractions. Pellets were
washed with Tris-HCl buffer containing 0.1% (v/v) Triton
and resuspended in Tris-HCl buffer to obtain the mem-
brane fractions. Whole cell extracts and the three subcel-
lular fractions were each mixed with SDS-PAGE sample
buffer, boiled, and analyzed on 12.5% SDS-polyacrylamide

J.-M. Betton / Research in Microbiology 153 (2002) 399–404
401
Fig. 1. Expression of MalE variants. A. Whole cell extracts were separated by SDS-polyacrylamide gel electrophoresis (12.5% acrylamide), corresponding to
8
1
0 bacteria. The gel was stained with Coomassie blue. Lanes: (1) pꢀ709,(2) p1H, (3) p1ꢀSS, (4) p1M-18, (5) p31H, (6) p31ꢀSS, (7) p31M-18. The band
corresponding to MalE is indicated by an arrow, and those corresponding to GroEL and DnaK by asterisks. B. Immunoblot analysis of whole cell extracts
from 10-fold diluted samples was performed using a specific anti-MalE antibody.
Table 1
about 60 and 70 kDa, in the lanes corresponding to p31ꢀSS
and p31M-18R. These bands represent the major heat-shock
proteins GroEL and DnaK (see below).
Relative expression levels of MalE variants
Plasmid Protein
Signal
sequence
SDS-PAGE
scan (%)
Immunoblot
scan (%)
The formation of inclusion bodies, which is a conse-
quence of a folding defect, can be biochemically determined
by soluble/insoluble fractionation based on separation by
centrifugation of native lysates [12]. Therefore, we deter-
mined the cellular location of the different MalE variants
after spheroplast preparation (Fig. 2). The periplasmic and
cytoplasmic location (both fractions define a soluble frac-
tion) of wild-type MalE, expressed from p1H and p1ꢀSS,
confirmed that our subcellular fractionation procedure was
correct. MalE31 expressed with a wild-type signal sequence
p1H
p1ꢀSS
p1M-18 MalE
p31H
p31ꢀSS MalE31
p31M-18 MalE31
MalE
MalE
wild-type
no
M-18R
wild-type
no
80
100
75
85
80
85
100
70
90
70
MalE31
a
M-18R
20
25
a
This value was obtained by subtracting the area of EF-Tu band
determined for the corresponding lane in the gel shown in Fig. 1A from
the mean value of EF-Tu bands determined from the other lanes.
predominant protein of E. coli. Thus, its quantification was
rendered less accurate. Nevertheless, the apparent molecular
weight of MalE or MalE31 expressed from p1M-18 or
p31M-18 indicates that this export-defectivesignal sequence
of both proteins is not processed, in good agreement with
previous kinetics studies of export [1]. Densitometry of an
immunoblot of duplicate gel, loaded with 10-fold diluted
samples, with a specific anti-MalE antibody gave similar
results to those from the Coomassie blue stained gel (Fig. 1B
and Table 1). Close inspection of the gel shown in Fig. 1A
revealed the presence of increased amounts of two bands of
(
from p31H) is found entirely in the membrane or insolu-
ble fraction as expected for aggregated proteins [12]. We
previously demonstrated by flotation gradient centrifugation
that aggregated MalE31 proteins form periplasmic inclusion
bodies which co-sediment with membrane vesicles rather
than membrane-associated proteins [3]. In contrast, MalE31
expressed either without a signal sequence (from p31ꢀSS)
or with an altered signal sequence (from p31M-18) displays
different soluble/insoluble partitions.

402
J.-M. Betton / Research in Microbiology 153 (2002) 399–404
3
.2. Stress responses induced by expression of MalE
variants
From Fig. 1 it is evident that cytoplasmic expression of
MalE31 from p31ꢀSS or p31M-18R increased the synthe-
sis of GroEL and DnaK. In contrast, no such elevated level
of heat-shock proteins was visible with periplasmic expres-
sion of MalE31. We quantitatively determined the extent of
induction of stress response caused by these variants, us-
ing two bacterial strains carrying either a single copy of
Plon::lacZ (SR1364) or PdegP::lacZ (SR1458) transcrip-
tional gene fusions. The lon gene encoding a heat-shock cy-
toplasmic protease is transcribed by the Eσ32 RNA poly-
merase, whereas degP encoding a heat-shock periplasmic
E
protease is transcribed by the Eσ RNA polymerase [11].
This gene differs from the former in that its transcription
E
is not only regulated by σ but also by the CpxRA path-
E
way. The sigma factor σ , encoded by the rpoE gene, is
a member of the extracytoplasmic function (ECF) subfam-
ily sigma factors, which function as effectors molecules re-
sponding to extracytoplasmic stimuli [18]. The cytoplas-
mic accumulation of MalE31 expressed from p31ꢀSS or
p31M-18R is reflected by a more than twofold increase in
β-galactosidase synthesis directed from the Plon promotor
(
Fig. 3). Surprisingly, wild-type MalE carrying the export-
defective signal sequence significantly induced the lon pro-
motor. The periplasmic aggregation of MalE31 from p31H
increased by a factor of two the β-galactosidase activity
measured from the PdegP::lacZ fusion, confirming our pre-
vious data [3]. It is interesting to note that the expression
of MalE31 from p31ꢀSS or p31M-18R caused a decreased
basal level of activity of the degP promotor (Fig. 3). This
observation suggests that a direct competition between both
heat-shock sigma factors may play a role in directing RNA
Fig. 2. Subcellular fractionation of MalE variants. Periplasmic, cytoplas-
mic, and membrane fractions were separated by SDS-polyacrylamide gel
electrophoresis, and Coomassie stained bands were scanned by densitom-
etry. Lanes: (1) pꢀ709,(2) p1H, (3) p1ꢀSS, (4) p1M-18, (5) p31H, (6)
p31ꢀSS, (7) p31M-18. An arrow indicates the band corresponding to MalE.
The proportions were calculated by summing the area of the MalE band in
the three subcellular fractions and are shown under the corresponding lanes.
Fig. 3. Stress responses induced by MalE variants. The activities of lon and
degP promoters, both fused to the lacZ gene, were assessed in E. coli strains
SR1364 and SR1458, respectively, each carrying the various plasmids
described in Fig. 1. Miller activities of the lacZ encoded β-galactosidase
are calculated using the average of four independent assays.

J.-M. Betton / Research in Microbiology 153 (2002) 399–404
403
3
2
E
polymerase predominantly either to σ -dependent or σ -
dependent promoters.
presence of a mutated signal sequence. In accord with this
idea, it has been proposed that signal sequences maintain
the export-competence conformation by interfering directly
with the folding of precursors [19]. Structurally, a nascent
precursor in an export-competent conformation may be sim-
ilar to a partially folded protein prone to aggregate. In-
deed, Wild et al. [25] showed that accumulation of pro-
tein precursors in strains lacking the SecB chaperone, the
primary chaperone involved in export of many periplasmic
and outer membrane proteins [9], generated a signal for in-
4
. Discussion
It was previously proposed that there is a kinetic partition-
ing of MalE precursor between the productive export and the
folding pathway [23]. After the precursor has folded, it can
no longer enter the export pathway. Since we never observed
misfolding of MalE31 when it was expressed with its wild-
type signal sequence in the cytoplasm, we concluded that the
MalE31 precursor enters efficiently and rapidly into the ex-
port pathway. Because MalE31 refolds slowly in vitro [21],
the export pathway could have a kinetic advantage over the
aggregation pathway. The mutational deletion or substitution
in signal sequences of the MalE31 precursors studied here
has been shown to strongly decrease the efficiency of MalE
export [2]. The presence of these altered signal sequences
in combination with the defective-folding malE31 mutation
results in the accumulation of MalE31 precursors. The con-
sequence is a doubly altered MalE31 precursor, leading to
strongly reduced rates of export and folding.
In the steady state, the cytoplasmic soluble fraction of
MalE31, when expressed from p31ꢀSS or p31M-18 (69%
and 37%), is higher than the periplasmic soluble fraction
of MalE31, when expressed from p31H (5%). This obser-
vation suggests that in the former case, the increased level
of GroEL and DnaK, resulting from induction of the cy-
toplasmic stress response by expression of MalE31ꢀSS or
MalE31M-18, can partially suppress the misfolding path-
way of these aggregation-prone proteins. Furthermore, it
appears that under these experimental conditions (tempera-
ture of growth, expression levels, bacterial strain, etc.), there
are insufficient or no classical heat-shock molecular chaper-
ones, such as cytoplasmic DnaK or GroEL, in the periplasm
to prevent or reduce aggregation of MalE31 in this com-
partment. Indeed, aside from specific molecular chaperones
such as the PapD family involved in pilus assembly, most
of the known periplasmic folding helpers thus far identi-
fied are folding catalysts: The protein disulfide isomerases
of the Dsb family and the peptidyl-prolyl isomerases [8].
An alternative explanation for the increased soluble yield of
MalE31 in the cytoplasm could be that the conformation of
nascent MalE31 polypeptide chains emerging from the large
ribosomal subunit is already directed towards the produc-
tive folding pathway, in contrast to their conformation when
they emerge inside the periplasm from the translocation ma-
chinery. Such beneficial associations of newly synthesized
polypeptide chains with ribosomes have been recently de-
scribed for the tailspike protein of phage P22 [7].
3
2
duction of the σ -stress response. Obviously, the increased
activity of the lon promotor in cells producing either the
MalEM-18 or MalE31M-18 precursor agrees with this ob-
servation. However, while both proteins are encountered ex-
clusively in their precursor sizes, the MalE precursor with
the altered signal sequence (expressed from p1M-18R) is
produced at a higher level than the MalE31 carrying both
the altered signal sequence and the malE31 mutation (ex-
pressed from p31M-18). This difference is likely explained
by a higher degradation of the latter protein. Because this
MalE31 variant displaying defective folding properties in-
3
2
duced the strongest σ -stress response, the heat-shock cy-
toplasmic proteases (such as Lon, HslUV, or Clp proteases)
could also degrade this misfolded protein, and its level of
production is dictated by the alternative fate between fold-
ing, degradation, and aggregation.
Finally, the stress response induced by the presence of
misfolded proteins in E. coli is compartmentalized into cy-
toplasmic and extracytoplasmic responses that are controlled
by two different sigma factors, σ32 and σ respectively [11].
We showed that MalE31 misfolding in the periplasm does
not induce a stress response via σ32 as it does when MalE31
is produced in the cytoplasm with altered signal sequences.
By this criterion, we were able to discriminate between cy-
toplasmic and periplasmic, the exact cellular location of the
folding defect of MalE31.
E
Acknowledgements
We are grateful to Nicole Jarrett for critical reading of
the manuscript. This material is based upon work supported
under the ‘Programme de Recherche Fondamentale en
Microbiologie, Maladies Infectieuses et Parasitaires’ from
the ‘Ministère de la Recherche’, under a Feodor Lynen
Fellowship to Sabine Hunke, and under grants from the
Institut Pasteur and the CNRS.
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