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Journal of Bacteriology, April 1999, p. 2267-2272, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Structure-Function Study of MalF Protein by Random
Mutagenesis
María Isabel
Tapia,
Michaël
Mourez,
Maurice
Hofnung, and
Elie
Dassa*
Unité de Programmation
Moléculaire et Toxicologie Génétique, CNRS URA
1444, Institut Pasteur, F75724 Paris Cedex 15, France
Received 13 October 1998/Accepted 28 January 1999
 |
ABSTRACT |
MalF is one of the two integral inner membrane proteins of the
maltose-maltodextrin transport system. To identify functional regions
in this protein, we characterized a collection of malF mutants obtained by random mutagenesis. We analyzed their growth on
maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK. Only 2 of the 21 MalF mutant proteins allowed growth on
maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the
cytoplasmic membrane. Having identified mutations leading to
reduced steady-state level, to partial mislocation, and/or to
misfolding, we were able to assign to some regions of MalF a role in
the assembly of the MalFGK2 complex and/or in the transport mechanism.
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TEXT |
The maltose-maltodextrin transport
system of Escherichia coli constitutes an excellent model to
study the folding, assembly, and functioning of an integrated system of
proteins involved in the acquisition of molecules by bacteria. This
system is, together with the histidine transport system of
Salmonella typhimurium, one of the most studied
periplasmic-binding-protein-dependent ATP-binding cassette
(ABC) transporters, reviewed in reference 1. Maltose
and maltodextrins enter the periplasm through LamB, an outer
membrane porin. The periplasmic maltose-binding protein (MBP or
MalE protein) binds substrates and interacts with MalF and MalG
(16), the two integral membrane proteins which
constitute, together with two copies of the MalK ATPase, the inner
membrane complex MalFGK2. This interaction triggers ATP
hydrolysis by MalK, allowing the MalFGK2 complex to
translocate the substrate released by MalE into the cytoplasm. The
exact mechanism of the transport is not known. Random linker insertion
mutagenesis proved to be a powerful tool to identify dispensable or
functionally important regions within LamB (2), MalE
(9), and MalG (6) proteins. Recently, a
transposon-mediated insertion mutagenesis approach was used to further
characterize the MalK (18) and MalG (22) proteins. The topology (3, 12, 13, 14), the assembly (29), and the membrane insertion (30) of the MalF
protein were studied in some detail. In contrast, little is known about the regions of MalF crucial for transport. In order to identify the
MalF regions important for structure and function, we mutagenized the
malF gene by random linker insertion. As mutations were
isolated independently of an activity test or phenotypic modification, potential bias toward the isolation of any particular class of mutant
was minimized, and silent mutations could be obtained.
Random mutagenesis, screening, sequencing, and phenotypes of
mutants.
E. coli strains, plasmids, media, and culture
conditions were previously described in reference
20. Random linker mutagenesis was performed as
described elsewhere (2, 6), except that a double-stranded
unphosphorylated BgIII linker, d(CAGATCTG)
(Biolabs), was used. Mutated pTAZFQ (malF under
the control of the tac promoter) plasmid was
transformed into the MalF
strain DHB4. Sixty-two of
533 screened clones had one insertion in or very close to
malF. Total cell extracts were prepared according to the
method described in reference 20. Thirty-one
of these plasmids were found to promote expression of a protein
recognized by the MalF antiserum (data not shown). Clones with no
immunodetectable MalF could result from the instability of the mutated
protein or plasmid, from a large deletion of the protein, or from an
early frameshift. We tried to restore the reading frame of the latter nonimmunodetectable mutants. We cut the plasmid DNAs with
BgIII and filled in or removed the cohesive ends by using
the Klenow fragment of E. coli DNA polymerase I or the
mung bean nuclease, respectively. The protein sequence
modifications generated by the linker insertions were deduced from DNA
sequence analysis. DNA sequencing (25) revealed that several
mutants were obtained more than once. They were probably nonindependent
clones arising during transformation. Finally, we obtained 21 mutants
from 15 independent different random BglII linker insertions
in malF. The positions of these primary insertions are
shown in Fig. 1, as related to the
topological model of MalF predicted by the PHDtoplogy program
(24). This model agrees with the experimentally
determined topological model (14) and was used to
predict the topology of the mutant proteins. Almost all the
random insertions resulting in immunodetectable MalF mutant proteins
were situated in or near hydrophilic regions (Fig. 1). As insertions
occurred at random, this suggests that hydrophilic domains are more
prone to modifications than are transmembrane (TM) segments. The
sequences of the 21 mutants are shown in Fig.
2A. In Fig. 2B, mutations are represented schematically, and the relative sizes of insertions and deletions are
shown. We obtained 12 immunodetectable mutant proteins without large
insertions or deletions. These mutations consisted in the insertion of
one to five amino acids determined by the linker accompanied by the
deletion of 4 to 19 amino acids, except in mutant 183, in which five
amino acids were inserted. In five cases (217, 308b, 352, 372b, and
453a), the mutation resulted in a truncated MalF, either by a large
deletion or by a shift in the translation reading frame, leading to a
premature stop. In one case (mutant 484a), the frameshift created a
replacement of the last 30 amino acids by 78 non-MalF amino acids. Two
mutants had large in-frame deletions including TM3 (mutant 62b) and TMs
2 and 3 (mutant 32).
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FIG. 1.
Topology model of MalF according to the PHDtopology
program from the EMBO server (24). Positions of the
different randomly inserted BglII sites are shown by arrows.
Numbers indicate the codon numbers preceding the BglII
sites. C, cytoplasmic domain; P, periplasmic domain. Boldface
letters correspond to the conserved peptidic motif EA----G---------I-LP
(7). Two independent insertions, but with different
downstream sequences due to a change in the reading frame, were found
after codon 308 (308a and 308b).
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FIG. 2.
Mutations in protein MalF. The derivatives of
malF were sequenced with the Sequenase version 2.0 DNA
sequencing kit (U.S. Biochemical and Amersham) with
[ -33P]dATP (ICN). Oligonucleotides (Eurogentec)
corresponding to 12 segments of the malF gene were used as
primers. The approximate location of the insertion was first determined
by restriction enzyme analysis, and then the appropriate primer was
chosen. In some cases, the sequencing of the antiparallel strand was
carried out to resolve ambiguities. (A) Sequences of wild-type MalF and
the collection of mutants described in this work. Predicted -helices
are shown underlined in the wild-type sequence. In mutant sequences,
amino acids inserted are shown in italics between the positions of
original MalF amino acids. Each mutant is named according to the last
nonmodified amino acid. Hyphens denote deletions. Stop codons in the
frameshift mutations are indicated by asterisks. (B) Mutations in MalF
protein, represented schematically in a linear fashion. Predicted TM
segments are boxed and numbered in roman numbers. The lengths of
rectangles are proportional to the sizes of deletions (solid) or
insertions (hatched).
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We analyzed the overall activity of MalF mutants by an in vivo genetic
complementation assay. The mutant
malF genes carried
by the
plasmid pTAZFQ were expressed in DHB4 (MalF
). Maltose
transport phenotypes of mutants were characterized
on MacConkey-2%
maltose or MacConkey-1% maltodextrin (malto-oligosaccharide
mixture
from Pfanstiehl Laboratories Inc.) plates. In the absence
of the
inducer isopropyl-
-
D-thiogalactopyranoside (IPTG), the
basal expression level of the wild-type
malF gene was
sufficient
to promote a Mal
+ Dex
+ phenotype.
Among the mutant proteins only two (mutants 35 and
163) complemented
the
malF3 mutation for maltose and maltodextrin
fermentation. The phenotypes were significantly improved neither
by
adding IPTG nor when MalG and/or MalK was co-overexpressed,
indicating
that none of the proteins was limiting. None of the
MalF mutants
displayed a MalE-independent phenotype (
4). Maltose
phenotypes were characterized by measuring the doubling time (DT)
of
mutants in liquid M63B1-maltose minimal medium (Table
1).
Under these conditions, only mutants
35 and 163 displayed DTs
similar to that obtained with wild-type MalF.
The growth rate
of the strains in glycerol-supplemented minimal medium
was similar
to that of control cells (wild-type
malF),
indicating that no
growth defect was caused by the expression of mutant
proteins
(data not shown).
Steady-state levels, subcellular localization, and stability of
MalF mutant proteins.
We examined the steady-state levels of the
MalF mutants (Table 1). All mutant proteins accumulated at lower
steady-state levels than that of the wild-type protein, indicating that
mutations led to reduced protein production or to unstable proteins.
Mutant 35 was functional in spite of its low protein level. This result suggests that the loss of activity of MalF mutants was
not due merely to a low steady-state level of protein in the cells. The
ability of the nonionic detergent Triton X-100 to solubilize the MalF
proteins was examined. This detergent specifically solubilizes
cytoplasmic membrane proteins (8) and has been used to show
the membrane localization of the MalFGK2 complex (20). All mutants except two (453b and 497b) were completely extracted with Triton X-100 (Fig. 3) and
therefore localized exclusively in the cytoplasmic membrane like
wild-type MalF. Mutants 453b and 497b were only partially extracted
with Triton X-100 (Fig. 3A). Hence, a fraction of each of these mutant
proteins consisted of aggregated rather than membrane-associated
proteins. The insertion of MalF into the membrane was not prevented by
the deletion of any of the TMs. These results agree with previously
reported findings showing that individual membrane-spanning sequences
act as autonomous insertion domains (14, 27) and with
earlier studies on polytopic cytoplasmic membrane proteins (6, 19,
22). Immunoblots of mutants with short deletions displayed a
single band recognized by the antibody (Fig. 3A, lanes 2 to 8).
By contrast, mutants affected in TMs (mutants 35, 372a, 484b,
497a, and 497b), with large in-frame deletions (mutants 32 and 62b), or
with frameshifts (mutants 308b, 352, 372b, 453a, and 484a) showed
several bands of higher electrophoretic mobility, which most likely
correspond to proteolytic degradation products of MalF mutant proteins
(Fig. 3).
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FIG. 3.
Subcellular localization of MalF mutant proteins. Cells
were induced at an A600 of 0.5 with 1 mM IPTG.
Cell fractionation was performed according to the method described in
reference 20, with the following modifications. The
protease inhibitor PEFABLOC (Interchim) was added at an 0.5 mM final
concentration to all buffers. Membrane extracts were recovered after
1 h of centrifugation at 20,000 × g. To
solubilize cytoplasmic membrane proteins, Triton X-100 was used as
described in reference 15. Equivalent volumes of
Triton-soluble (s) and Triton-insoluble (i) fractions were mixed with
an equal volume of double-strength sodium dodecyl
sulfate-polyacrylamide gel electrophoresis loading buffer, boiled for 5 min in a water bath, loaded onto sodium dodecyl sulfate-12%
(wt/vol) polyacrylamide gel electrophoresis gels, and subjected to
immunoblotting. Nitrocellulose membranes were probed with the specific
antibody and with a horseradish peroxidase anti-rabbit immunoglobulin
conjugate (Bio-Rad). Immune complexes were revealed by ECL Western
blotting detection reagents (Amersham). Relative intensities were
quantified by scanning the ECL films with an Image Master VDS apparatus
(Pharmacia Biotech). The solid arrowheads indicate the full-length MalF
mutant proteins. The empty triangles indicate cross-reacting proteins
that are visible in all lanes. The amount of protein loaded in
immunoblot B was threefold higher than that loaded in immunoblot A. WT,
wild type. MW, molecular weight markers, in thousands.
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Subcellular localization of MalK.
Both in MalF and MalG, the
conserved region EA----G---------I-LP is very probably important for
the interactions between the hydrophobic membrane proteins and the MalK
ATPase (20). Mutants affected in this region were not able
to stably maintain MalK in the membrane. We analyzed the cellular
localization of MalK in cells containing MalF mutant proteins unable to
grow on maltose and maltodextrin, in order to examine if regions
other than the conserved region were important for the interaction with
MalK. Strain ED169 was transformed with recombinant or
wild-type pTAZFGQ and pACYK. Plasmid genes were induced
with 10 µM IPTG. In MalF+ MalG+ cells (ED169
transformed with nonmutated pTAZFGQ and pACYK), we found about 90%
MalK in the particulate fraction, from which it was completely
extracted with Triton X-100. In MalF MalG
cells (ED169 transformed with pTAZQ and pACYK), we found about 100%
MalK in the cytoplasmic fraction. The membrane association of MalK was
altered in mutant 308a (47% of MalK in the cytoplasm) and more
drastically in mutant 372a (98% of MalK in the cytoplasm) (Fig.
4; Table 1). Mutant 497a behaved like
wild-type MalF, and in the other mutants tested, the amount of
cytoplasmic MalK ranged from 19 to 36%.
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FIG. 4.
Subcellular localization of MalK. MalK subcellular
localization experiments were performed as described elsewhere
(20), except that strain ED169 instead of strain ED170 was
used and extracts were centrifuged at 20,000 × g
instead of 200,000 × g. Under these conditions, all
MalK protein was solubilized by Triton X-100, and thus, no additional
fractionation of the Triton-insoluble fraction with urea was made.
Triton-soluble (s), cytoplasmic (c), and Triton-insoluble (i) fractions
were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and subjected to immunoblotting. The volume loaded for
the Triton-soluble and Triton-insoluble fractions was double that for
the cytoplasmic fractions.
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Potential functional domains of the MalF protein.
In several
cases, the insertion of the linker introduced a change in specific
domains of MalF. We will discuss these changes, with a focus on short
deletions, in order to assign tentatively a function to these domains.
The first periplasmic loop (P1) was changed in mutation 35, together with the last amino acids of TM1. This mutation did not
affect the utilization of maltose and maltodextrins and mapped
in
a region of MalF that had been previously defined as highly
permissive
(
11). The mutation introduced at this point seemed
to affect
the stability of the protein. The amount of nondegraded
mutant protein
inserted in the membrane, even under noninducing
conditions,
would be enough to account for the observed Mal
+
Dex
+ phenotype. This region would not be crucial for
the assembly
of the MalFGK
2 complex or for the constitution
of the maltose
translocation pathway but would contribute to the
stability of
the
protein.
The large periplasmic domain P2 is characteristic of MalF proteins
in enterobacteria. MalF proteins in other bacteria lack
this domain and
have in some cases six TMs rather than eight (
17).
Although
its sequence in enterobacterial MalF is less conserved
than that of
other parts of the protein (
5,
26), our results
strongly
suggest that P2 is essential for function. Mutations
in P2 had little
influence on the steady-state level of MalF,
and they did not cause the
appearance of proteolytic products.
For the five mutations
affecting P2, only mutant 163 was competent
for
maltose-maltodextrin transport. The other mutations affected
MalK
localization. Mourez et al. (
20) showed that both MalF
and
MalG are needed to stably maintain MalK in the membrane and
that
residues in C4 of MalF are essential for the binding of MalK
to MalFG.
The dislocation of MalK provoked by the above mutations
could be
explained by assuming that P2 participates in anchoring
MalK into
the membrane. According to the proposed model of the
eukaryotic ABC
transporter P-glycoprotein determined by electron
microscopy and
image analysis (
23), it is possible that extracytoplasmic
domains of ABC transporters are in direct contact with
cytoplasmic
domains. Alternatively, the effect might be indirect,
the mutation
affecting either the conformation of C4 or the interaction
of
MalF with MalG. The latter hypothesis is consistent with the finding
that the conformation or the accessibility of MalF, and most notably
of
the P2 domain, is modified depending on the presence of MalG
(
21,
28).
Loop P4 was partly deleted in mutant 453b. The mutation led to a high
steady-state level of protein, which was defective in
transport and
partially mislocated since a small fraction of the
protein was not
extracted from membranes with Triton X-100. The
almost correct
subcellular localization of MalK in the mutant
suggests that the
interaction of MalF with MalG is not dramatically
affected. Hence, the
mutation probably affected transport either
by the modification of
a substrate binding site or by hampering
an interaction with MalE.
Interestingly, mutations in the last
periplasmic loop of MalG have
been proposed to affect the recognition
of substrates
(
6) or the interaction with MalE (
22). If these
interpretations are correct, the last periplasmic loops of MalF
and
MalG might cooperate in such
functions.
Cytoplasmic loop C2 was altered in mutant 62a. Although nonfunctional,
this mutant behaved like wild-type MalF with respect
to all other
criteria examined. This mutation would affect a
transport-specific
function.
C3 was partially deleted in mutant 308a. The mutant protein was well
expressed and correctly localized into the membrane,
but 47% of MalK
was found in the cytoplasm. Like loop P2, C3 might
be directly or
indirectly implicated in the attachment of MalK
to the
membrane.
Mutants in TMs displayed in general a low steady-state level of
protein. This was probably due to proteolytic degradation.
The deletion
of TM6 (mutant 372a) led to the complete dislocation
of MalK. This
could be due to the fact that C4 would probably
be translocated to the
periplasm in this mutant. By contrast,
the deletion of C5 and of
the C-terminus of TM8 (mutant 497a)
did not affect the localization of
MalK. Therefore, the C-terminal
part of MalF would not be essential for
the interaction with MalK
or MalG. The properties of mutant 497a are
similar to those of
mutant 453b (P4), suggesting that residues involved
in the transport
mechanism or in the translocation pathway of
substrates are present
in P4, in TM8, and in C5. This conclusion is in
agreement with
the results of reference
10, which
reported that several amino
acid substitutions in TM8 led to
Mal
+ Dex
phenotypes.
In summary, the incapacity of some MalF mutant proteins to participate
in maltose transport could be due at least partially
to a defective
insertion into the membrane, increased rates of
proteolysis, or partial
aggregation in the cytoplasm. By contrast,
mutations in C2, P4, TM8,
and C5 probably affected the transport
mechanism while mutations in C3
and P2 probably affected the assembly
of the MalFGK
2
complex. Regions potentially assigned to specific
functions will be
further dissected by site-specific mutagenesis
in order to refine our
model.
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ACKNOWLEDGMENTS |
M.I.T. was supported by a European Commission Research Training
Grant (Biotechnology Programme, contract number BIO4-CT96-5068).
We are grateful to Alain Charbit, Pierre Martineau, and Jean-Michel
Betton for their helpful technical advice and to Jesus A. G. Ochoa
de Alda and Wolfgang Köster for careful reading of the manuscript
and for helpful suggestions. We thank Beth Traxler and Erwin Schneider
for the gift of MalF and MalK antibodies. We thank Muguette
Jéhanno for the help in MalK localization.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
Programmation Moléculaire et Toxicologie Génétique,
CNRS URA 1444, Institut Pasteur, 25 rue du Dr. Roux, F75724 Paris Cedex
15, France. Phone: 33 (0)1 45 68 88 31. Fax: 33 0(1) 45 68 88 34. E-mail: elidassa{at}pasteur.fr.
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Journal of Bacteriology, April 1999, p. 2267-2272, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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