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INTRODUCTION |
The maltose transport complex of Escherichia coli is a
member of the ATP-binding cassette
(ABC)1 transporter
superfamily (1, 2), which includes bacterial binding
protein-dependent transporters, the cystic fibrosis
transmembrane conductance regulator, and the P-glycoprotein of
multidrug-resistant tumor cells. Each member of this family has two
conserved nucleotide binding domains. The binding
protein-dependent maltose transport complex (MTC) of
E. coli comprises four protein subunits (3). One copy each
of MalF and MalG probably form a channel in the cytoplasmic membrane
through which maltose passes. Two copies of the peripheral membrane
ATP-binding protein MalK are associated with MalF and MalG. The
hydrolysis of ATP by MalK is presumed to energize the process of sugar
transport. Additionally, MalK plays a role in the regulation of maltose
transport through two distinct pathways. MalK regulates the expression
of mal genes through an unknown mechanism that is dependent
on the mal transcriptional activator MalT (4). MalK also
interacts with unphosphorylated enzyme IIAglc
(EIIAglc) of the phosphenolpyruvate:sugar
phosphotransferase system, which lowers the activity of the MTC and
decreases maltose transport (5).
Previous studies of the MTC have provided evidence that the two copies
of MalK interact functionally within the complex. It has been shown
that the MTC hydrolyzes ATP with positive cooperativity (6), and the
mutation of the ATP-binding site in a single MalK subunit in the MTC
severely impairs transport (7).
We are characterizing the in vivo tetramerization of the Mal
proteins as a model for membrane protein assembly. In this paper, we
provide biochemical evidence that MalK forms a dimer in the absence of
the MalF and MalG proteins. We have utilized several MalK mutants
carrying transposon-mediated in-frame epitope insertions that were
previously characterized for their ability to assemble into the complex
and transport maltose (8). Dimerization of several of the mutant
proteins with wild-type MalK was shown using coimmunoprecipitation
techniques. Furthermore, the oligomerization of MalK was confirmed
in vivo using the
repressor fusion assay (9). The
dimerization of MalK may represent an initial step in the assembly of
the maltose transport complex.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Plasmids--
The bacterial strains and
plasmids that were used in this study are listed in Table
I. Plasmid preparation, cloning, and transformation were carried out as described previously (10). To make
plasmid constructs for co-immunoprecipitation, DNA fragments containing
malK insertion mutants with the IPTG-inducible
trc promoter and lacIq from
pTrc99A-based plasmids were subcloned into pAYC184 using SphI and HindIII. To make the
repressor
cI-malK fusion, polymerase chain reaction-amplified
wild-type malK gene was subcloned into pJH391 digested with
SalI and BamHI. This construct, pKK700, produces a protein consisting of the N-terminal 132 amino acids of the cI
repressor fused to the N terminus of MalK.
Media--
Rich (LB), minimal (M63), and MacConkey media have
been described (11). Minimal media were supplemented with thiamine and all of the amino acids except cysteine and methionine and with glycerol
at 0.2%. Antibiotics were used at final concentrations of 100 µg/ml
for ampicillin and 30 µg/ml for chloramphenicol.
Preparation of Labeled Whole Cell Extracts--
Strains were
grown in supplemented M63 minimal medium at 37 °C with aeration to
an A600 of 0.3. Strains were induced with 1 mM IPTG for 5 or 15 min and then 1 ml of culture was
labeled by the addition of 120 µCi of [35S]methionine
for 10 min. Labeling was stopped by the addition of 0.05% cold
methionine, and the cultures were immediately placed on ice. Cells were
washed one time with and then resuspended in 0.5 ml of 50 mM Tris-HCl, pH 8.0, 1 mM EDTA (Buffer A). The
resuspended cells were lysed with two 15-s bursts of a probe sonicator.
Lysates were aliquoted and stored at -80 °C.
Immunoprecipitation of MalK Species--
The
coimmunoprecipitation procedure was adapted from a method described by
Davidson and Nikaido (3). Whole cell extracts were solubilized with 1%
dodecyl maltoside for 20 min on ice. The insoluble material was removed
by centrifugation for 10 min at 16,000 × g at 4 °C
in a microcentrifuge. This insoluble fraction (Fig. 1A, lane
2) likely represents a mixture of unbroken cells, insoluble
protein, and residual soluble material after removal of the soluble
extract. The remaining soluble extracts were diluted 1:10 in a buffer
containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.01% dodecyl maltoside (Buffer B). The
insertion-specific antiserum was added and incubated on ice for 1-2 h
or overnight. To isolate immune complexes, 25 µl of 50% protein
A-Sepharose CL-4B (Amersham Pharmacia Biotech) in Buffer B was added to
the extracts and incubated on ice for 30 min, with mixing every 5 min.
The protein A-Sepharose beads were sedimented in a microcentrifuge and
washed three times with 0.5 ml of Buffer B. The beads were heated to
65 °C for 20 min in 25 µl of gel loading buffer before the
proteins were separated by SDS-PAGE on 10% gels.
The procedure to immunoprecipitate epitope-tagged MalK alone was
adapted from a procedure used by Traxler and Beckwith (12). Whole cell
lysates were solubilized with 2% SDS followed by heating at 65 °C
for 20 min. This extract was then diluted 1:17 in Buffer C (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 2% Triton X-100) and centrifuged for 10 min at
16,000 × g at 4 °C in a microcentrifuge to remove
insoluble material. Insertion-specific antiserum was added to the
soluble extract and incubated overnight at 4 °C. Immune complexes
were isolated as described above except that Buffer C was used for
washing the protein A-Sepharose beads.
Quantitation--
Phosphorimages were scanned using a Molecular
Dynamics PhosphorImager SF, and the data were analyzed with the
Molecular Dynamics ImageQuant software, version 3.1. Mutant and
wild-type MalK species were readily detected and quantitated in the
induced whole cell extracts (Fig. 1A). These quantitations
were corrected for background labeled cellular proteins.
cI Repressor Fusion Assay--
Strains, vector plasmids, and
KH54 were provided in the strain kit for repressor fusions courtesy
of Jim Hu. The cI-malK fusion construct in strain KTK51
produces a protein that is approximately 12 kDa larger than MalK as
detected by Western analysis using a MalK-specific antiserum provided
by Howard Shuman (data not shown). The level of the cI repressor-MalK
fusion in uninduced KTK51 was lower than that of MalK expressed from
the normal chromosomal locus in BT8. These strains were then tested for
immunity to bacteriophage
by cross-streak analysis without
IPTG.
Proteolysis Test of MalFGK2 Complex
Assembly--
Cultures of BT8 transformed with the pLK plasmids were
grown with aeration at 37 °C in supplemented M63. During the final 2 h of growth before labeling, 1 mM IPTG was added to
induce expression of the malK mutations, and 2 mM cAMP was added for the last 5 min. Cells were
pulse-labeled for 30 s with 40-50 µCi/ml
[35S]methionine and then mixed with 0.05% cold
methionine for 10 min of continued incubation. After labeling, cells
were converted to spheroplasts as described previously (12).
Proteolysis was done on 0.5-ml portions of spheroplasts for 20 min at
0 °C with 25 µg/ml trypsin and stopped as described previously
(12). After proteolysis, spheroplasts were harvested and resuspended in
50 µl of 50 mM Tris, pH 7.6, 2% SDS, 1 mM
EDTA, and heated to 65 °C for 20 min. These samples were diluted
1:17 in Buffer C. MalF protein was immunoprecipitated with a specific
antiserum, using the previously described method (12), and results were
analyzed after SDS-PAGE on 12.5% resolving gels. Results presented are an average of 2-4 experiments. All samples were checked for integrity of spheroplasts during proteolysis by SDS-PAGE of extracts prior to
immunoprecipitation (data not shown).
Chemicals and Enzymes--
General chemicals and media supplies
were purchased from Difco, Fisher Scientific, and Sigma. IPTG was
purchased from Bachem. Maltose was bought from Pfanstiehl. Lysozyme,
phenylmethylsulfonyl fluoride, and cAMP were from Sigma, and trypsin
was from Worthington. Prestained protein molecular weight markers and
acrylamide were obtained from Bio-Rad. Soybean trypsin inhibitor, SDS,
and n-dodecylmaltoside were purchased from Boehringer
Mannheim. [35S]Methionine was purchased from NEN Life
Science Products (EXPRE35S35S label).
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RESULTS |
Coimmunoprecipitation of a MalK Oligomer--
The oligomeric state
of MalK was analyzed in extracts prepared from E. coli
strains (containing a chromosomal deletion of the malFGK
region) producing both wild-type and epitope-tagged MalK species.
MalK550 contains an in-frame insertion of 31 mostly hydrophilic amino
acids, retains MalK functions, and is recognized by a polyclonal
antiserum specific for the insertion (8, 19). Cells expressing both
malK550 and malK+, each from a
plasmid under the control of the trc promoter, were induced
briefly with IPTG, labeled with [35S]methionine, and
sonicated. Based on the observation that MalFGK2 tetramers
are stable in dodecyl maltoside (3), these extracts were solubilized
with this nonionic detergent. Although previous studies have
demonstrated that MalK tends to form inclusion bodies when highly
expressed in E. coli (4, 20), more than 90% of the MalK
species in our extracts was present in the soluble fraction (Fig.
1A), likely due to the short
induction time used. These solubilized extracts were used for
coimmunoprecipitation studies.

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Fig. 1.
Coimmunoprecipitation of MalK550 and
wild-type MalK. A, BT6 containing the compatible
plasmids pBT100 and pKK16 was labeled with
[35S]methionine. The sonicated whole cell extract was
solubilized with 1% dodecyl maltoside. Any insoluble material was
removed by centrifugation and then resuspended in a volume equivalent
to the original sample. Proteins were separated by SDS-PAGE and
visualized with a phosphorimager. Lane 1, sonicated whole
cell extract; lane 2, insoluble fraction; lane 3,
soluble fraction. B, dodecyl maltoside-solubilized extracts
of BT6 with plasmids pBT100 or pTrc99A and pKK16 or pACYC184 were
incubated with insertion-specific antiserum against MalK550, and immune
complexes were recovered by incubation with protein A-Sepharose.
Lane 1, extract with both MalK550 and wild-type MalK;
lane 2, extract with MalK550 only; lane 3,
extract with wild-type MalK only. C, same as B
except that extracts were solubilized with 1% SDS and diluted in 2%
Triton X-100. D, control immunoprecipitations of dodecyl
maltoside solubilized extract containing both MalK550 and wild-type
MalK. Lane 1, with insertion-specific antiserum; lane
2, with plain protein A-Sepharose beads; lane 3,
immunoprecipitation with anti-human Hck (N-30) (Santa Cruz
Biotechnology). The positions of migration of molecular mass markers
(in kDa) are indicated.
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The soluble fractions were incubated with the insertion-specific
antiserum. Under these conditions, both MalK550 and wild-type MalK were
immunoprecipitated from strains expressing both proteins (Fig.
1B, lane 1). MalK550 was easily distinguished from wild-type MalK due to its reduced electrophoretic mobility on SDS-PAGE gels. Only
MalK550 was immunoprecipitated from a strain expressing MalK550 alone,
and no proteins were immunoprecipitated from a strain expressing only
wild-type MalK (Fig. 1B, lanes 2 and 3). These
results show that wild-type MalK is associated with MalK550 in these
solubilized whole cell extracts. Similar results were obtained with a
related MalK insertion mutant, MalK556, which has similar phenotypes to MalK550 (data not shown).
Immunoprecipitations also were carried out on extracts solubilized with
2% SDS and diluted in 2% Triton X-100. Only MalK550 was recovered in
these reactions (Fig. 1C), showing that the interaction between MalK species can be disrupted by denaturation in ionic detergent and that the antiserum is specific for MalK550. The heteromeric complex also does not reform in vitro after
denaturation and partial renaturation. In order to show that the
observed MalK-MalK550 oligomer is due to specific interaction rather
than nonspecific aggregation during sample preparation, the procedure
was carried out using an equivalent amount of an irrelevant affinity
purified antiserum, anti-human Hck (N-30) (Santa Cruz Biotechnology)
and using protein A-Sepharose beads alone (Fig. 1D, lanes 2 and 3). The hetero-oligomeric complex was recovered only in
the immunoprecipitation containing the insertion-specific antiserum
(Fig. 1D, lane 1).
The immunoprecipitation also was carried out with extracts solubilized
with 1% Triton X-100 instead of dodecyl maltoside. Again, we observed
more than 90% of MalK species in the soluble fraction and the
coimmunoprecipitation of the hetero-oligomeric complex (data not
shown), indicating that the solubility of MalK and its oligomeric
association are maintained in both nonionic detergents.
The MalK Oligomer Is a Dimer--
In order to determine the
oligomeric state of MalK, the amount of immunoprecipitated MalK550 and
wild-type MalK was quantitated and analyzed using an equation
describing the distribution of random pairs. If MalK is a dimer, cells
expressing a mixture of MalK550 and wild-type MalK would contain three
species of dimer: MalK550 homodimer, wild-type MalK homodimer, and
MalK550/wild-type MalK heterodimer. The insertion-specific antiserum
would immunoprecipitate MalK550 homodimers and MalK550/wild-type MalK
heterodimers only. Assuming random mixing of MalK550 and wild-type MalK
molecules, the distribution of heterodimer and both homodimers in the
extract is described by the binomial expansion
p2 + 2pq + q2 = 1, where p and q represent the proportions of
MalK550 and wild-type MalK, respectively. The pq term
represents the proportion of heterodimer, and p2
and q2 represent the proportion of each
homodimer. Using this equation, the relative amounts of each molecule
immunoprecipitated by the insertion-specific antiserum can be predicted
based on the relative amounts of MalK550 and wild-type MalK present in
the whole cell extracts. These amounts were determined, and the above
equation was used to predict the relative amounts of MalK550 and
wild-type MalK that should be immunoprecipitated (Table
II). Similar binomial expansions were
used to predict the relative proportions that would be present in the
immunoprecipitations if the oligomer was a trimer or a tetramer. The
relative amounts of the two MalK species actually present in the
immunoprecipitations were found to agree with values predicted for a
dimeric species (two independent analyses are shown). This suggests
that MalK550 and wild-type MalK molecules are present in the extract as
randomly associated dimers.
In separate studies, the ratio of the relative proportions of MalK550
and wild-type MalK that were expressed in the cells was altered by
switching the vectors carrying the malK alleles. In these
experiments, the ratio of MalK550 to wild-type MalK in whole cell
extracts was 4:1, in contrast to ratios close to 3:7 in the experiments
above. Again, wild-type MalK coimmunoprecipitated with MalK550 using
the insertion-specific antiserum. Consistent with the prediction of the
binomial expansion, the amount of wild-type MalK recovered was
substantially smaller than in the above experiments and close to that
expected for dimers. However, the difference between the predicted
ratios for dimers, trimers and tetramers is not significant due to the
small population of wild-type MalK (data not shown).
MalK550 and Wild-type MalK Hetero-oligomer Can Be Formed in
Vitro--
In order to assay for the association and dissociation of
MalK in vitro, sonicated whole cell extracts were prepared
from two different strains expressing either MalK550 or wild-type MalK. These extracts were mixed and incubated at 37 °C for various times and then were solubilized with dodecyl maltoside and immunoprecipitated as before, using the insertion-specific antiserum. A small amount of
hetero-oligomer was recovered (Fig. 2).
The proportion of wild-type MalK relative to MalK550 that was
coimmunoprecipitated increased 47% over 1.5 h. The MalK oligomer
can form in vitro from two separate populations of MalK
molecules.

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Fig. 2.
Formation of MalK hetero-oligomers in
vitro. Sonicated [35S]methionine-labeled
whole cell extracts of BT6 each expressing either MalK550 or wild-type
MalK were mixed and incubated at 37 °C for various times. These
mixtures were then solubilized with dodecyl maltoside and subjected to
immunoprecipitation with the insertion-specific antiserum. Labeled
protein samples were separated by SDS-PAGE and visualized by
autoradiography. Lane 1, mixed extracts, 30-min incubation;
lane 2, mixed extracts, 1-h incubation; lane 3,
mixed extracts, 2-h incubation; lane 4, MalK550 extract
alone, 1-h incubation; lane 5, wild-type MalK extract alone,
1-h incubation.
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MalK Oligomerization Assayed Using the
Repressor Fusion
System--
To assay for MalK oligomerization in vivo,
full-length malK was fused to the coding region for the
N-terminal DNA binding domain of the bacteriophage
cI repressor. A
strain expressing this cI repressor-MalK fusion and various control
strains were cross-streaked against
. The ability of a fusion
protein to confer immunity to
indicates the formation of an active
dimeric repressor protein (9). Although the control strains were
-sensitive, the cI repressor-MalK fusion protein (expressed from
pKK700) conferred resistance to
(Fig.
3), providing additional evidence for the formation of MalK dimers in vivo. The cI-malK
fusion plasmid restores a Mal+ phenotype to the
malK strain HS3169 on maltose MacConkey plates, indicating that the fusion protein is functional for transport. There
were no bands other than the cI repressor-MalK fusion protein visible
on a Western blot developed with a MalK-specific antiserum (data not
shown), indicating that the maltose transport function was conferred by
the fusion protein and not by a MalK breakdown product.

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Fig. 3.
Cross-streak analysis. Derivatives of
E. coli strain HS3169 transformed with various plasmids were
streaked across phage KH54 on an LB plate containing ampicillin. The
strain in which the N terminus of the cI repressor is present as a
dimer is resistant to phage, and the other strains are sensitive to
phage. The bacterial streaks are labeled according to the plasmid in
each strain. pBR322 contains the cloning vector pBR322. The pKK700
plasmid construct expresses a fusion of the N terminus of the cI
repressor to full-length wild-type MalK. The pOAC100 plasmid construct
expresses the N-terminal 131 amino acids of the cI repressor protein.
The pBT100 plasmid expresses full-length wild-type MalK.
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Insertion Mutants That Cannot Assemble into MalFGK2
Complexes Still Produce Dimers--
Previously, two other MalK
insertion mutants, MalK552 and MalK554, were characterized, and both
were found to be deficient in maltose transport and in MTC assembly
(8). These mutants were tested with the coimmunoprecipitation assay in
order to determine whether their assembly defects are due to an
inability to dimerize. MalK552 and MalK554 were each expressed with
wild-type MalK and subjected to immunoprecipitation with the
insertion-specific antiserum. Both assembly incompetent mutants form
hetero-oligomers (Fig. 4). This result
suggests that although both of these MalK mutants are unable to
assemble into a functional transport complex, these phenotypes are not
due to a dimerization defect. These mutants are dissimilar in that the
malK552 gene is dominant to wild-type malK when
expressed at high levels, whereas malK554 is recessive. It
is likely that the behavior of these mutants is due to a difference in
some other intermolecular interaction in the MTC assembly pathway. Therefore, we tested the different dominance phenotypes of these malK alleles in a biochemical assay that measures
MalFGK2 assembly.

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Fig. 4.
Coimmunoprecipitation of MalK552 and MalK554
with wild-type MalK. Labeled extracts of strain BT6 transformed
with compatible plasmids (pBT100 and pKK268 in lane 1, pBT100 and pKK442 in lane 2) were solubilized with 1%
dodecyl maltoside and immunoprecipitated with the insertion-specific
antiserum. Protein samples were separated by SDS-PAGE and visualized
with a phosphorimager. Lane 1, extract of BT6 expressing
both MalK552 and wild-type MalK; lane 2, extract of BT6
expressing both MalK554 and wild-type MalK. The positions of migration
of the MalK insertion mutants (MalKi31) and wild-type
(wtMalK) are indicated.
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We have described a protease sensitivity assay to measure the
oligomerization of the MTC in vivo (12). Briefly, the MalF component of the complex is initially inserted into the cytoplasmic membrane in a form that is sensitive to cleavage by trypsin in its
periplasmic domains. If the protein assembles with both MalG and MalK,
MalF becomes trypsin-resistant. We compared the abilities of the
insertion mutant proteins expressed from the dominant
malK552 and the recessive malK554 mutations to
compete with wild-type MalK for complex assembly in a Mal+
strain with this assay. We found that although the MalK554 mutant did
not inhibit the acquisition of protease resistance of MalF relative to
the positive control (the assembly and transport proficient MalK556
insertion mutant), MalK552 had a strong inhibitory effect (Table
III). Although both of the assembly
defective MalK insertion mutants apparently dimerize, the two mutants
are different in their abilities to compete with wild-type MalK for
MalFGK2 assembly. The difference in the genetic dominance
of the mutations is revealed in the proteolysis assay.
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DISCUSSION |
We have shown that the MalK protein forms a dimer in the absence
of the two integral membrane proteins, MalF and MalG, that are
associated with MalK in the maltose transport complex in E. coli. A mixed oligomer of two distinguishable forms of the MalK protein was isolated from extracts of strains that do not express MalF
and MalG (Fig. 1). Quantitation of the immunoprecipitated MalK proteins
showed that a dimer was isolated and not a higher order oligomer (Table
II). The fact that an equation that describes the random distribution
of homodimers and heterodimers accurately predicted the results of the
coimmunoprecipitation assays shows that a very high proportion of the
epitope-tagged MalK protein is present in the solubilized extract as a
dimer. Based on these data, we suggest that MalK forms a dimer prior to
assembling into the membrane bound complex and that this dimerization
may represent a step in an ordered assembly pathway of the MTC.
The formation of heterodimers by mixing whole cell extracts each
containing either tagged or wild-type MalK shows that the dimerization
can occur in vitro (Fig. 2). The inefficiency of heterodimer
formation in this mixing experiment may indicate that the MalK dimer is
relatively stable, resulting in a scarcity of free monomer and/or a
slow rate of dimer dissociation. This observation also implies that the
dimers observed in Fig. 1 were formed in vivo. The formation
of dimers in vitro can be used as an assay to determine what
factors may be necessary for MalK association and dissociation. It is
possible that the small amount of dimer being formed in this assay is
produced from MalK monomers present in the whole cell extracts.
However, we consider this unlikely, as the calculations that were
discussed above showed that the amount of MalK monomer present in these
extracts is likely to be small.
Additional evidence for dimerization was provided by the
cI
repressor fusion assay in vivo (Fig. 3). The cI
repressor-MalK fusion protein was expressed in the presence of the MalF
and MalG proteins of the MTC. This shows that the dimerization that was observed in vitro occurs in vivo and therefore is
likely to be biologically significant. This conclusion is further
supported by the fact that the cI repressor-MalK fusion protein
complements the transport defect of a
malK strain.
Heteromeric complexes were also identified between wild-type MalK and
the MalK552 and MalK554 insertion mutants (Fig. 4). These mutant
proteins are unable to assemble into MalFGK2 complexes with
protease-resistant MalF in the absence of wild-type MalK (9).
Therefore, the assembly defect of these MalK mutants seems to be
unrelated to dimerization and likely occurs at a subsequent step in
complex assembly. For transport activity, the malK552 allele
is dominant to wild-type malK and MalK552 also prevents the
formation of MalFGK2 complexes containing
protease-resistant MalF (Table III). The insertion in MalK552 might
allow stable formation of MalK/MalK552 heterodimers that are
incompetent for late stages of complex tetramerization. Paradoxically,
the malK554 mutation is recessive to
malK+ even though the MalK554 protein can
dimerize with wild-type MalK. One possible explanation for these
results is that a MalK/MalK554 complex may be destabilized at a
subsequent stage of complex assembly. This situation would liberate the
wild-type MalK to interact anew with the intracellular population of
MalK molecules. The assembly of a stable tetramer of wild-type MalFGK
proteins could then "select" for the rarer wild-type MalK
homodimers (resulting in protease-resistant MalF and maltose
transport). Further studies will identify the stage of assembly that is
blocked with these mutants and contribute to our understanding of the
assembly pathway.
The dimer form of the nucleotide binding domain protein, HisP, from the
ABC transporter histidine permease in Salmonella typhimurium has also been described by Nikaido et al. (21). They found
that purified HisP hydrolyzes ATP with a nonlinear dependence on
protein concentration, in a manner suggesting that the active form of HisP is a dimer. In gel filtration analysis of purified HisP, only
about 3% of the soluble protein was in the dimer peak, whereas the
rest of the protein eluted as a monomer. In contrast to this, there
appears to be a high proportion of MalK dimers in our solubilized extracts. The discrepancies between the results with the MalK and HisP
proteins may be due to differences in assay conditions. Purified
proteins were assayed in the HisP system, whereas complex cell extracts
were assayed in the MalK experiments. Although MalK and HisP are
homologues, they have distinct biological roles and may therefore have
different dimerization and assembly characteristics. In addition to
assembling into a membrane bound transport complex and hydrolyzing ATP
as HisP does, MalK also regulates the expression of mal
genes and the activity of the transporter (4, 5). All of the MalK
insertion mutants examined in this study are proficient for the
MalK/MalT-dependent mal gene regulation (8). It
is possible that the dimerization of MalK may also be important in this process.
The in vivo assembly pathway of the MTC or other members of
the ABC transporter superfamily is unknown. The finding that MalK efficiently forms a dimer in the absence of MalF and MalG implies that
a dimer of the nucleotide binding domain proteins of these transporters
may be a first step in their assembly pathways. However, Liu and Ames
(22) recently reported that HisP molecules are recruited individually
to the HisQM complex in vitro. Clearly, there are important
differences between the in vitro reconstitution studies with
the HisQMP2 complex and our studies of MalK dimerization in
whole cell extracts. If the different oligomerization characteristics that are observed with HisP and MalK are due to biological differences between these proteins, distinct assembly pathways of different ABC
transporters may exist. Alternatively, either the MalK dimer or monomer
may be proficient for complex assembly. This idea is consistent with a
model that supposes an efficient assembly process in which the complex
incorporates each subunit of the MTC as they become available and not
necessarily in a strictly ordered pathway. Our results suggest that the
dimer form of MalK is the physiologically relevant species for assembly
in vivo.
The present studies provide a basis for future work in which the
protein domains responsible for dimerization can be determined. Subsequently, the role that dimerization plays in the assembly of the
MTC and in the regulatory roles of MalK can be explored experimentally.
The coimmunoprecipitation techniques can be employed to isolate higher
order assembly intermediates of the MTC and could also be valuable in
the characterization of other systems.