INTRODUCTION

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Integral membrane proteins play a central role in the ATP-binding cassette (ABC) transporter superfamily, whose prokaryotic and eukaryotic members traffic a variety of substrates such as ions, sugars, amino acids, peptides, and proteins (15). This large family of transporters is defined by a conserved cytoplasmic ATPase component and integral membrane domains which interact to carry out the specific transport process (4, 15). Among the eukaryotic members are such medically relevant proteins as the P-glycoprotein implicated in multidrug-resistant cancer cells, the cystic fibrosis transmembrane regulator protein, and the human peroxisomal adrenoleukodystrophy protein (2, 34, 35). Among the prokaryotic members of the ABC superfamily are the periplasmic binding protein-dependent transporters. These family members are characterized by a conserved region of the integral membrane component(s) in addition to the conserved cytoplasmic ATPase (4). One member of this prokaryotic subgroup, the maltose transport complex of Escherichia coli, presents a useful model for the integral membrane folding and assembly interactions required for ABC transporters. The maltose transport complex consists of the integral membrane proteins MalF and MalG and a peripheral cytoplasmic membrane ATPase, MalK (reviewed in reference 24). These three proteins copurify (11), forming a MalFGK₂ tetrameric complex which acts in concert with the periplasmic maltose-binding protein (MBP), the product of malE, to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane.

MalF has been shown to have eight transmembrane (TM) domains (5), whereas MalG possesses six TM domains (6, 10). Following independent insertion of these proteins into the membrane (22a, 31), assembly of the MalFGK₂ complex is likely mediated by interactions among discrete domains of MalF, MalG, and MalK, resulting in tetramerization (20, 26).

Although the specifics of these interactions are unknown, a combination of biochemistry and genetics has allowed for a partial characterization of the complex. Shuman and colleagues isolated and characterized MalF and MalG mutants which enable the MalFGK₂ complex to transport maltose in the absence of MBP (7, 32). These analyses have pointed toward a direct interaction between MBP and periplasmic portions of MalG and MalF (16), between MalG and MalF themselves (7), and between MalK and both MalF and MalG (12). Davidson and Nikaido purified the MalFGK₂ complex and demonstrated extensive chemical cross-linking between MalG and MalF and among MalG, MalF, and MalK (11). Traxler and Beckwith observed that periplasmic loops of MalF become protease resistant only in the presence of MalG and MalK, also suggesting that specific interactions occur among the proteins in the context of an assembled complex (31). Finally, a potentially important MalG-MalK protein interaction signal has been identified in the hydrophilic cytoplasmic loop between the fourth and fifth TM domains of MalG (reference 9; Fig. 1). This motif is conserved in MalF and in other binding protein-dependent transporters of the ABC superfamily (9, 28) and has been hypothesized to mediate interactions with the conserved ATPase subunit of the complex (17, 22).

View larger version (62K): [in this window] [in a new window]   FIG. 1.   Topology model of MalG. Hydropathy plots and fusion protein analyses (6, 10) suggest that the N and C termini of the 296-residue protein are cytoplasmically localized. The shaded boxes represent putative TM domains, and the shaded amino acids are conserved in integral membrane proteins of periplasmic binding protein-dependent ABC transporters (9, 28). The location of each 31-residue insertion is shown by an arrowhead. The black arrowhead represents an insertion which did not significantly affect MalG transport function, the gray arrowhead depicts partial transport function, and the white arrowheads represent loss of transport ability for the corresponding insertion mutants. Each numbered disc shows the mutant classification of the adjacent insertion mutant (see Discussion for details).

Recently, a transposon-mediated insertion mutagenesis technique was developed and used to characterize both permissive and nonpermissive regions of the integral membrane protein LacY (19), as well as the cytoplasmic MalK and LacI proteins (18, 23). These analyses not only identified tolerant hydrophilic regions of each protein but also defined several distinct mutant classes (18, 19, 23). In particular, the phenotypes attributable to the lacI insertion mutations that we isolated were strikingly similar to those of previously characterized amino acid substitutions mapping to the same sites in lacI. Here, we describe the results of this insertion mutagenesis on the MalG protein. This analysis provides a unique in vivo view of the requirements for proper MalG protein folding and of the interactions necessary for MalFGK₂ assembly and maltose transport.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The E. coli strains used in this study are described in Table 1, and the plasmids used are described in Table 2. The malG::i31 alleles were crossed from their pmalG plasmids onto DBK261 (14) by homologous recombination between upstream promoter regions and between downstream bla sequences during infections of BT45 containing the various plasmids. Strain BT10 (lacI) was then lysogenized with these malG::i31-transducing phages to create strains BN30 to BN42, which constitutively express the malG alleles (Table 1).

Media and chemicals. The minimal (M63), rich (LB), and MacConkey media used were described previously (19, 21). The following medium supplements were used at the indicated concentrations: sucrose, 5% (wt/vol); maltose, 0.2 or 1% for minimal or MacConkey medium, respectively; glycerol, 0.2% for minimal medium; maltodextrin (from Pfanstiehl), 0.2% for minimal medium (2 mM [final concentration] maltodextrins between maltotetrose and maltoheptose); chloramphenicol, 30 µg/ml; kanamycin, 30 µg/ml; ampicillin, 100, 75, or 25 µg/ml for high-, low-, or single-copy-number conditions, respectively; tetracycline, 15 or 10 µg/ml for high- or low-copy-number conditions, respectively; 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal), 40 µg/ml; 5-bromo-4-chloro-3-indolylphosphate, p-toluidine salt (X-P), 40 µg/ml; and IPTG, 1 mM except where noted otherwise.

DNA techniques. Standard DNA preparations and manipulations were used as previously described (27). To locate the insertion site of a transposon element, plasmid DNA was sequenced by the dideoxynucleotide termination method with Sequenase (United States Biochemical), using double-stranded DNA templates and the TnlacZ-II oligonucleotide primer (19).

Transposon mutagenesis. E. coli strains CC118 and CC191 containing malG on plasmid pBDN4 were infected with a replication-deficient lambda phage carrying the TnlacZ/in or TnphoA/in transposon, using the method developed by Manoil and Bailey (19; see references 18 and 23 for adaptations). Transposition of the ISlacZ/in or ISphoA/in element into pBDN4 was selected on ampicillin- and chloramphenicol-containing medium (with resistances specified by the plasmid and insertion sequence [IS] element, respectively). In-frame transposition events into malG were screened by addition of the color indicator X-Gal or X-P to the selective medium. All but 93 nucleotides from each inserted IS element were then removed by BamHI digestion to create the in-frame 31-codon insertion, shown here in the one-letter amino acid designation: (S, P, A, or T)DSYTQVASWTEPFPFSIQGDPRSDQET(G, A, V, E, or D)XX. The amino acids denoted as X are specified by the two codons directly 5' of each insertion event; these codons are duplicated as part of the transposition event.

Maltose transport assays. Mal phenotypes of the MalG insertion mutants were initially assayed in strain BN20 transformed by plasmids expressing the various malG alleles (Table 2). Colony morphologies of the resulting strains were examined on maltose-MacConkey and maltose minimal medium in the absence of IPTG. In addition, the phenotypes of selected MalG mutants produced by strains BN30 to BN42 were examined in a similar fashion.

Proteolysis assay of MalFGK₂ complex assembly. The ability of each MalG insertion mutant to assemble into a stable MalFGK₂ complex was tested by assaying trypsin sensitivity of MalF in each mutant strain as described previously (18, 31). E. coli BN27 (pcnB) was transformed with the different malG insertion mutant plasmids, pTrc99A or pGAP1 as a negative control and pBDN4 as a positive control. The resulting strains were grown in M63 medium (with glycerol, ampicillin [75 µg/ml], and all amino acids except cysteine and methionine) at 37°C. At an A₆₀₀ of between 0.2 and 0.3, expression of malG was induced in each strain by the addition of 1 mM IPTG for 2 h, whereupon the cells were converted to spheroplasts (31). Strains BN30 to BN42 were examined in a similar fashion, except that 25 µg of ampicillin per ml was used in the M63 medium and no IPTG was added prior to spheroplast conversion. MalF protein in the cell extracts was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by Western blot analysis.

Western blot analysis. Western blot analysis was performed as previously described (23, 31). Protein levels were normalized prior to gel loading by use of the Bio-Rad protein assay, and the MalG mutant proteins were detected with a 1:1,000 dilution of polyclonal antibody specific for the 31-codon insert (19). For Western blot analyses following proteolysis assays, protein levels were normalized according to final A₆₀₀ (BN27-transformed strains) or according to Bio-Rad protein assays (strains BN30 to BN42). MalF protein was detected by a polyclonal antibody specific for MalF (31).

	RESULTS

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Isolation of malG insertion mutations. We wished to investigate the role of various regions of the MalG protein in maltose transport function and in maltose transport complex assembly. As one approach to characterizing MalG, we utilized the TnlacZ/in and TnphoA/in transposon mutagenesis technique as described by Manoil and Bailey (19; see also references 18 and 23) to generate 31-residue insertion mutations in malG. The first step of this transposon mutagenesis involves the recovery of translational malG-lacZ or malG-phoA fusions created from the transposition of the ISlacZ/in or ISphoA/in transposon element, respectively. From approximately 140,000 colonies possessing plasmid-linked ISlacZ/in insertions, we identified about 250 malG-lacZ fusion candidates based on blue colony phenotypes on X-Gal-containing medium. Likewise, from approximately 36,000 colonies possessing plasmid-linked ISphoA/in insertions, we identified about 50 malG-phoA fusion candidates on X-P-containing medium. After analyzing these fusions by restriction enzyme digestion, we sequenced 28 malG-lacZ candidates and 11 malG-phoA candidates and recovered 18 different in-frame fusions to malG. The second step of the transposon mutagenesis, a BamHI-mediated removal of all but 93 nucleotides of the IS element, yielded 18 different 31-codon insertion mutations in malG. Each of these mutations was given an allele number (Table 2); the positions of the 18 insertions as they relate to MalG membrane topology are shown in Fig. 1.

Transport activity of MalG insertion mutants. The transport phenotypes of the 18 insertion mutants were initially characterized on maltose-containing media. When examined in strain BN20, only periplasmic insertion mutant MalG566 retained transport activity similar to that of wild-type MalG. We additionally tested 12 of the MalG insertion mutants at single-copy-number (strains BN30 to BN41, with BN42 as a positive control), since constitutive malG expression in a lacI strain may allow the identification of weakly active mutants. Consistent with this idea, we observed an increase of approximately 1.5- to 2-fold in relative MalG mutant protein levels in the six constitutive malG expression strains tested compared to their uninduced BN20/pmalG counterparts (data not shown).

MalG mutant protein production. We determined the relative protein abundance of the 18 MalG insertion mutants by Western blot analysis using antiserum directed against the IS (Fig. 2). In addition to the MalG insertion mutant band migrating at about 29 kDa, a background band migrating at about 33.3 kDa was detected. Of the 18 MalG insertion mutants, 12, including 2 mutants active in transport and 10 deficient in transport, accumulated protein to significant levels. These results indicate that the loss of transport activity by 10 MalG insertion mutants is not attributable to an absence of MalG mutant protein in the cells. Observed mobility differences among several of the mutant MalG proteins are not attributable to transposon-mediated rearrangements in malG since restriction digest analysis confirmed the expected size and location of each malG insertion (data not shown). Mobility differences have also been observed among similar 31-residue insertion mutants of MalK and LacI (18, 23).

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Western immunoblot analysis of MalG mutant proteins. Proteins from BN20-derived cell extracts were detected by incubation with affinity-purified antibody directed against the 31-residue insert (19). MalG+ (from BN20/pBDN4) served as the negative control since it lacks the insert. The background band of approximately 33 kDa appeared in all lanes. The locations of the size standards, in kilodaltons, are shown on the left.

MalG mutant assembly competence. We wished to ascertain whether individual MalG mutants were compromised in the ability to assemble the MalFGK₂ complex. To this end, we used the MalF protease sensitivity assay as described by Traxler and Beckwith (31). Briefly, periplasmic loops of MalF are cleaved by exogenous proteases such as trypsin in the absence of proper MalFGK₂ complex formation. Upon complex formation, however, MalF becomes resistant to these same proteases. Therefore, by assaying MalF protease susceptibility in the presence of individual MalG mutants, we can gauge the ability of these mutants to form a stable MalFGK₂ complex.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   MalFGK2 assembly competence of selected MalG mutants. The Western blots show the abilities of different MalG insertion mutants to assemble into a MalFGK2 complex in which MalF is protected from trypsin proteolysis. The blots shown are representative of at least three proteolysis assays for each insertion mutant; 10 µg of protein from each of strains BN30 to BN39, BN41, BN42 (malG+), and BT10 [malG(Am)] was loaded per lane. The position of full-length MalF is indicated. The two prominent bands directly below full-length MalF are high-molecular-weight tryptic MalF peptides (31). Numbers above the blots refer to the different malG alleles tested. , no-protease controls; +, 25 µg of trypsin added. A faint band corresponding to intact MalF is observed in the proteolyzed BT10 sample; this low-level protection may be due to incomplete spheroplasting or read-through expression of the chromosomal malG(Am) allele in this background. MalG581 in strain BN40, which is not shown, protected MalF from proteolysis at levels similar to those for MalG582 in BN41.

Mutant MalG-MBP interactions. Shuman and colleagues have identified mutations in malF and malG which allow maltose transport in E. coli cells in the absence of MBP (7, 32). One of these mutant strains, NT205 (malE malF500 malG⁺ malK⁺), grows well on maltose minimal medium but is Mal when transformed with a plasmid producing wild-type MBP (32). This transport block is thought to be due to a nonproductive association between the MBP-independent MalF500GK₂ complex and wild-type MBP (32, 33). We reasoned that if this nonproductive interaction prevents maltose transport, a class of MalG mutant that disrupts the MBP interaction but which retains MalFGK₂ assembly competence and is otherwise active in transport might be detected.

MalG mutant dominance assays. To test the MalG insertion mutants for potential transdominant negative phenotypes, we initially transformed the Mal⁺ strain BT60 with each of the pmalG plasmids and observed the colony phenotypes on MacConkey-maltose agar. All of the mutant malG alleles were recessive to malG⁺, even when their expression was induced with 10 µM IPTG. Further induction of malG allele expression from the high-copy-number plasmids severely impaired colony formation on the agar plates. We additionally examined MalG mutant transdominance in strain NT205, which possesses a 3-log reduction in external maltose-binding affinity compared to a wild-type transport system (32). We reasoned that the decreased transport activity of NT205 might allow us to detect partial dominance effects of MalG mutants not observable in the wild-type background. We transformed NT205 with each of the 12 pmalG plasmids expressing stable MalG protein, plus pmalG567 (low-abundance protein) and pBDN4 (wild-type MalG) as negative controls, and again observed the colony morphologies on maltose-MacConkey medium. In the absence of IPTG, transdominance over malG⁺ was observed only for allele malG571, resulting in a Mal phenotype. In the presence of 10 µM IPTG, however, we observed transdominance over malG⁺ for malG571, malG572, malG577, malG578, malG579, malG581, and malG582 (summarized in Table 3).

	DISCUSSION

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As one step toward a more complete view of the participation of the MalG protein in MalFGK₂ complex assembly and in maltose transport, we have used a transposon-mediated insertion mutagenesis strategy to generate 18 insertion mutations of 31 codons each in the malG gene. A summary of the resulting mutant protein phenotypes is shown in Table 3. We have identified two distinct periplasmic regions of MalG which are mutationally tolerant to various degrees, suggesting that the immediately surrounding regions do not play a vital role in assembly of the MalFGK₂ complex or in the transport process. In addition, we have identified several hydrophilic regions of MalG which are not critical for complex assembly but which are necessary for transport function; this distinction will enable a further examination of transport-specific protein interactions mediated by these regions. In particular, the identification of a MalG periplasmic region which is likely involved in interactions with MBP will allow a more extensive characterization of MalG-MBP interactions. Our insertion mutagenesis results also support the idea that a discrete set of hydrophilic domains of MalG, including portions of the third cytoplasmic and third periplasmic domains, are important for assembly-specific interactions. To facilitate the following discussion, we have grouped the MalG insertion mutants into four classes based on similar phenotypes (Table 3).

Class I mutants define tolerant regions of MalG. Class I mutants MalG566 and MalG578 retain both assembly competence and significant maltose transport function, suggesting that the periplasmic regions of MalG affected by these insertions are surface exposed within the context of the functional complex. The region around residue Glu68 of periplasmic loop 1, where the insertion junction of MalG566 maps, seems to be particularly tolerant of large insertions, as MalG566 retained about 43% of wild-type MalG transport activity at 37°C. MalG578 maps to the third periplasmic loop of MalG after residue Thr244 and possesses partial assembly competence, a low but significant level of transport function (3% of wild-type MalG activity at 37°C), and a temperature-sensitive transport phenotype observed at 30 and 42°C. The temperature-sensitive phenotype seems to be a result of altered transport-specific interactions, since the partial assembly competence of MalG578 (measured by MalF protease protection) was the same at 30, 37, and 42°C in strain BN37 (data not shown). This partial assembly competence is also supported by the observation that malG578 is transdominant to malG⁺ in strain NT205. One possible explanation for these results is that MalG578 is partially defective for transport-specific interactions with MalF.

Class II mutants are impaired in transport-specific interactions. Class II mutants MalG565, MalG571, MalG572, MalG580, MalG581, and MalG582 all possess a Mal phenotype but produce significant levels of protein and protect MalF from proteolysis to various degrees. Therefore, these mutants are specifically defective for maltose transport but retain the ability to assemble the MalFGK₂ complex. MalG571, MalG581, and MalG582, whose insertions map to cytoplasmic regions of MalG, may disrupt transport-specific contacts with MalK or with cytoplasmic regions of MalF. In contrast, insertion mutants MalG565, MalG572, and MalG580 may disrupt transport-specific interactions with periplasmic regions of MalF or with MBP.

+/

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Class III mutants are MalFGK₂ assembly deficient. Class III mutants MalG577 and MalG579, mapping to the third periplasmic loop of MalG (Fig. 1), fail to transport maltose and are defective in complex assembly, as demonstrated by the MalF protease sensitivity assay (Fig. 3). We additionally observed that malG577 and malG579 alleles are transdominant to malG⁺ in strain NT205. These mutant phenotypes may be explained by oligomerization of the mutant MalG proteins with MalK but not with MalF500 (or MalF), thus forming a "dead-end" partial complex and preventing transport. A similar albeit stronger transdominant negative mutant blocking MalFGK₂ assembly was identified previously in MalK (18). We therefore propose that the third periplasmic domain of MalG defined here by insertion mutants MalG577 and MalG579 is a previously unidentified assembly motif, specifying interactions with MalF.

Class IV mutants are likely defective in protein stability. Class IV mutants MalG567, MalG568, MalG569, MalG570, MalG573, and MalG576 displayed a Mal phenotype, produced protein at low or undetectable levels in strain BN20 (Fig. 2), and failed to protect MalF from trypsin proteolysis in strain BN27. As the insertion junctions of these mutants map in or near putative TM domains, their primary defect is likely one of improper intramolecular folding, leading to protein instability and degradation by endogenous cellular proteases (19).