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Selected Publications with Abstracts

Author: Kennedy-K. Traxler-B.
Title: MalK forms a dimer independent of its assembly into the MalFGK2 ATP-binding cassette transporter of Escherichia coli.
Source: Journal of Biological Chemistry. 274(10): 6259-6264, 05 Mar 1999.
The maltose transport complex (MTC) is a member of the ATP-binding cassette superfamily of membrane transport proteins and is a model for understanding the folding and assembly of hetero-oligomeric membrane protein complexes. The MTC is made up of two integral membrane proteins, MalF and MalG, and a peripheral membrane protein, MalK. These proteins associate with a stoichiometry of 1:1:2 to form the complex MalFGK2. In our studies of the oligomerization of this complex, we have shown that the ATP-binding component, MalK, forms a dimer in the absence of MalF and MalG. Epitope-tagged MalK coimmunoprecipitated with wild-type MalK, indicating that the MalK protein forms an oligomer. The relative amounts of tagged and wild-type MalK that were present in the whole cell extracts and in the immunoprecipitated complexes show that the MalK oligomer is a dimer. These hetero-oligomers can also be formed in vitro by mixing two extracts, each containing either tagged or wild-type MalK. The dimerization of MalK was also demonstrated in vivo using the bacteriophage lambda repressor fusion assay. The formation of a MalK dimer in the absence of MalF and MalG may represent an initial step in the assembly pathway of the MTC.

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Author: Nelson-B-D. Traxler-B.
Title:Exploring the role of integral membrane proteins in ATP-binding cassette transporters: analysis of a collection of MalG insertion mutants.
Source: Journal of Bacteriology. 180(9): 2507-14, 1998 May.
The maltose transport complex of Escherichia coli is a well-studied example of an ATP-binding cassette transporter. The complex, containing one copy each of the integral membrane proteins MalG and MalF and two copies of the peripheral cytoplasmic membrane protein MalK, interacts with the periplasmic maltose-binding protein to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane. To investigate the role of MalG both in MalFGK2 assembly interactions and in subsequent transport interactions, we isolated and characterized 18 different MalG mutants, each containing a 31-residue insertion in the protein. Eight insertions mapping to distinct hydrophilic regions of MalG permitted either assembly or both assembly and transport interactions to occur. In particular, we isolated two insertions mapping to extracytoplasmic (periplasmic) regions of MalG which preserved both assembly and transport abilities, suggesting that these are permissive sites in the protein. Another periplasmic insertion seems to affect only transport-specific interactions between MalG and maltose-binding protein, defining a novel class of MalG mutants. Finally, four MalG mutant proteins, although stably expressed, are unable to assemble into the MalFGK2 complex. These mutants contain insertions in only two different hydrophilic regions of MalG, consistent with the notion that a restricted number of domains in this protein are critical complex assembly determinants. These MalG mutants will allow us to further explore the intermolecular interactions of this model transporter.

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Author: Nelson-B-D. Manoil-C. Traxler-B.
Title: Insertion mutagenesis of the lac repressor and its implications for structure-function analysis.
Source: J-Bacteriol. 1997 Jun. 179(11). P 3721-8.
Journal Title: JOURNAL OF BACTERIOLOGY.
Abstract: We recently developed a simple technique for the generation of relatively large (31-codon) insertion mutations in cloned genes. To test whether the analysis of such mutations could provide insight into structure-function relationships in proteins, we examined a set of insertion mutants of the Escherichia coli lac repressor (LacI). Representatives of several LacI mutant classes were recovered, including mutants which exhibit fully active, inducer-insensitive, or weak dominant-negative phenotypes. The various properties of the recovered mutants agree with previous biophysical, biochemical, and genetic data for the protein. In particular, the results support the prior designation of mutationally tolerant spacer regions of LacI as well as proposed differences in dimerization interactions among regions of the protein core domain. These findings suggest that the analysis of 31-codon insertion mutations may provide a simple approach for characterizing structure-function relationships in proteins for which high- resolution structures are not available.

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Author: Lippincott-J. Traxler-B.
Title: MalFGK complex assembly and transport and regulatory characteristics of MalK insertion mutants.
Source: J-Bacteriol. 1997 Feb. 179(4). P 1337-43.
Journal Title: JOURNAL OF BACTERIOLOGY.
Abstract: MalK is a peripheral cytoplasmic membrane protein that has multiple activities in Escherichia coli. It associates with integral cytoplasmic membrane proteins MalF and MalG to form the maltose transport complex (MalFGK), a member of the ATP- binding cassette (ABC) superfamily of proteins. In addition, MalK participates in two different regulatory pathways which modulate mal gene expression and MalFGK transport activity. We have created a set of malK mutations for analysis of the protein's structure and folding. These mutations, distributed throughout malK, are all similar insertions of 31 codons. The ability of each mutant to function in maltose transport and MalK-dependent regulation was characterized. Furthermore, we have exploited a sensitive biochemical assay to classify our MalK insertion mutants into two additional categories: MalFGK complex assembly proficient and complex assembly defective. The regions containing the insertions in the assembly-proficient class should correspond to areas within MalK that are surface exposed within the MalFGK complex. Affected regions in assembly-deficient mutants may be involved in critical structural contacts within the complex. One mutant apparently blocks assembly at an intermediate stage prior to oligomerization of the final MalFGK complex. This work contributes to the analysis of ABC transport proteins and to the study of the assembly process for hetero-oligomeric membrane proteins.
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Author: Manoil-C. Traxler-B.
Title: Membrane protein assembly: genetic, evolutionary and medical perspectives.
Source: Annu-Rev-Genet. 1995. 29. P 131-50.
Journal Title: ANNUAL REVIEW OF GENETICS.
Abstract: Lipid bilayers are delicate structures that are easily disrupted by a variety of amphipathic molecules. Yet the viability of a cell requires the continued assembly of large amphipathic proteins within its membranes without damage. The need to minimize bilayer disruption may account for a number of fundamental features of membrane protein assembly. These include the use of redundant sequence information to establish the topologies and folded structures of membrane proteins, and the existence of efficient mechanisms to rid cells of misassembled proteins. Most missense mutations that inactivate a membrane protein probably do so by altering the folding of the membrane-inserted structure rather than by rearranging the topology or by changing key residues involved directly in function. Such misfolded membrane proteins may be toxic to cells if they escape cellular safeguards. This toxicity may underlie some human degenerative diseases due to mutant membrane proteins.
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Author: Traxler-B. Murphy-C.
Title: Insertion of the polytopic membrane protein MalF is dependent on the bacterial secretion machinery.
Source: J-Biol-Chem. 1996 May 24. 271(21). P 12394-400.
Journal Title: JOURNAL OF BIOLOGICAL CHEMISTRY.
Abstract: We examined the dependence of protein export and membrane protein insertion on SecE and SecA, two components of the secretion (Sec) apparatus of Escherichia coli. The magnitude of the secretion defect observed for signal sequence-containing proteins in cells depleted of SecE is larger and more general than that in many temperature- or cold-sensitive Sec mutants. In addition, we show that the proper insertion of the polytopic MalF protein (synthesized without a signal sequence) into the cytoplasmic membrane is also SecE-dependent. In contrast to an earlier study (McGovern, K., and Beckwith, J. (1991) J. Biol. Chem. 266, 20870-20876), the membrane insertion of MalF also is inhibited by treatment of cells with sodium azide, a potent inhibitor of SecA. Therefore, our data strongly suggest that the cytoplasmic membrane insertion of MalF is dependent on the same cellular machinery as is involved in the export of signal sequence-containing proteins. We propose that the mechanism of export from the cytoplasm is related for both signal sequence- containing and cytoplasmic membrane proteins, but hydrophobic membrane proteins such as MalF may have a higher affinity for the Sec apparatus.
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Author: Traxler-B. Boyd-D. Beckwith-J.
Title: The topological analysis of integral cytoplasmic membrane proteins.
Source: J-Membr-Biol. 1993 Feb. 132(1). P 1-11.
Journal Title: JOURNAL OF MEMBRANE BIOLOGY.
Abstract: We review three general approaches to determining the topology of integral cytoplasmic membrane proteins. (i) Inspection of the amino acid sequence and use of algorithms to predict membrane spanning segments allows the construction of topological models. For many proteins, the mere identification of such segments and an analysis of the distribution of basic amino acids in hydrophilic domains leads to correct structure predictions. For others, additional factors must come into play in determining topology. (ii) Gene fusion analysis of membrane proteins, in many cases, leads to complete topological models. Such analyses have been carried out in both bacteria and in the yeast Saccharomyces cerevisiae. Conflicts between results from gene fusion analysis and other approaches can be used to explore details of the process of membrane protein assembly. For instance, anomalies in gene fusion studies contributed evidence for the important role of basic amino acids in determining topology. (iii) Biochemical probes and the site of natural biochemical modifications of membrane proteins give information on their topology. Chemical modifiers, proteases and antibodies made to different domains of a membrane protein can identify which segments of the protein are in the cytoplasm and which are on the extracytoplasmic side of the membrane. Sites of such modifications as glycosylation and phosphorylation help to specify the location of particular hydrophilic domains. The advantages and limitations of these methods are discussed.
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Author: Dash-P-K. Traxler-B-A. Panicker-M-M. Hackney-D-D. Minkley-E-G-Jr.
Title: Biochemical characterization of Escherichia coli DNA helicase I.
Source: Mol-Microbiol. 1992 May. 6(9). P 1163-72.
Journal Title: MOLECULAR MICROBIOLOGY.
Abstract: The gene product of F tral is a bifunctional protein which nicks and unwinds the F plasmid during conjugal DNA transfer. Further biochemical characterization of the Tral protein reveals that it has a second, much lower, Km for ATP hydrolysis, in addition to that previously identified. Measurement of the single-stranded DNA-stimulated ATPase rate indicates that there is co-operative interaction between the enzyme monomers for maximal activity. Furthermore, 18O-exchange experiments indicate that Tral protein hydrolyses ATP with, at most, a low-level reversal of the hydrolytic step during each turnover.
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Author: Traxler-B. Beckwith-J.
Title: Assembly of a hetero-oligomeric membrane protein complex.
Source: Proc-Natl-Acad-Sci-U-S-A. 1992 Nov 15. 89(22). P 10852-6.
Journal Title: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA.
Abstract: The maltose transporter of Escherichia coli is a hetero- oligomeric complex located in the cytoplasmic membrane of the cell. The in vivo assembly of this complex has been examined by using an assay based on the proteolytic sensitivity of one of its components, MalF. Immediately after synthesis and insertion into the membrane, MalF is sensitive to exogenously added proteases. In a time- and complex assembly-dependent fashion, MalF becomes protease resistant. Using this assay, we show that MalF is inserted into the membrane independently of other components of the transport complex. The assembly of the maltose transport complex occurs subsequently from a pool of freely diffusing protein in the membrane. This assembly process is efficient and occurs with rapid kinetics.
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Author: Traxler-B. Lee-C. Boyd-D. Beckwith-J.
Title: The dynamics of assembly of a cytoplasmic membrane protein in Escherichia coli.
Source: J-Biol-Chem. 1992 Mar 15. 267(8). P 5339-45.
Journal Title: JOURNAL OF BIOLOGICAL CHEMISTRY.
Abstract: The topology of integral cytoplasmic membrane proteins can be analyzed using alkaline phosphatase fusions by determining which constructs have low and which have high specific activity. We show that in all cases the enzymatic activity is due to the fraction of the alkaline phosphatase moiety of the fusion protein localized to the periplasm. We present evidence that these fusions can also be used to analyze the process of assembly of cytoplasmic proteins into the membrane. The rate of acquisition of protease resistance of the alkaline phosphatase moiety of such hybrid proteins is compared for fusions to periplasmic and cytoplasmic domains. We show that this process, which is assumed to be representative of export of alkaline phosphatase, is significantly slower for fusions to cytoplasmic and certain periplasmic domains than for most periplasmic domains. These results are discussed in the context of the normal assembly of integral membrane proteins.
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Author: Bradshaw-H-D-Jr. Traxler-B-A. Minkley-E-G-Jr. Nester- E-W. Gordon-M-P.
Title: Nucleotide sequence of the traI (helicase I) gene from the sex factor F.
Source: J-Bacteriol. 1990 Jul. 172(7). P 4127-31.
Journal Title: JOURNAL OF BACTERIOLOGY.
Abstract: A 6.9-kilobase region of the Escherichia coli F plasmid containing the 3' half of the traD gene and the entire traI gene (encodes the TraI protein, DNA helicase I and TraI, a polypeptide arising from an internal in-frame translational start in traI) has been sequenced. A previously unidentified open reading frame (tentatively trbH) lies between traD and traI.
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Author: Traxler-B-A. Minkley-E-G-Jr.
Title: Evidence that DNA helicase I and oriT site-specific nicking are both functions of the F TraI protein.
Source: J-Mol-Biol. 1988 Nov 5. 204(1). P 205-9.
Journal Title: JOURNAL OF MOLECULAR BIOLOGY.
Abstract: Site-specific and strand-specific nicking at the origin of transfer (oriT) of the F sex factor is the initial step in conjugal DNA metabolism. Then, DNA helicase I, the product of the traI gene, processively unwinds the plasmid from the nick site to generate the single strand of DNA that is transferred to the recipient. The nick at oriT is produced by the combined action of two Tra proteins, TraY and TraZ. The traZ gene was never precisely mapped, as no available point mutation uniquely affected TraZ-dependent oriT nicking. With several new mutations, we have demonstrated that TraZ activity is dependent upon traI DNA sequences. The simplest interpretation of this finding is that the F TraI protein is bifunctional, with DNA unwinding and site-specific DNA nicking activities.
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