Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli

Zhang, Yianbiao; Broome-Smith, Jenny K.

doi:10.1016/0378-1119(90)90340-w

Cited by 39 publications

(30 citation statements)

References 15 publications

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“…This was then linearized with Bgl II, and a 12-mer BamHI linker was inserted, finally the mature blaM gene on a PvuII-EcoRV fragment from pYZ5 (Zhang and Broome-Smith, 1990) was inserted into the BamHI site after treatment with the Klenow fragment of DNA polymerase I. The fasA-blaM junction was confirmed by sequencing, and the fasA-blaM fusion was then cloned into the chloramphenicol-resistant suicide vector pRE112 (R. A.…”

Section: Plasmid Constructionsmentioning

confidence: 99%

Differential regulation of fasA and fasH expression of Escherichia coli 987P fimbriae by environmental cues

Edwards

Schifferli

1997

Molecular Microbiology

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SummaryAn early process in the pathogenesis of enteric bacteria is colonization of the intestinal epithelium leading to local multiplication, pathophysiological interactions with the host and further spreading. Attachment is typically mediated by bacterial fimbriae, which are selectively expressed during growth in the intestine. Here we report an analysis of the regulation of 987P fimbrial expression of enterotoxigenic Escherichia coli (ETEC). Expression of both fasH, the transcriptional activator of the 987P fimbrial genes, and fasA, the major fimbrial subunit, is regulated in response to a variety of environmental stimuli. We have found that expression of fasH is regulated in response to the carbon status of the growth medium by the cAMP-CRP complex. Moreover, fasH is regulated in response to both the nitrogen status of the growth medium and the external pH. Expression of fasA is activated by FasH, and is also selectively regulated in response to growth temperature by HNS. Regulation of fimbrial expression by carbon and/or nitrogen gradients is proposed to provide a mechanism that allows preferential colonization of different segments of the intestine by various enteropathogens, such as ETEC, enteropathogenic E. coli and Vibrio cholerae.

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Section: Plasmid Constructionsmentioning

confidence: 99%

Differential regulation of fasA and fasH expression of Escherichia coli 987P fimbriae by environmental cues

Edwards

Schifferli

1997

Molecular Microbiology

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“…The smaller NcoI/EcoRI fragment (805 bp) containing the native anaerobic promoter was discarded. The 205-bp fragment containing the lac promoter was obtained by a PCR reaction using the DS-18 (atatgaattcGAATTAATTCTCACTCATTAGG)/DS-19 (CTAGAA-GAAGCTTGGGATC) primer pair and pYZ4 DNA (pBR322-derived vector) as a template (22). The PCR product was digested with EcoRI and NcoI and ligated to the gel-eluted 7762-bp fragment to generate pDMS189.…”

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confidence: 99%

Multiple Roles for the Twin Arginine Leader Sequence of Dimethyl Sulfoxide Reductase of Escherichia coli

Damaraju

Turner²,

Simala‐Grant³

et al. 2000

Journal of Biological Chemistry

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Dimethyl sulfoxide (Me 2 SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under anaerobic growth conditions and is required for anaerobic growth with Me 2 SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support growth on glycerol-Me 2 SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membranebound DmsABC with greatly reduced enzyme activity and inefficient anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.

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“…To clarify the transmembrane topology of Tap1 we used an experimental approach, the toplogy reporter-protein system, which has been used extensively to monitor the transmembrane topology of membrane proteins (12), including many from eukaryotic cells (15,(17)(18)(19)(20)(21)(22). Although this approach requires heterologous expression in E. coli, this species has previously been used to express functional eukaryotic transport proteins (23,24), and several eukaryotic polytopic membrane proteins have been shown to fold correctly in the E. coli membrane (19,22).…”

Section: Resultsmentioning

confidence: 99%

Membrane Topology of the ATP-binding Cassette Transporter Associated with Antigen Presentation (Tap1) Expressed in Escherichia coli

Gileadi

Higgins

1997

Journal of Biological Chemistry

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The ATP-binding cassette transporters associated with antigen presentation (Tap1 and Tap2) mediate the transport of peptide fragments across the endoplasmic reticulum membrane of mammalian cells. Tap1 and Tap2 are closely related to one another and are believed to function as a heterodimer. Each protein possesses a hydrophobic domain predicted to span the membrane multiple times and a highly conserved nucleotide-binding domain. We have assessed the transmembrane topology of Tap1 by expressing a series of fusions to a reporter protein, the mature form of ␤-lactamase in Escherichia coli. From these data a topological model can be derived in which Tap1 spans the membrane eight times, with several large loops exposed in the lumen of the endoplasmic reticulum and with both the N and C termini (including the nucelotide-binding domain) residing in the cytoplasm. Cytotoxic T lymphocytes (CTLs)1 continually survey cells for changes in cytosolic content. Antigens from cytoplasmic proteins are presented to the CTLs at the cell surface in the form of peptide fragments complexed with major histocompatibility class I and ␤ 2 -microglobulin molecules (1). These trimeric complexes are recognized by the T cell receptor on the CTLs. To assemble this trimeric complex, the peptide fragments which are normally generated by the proteasome in the cytoplasm, must be translocated into the lumen of the endoplasmic reticulum (ER). Two proteins, Tap1 and Tap2, are required for this transport process (2, 3). Tap1 and Tap2 each consist of a hydrophobic domain predicted to span the membrane multiple times, and an ATP-binding domain, which is believed to couple the energy of ATP hydrolysis to peptide transport. Tap1 and Tap2 function as a heteromer (4 -6) and are members of the ATP-binding cassette (ABC) superfamily of transporters (7).The transmembrane domains of ABC transporters typically (although there are exceptions; see below) consist of 12 clearly defined, putative membrane-spanning segments, which could, potentially, span the lipid bilayer. For a number of ABC transporters, both prokaryotic and eukaryotic, this predicted topology has been confirmed experimentally (8 -10). The N-terminal hydrophobic domains of Tap1 and Tap2 appear to differ from those of other ABC transporters in that the potential membrane-spanning segments are not clearly defined by conventional algorithms and appear to number more than 12 (for the Tap1-Tap2 complex). To clarify this situation we have analyzed the transmembrane topology of the human Tap1 protein using a genetic approach in which a reporter protein (the mature form of ␤-lactamase) was fused to a series of defined points along the length of the Tap1 protein. The orientation of the ␤-lactamase with respect to the membrane was assessed by its ability to confer ampicillin resistance when expressed in Escherichia coli. This approach, and the related phoA method, have been used, successfully, to study many other membrane proteins (11-13). The data generate a model in which Tap1 spans the membrane eight times...

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Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli

Cited by 39 publications

References 15 publications

Differential regulation of fasA and fasH expression of Escherichia coli 987P fimbriae by environmental cues

Differential regulation of fasA and fasH expression of Escherichia coli 987P fimbriae by environmental cues

Multiple Roles for the Twin Arginine Leader Sequence of Dimethyl Sulfoxide Reductase of Escherichia coli

Membrane Topology of the ATP-binding Cassette Transporter Associated with Antigen Presentation (Tap1) Expressed in Escherichia coli

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