PMID:9733683
| Citation |
Golby, P, Kelly, DJ, Guest, JR and Andrews, SC (1998) Topological analysis of DcuA, an anaerobic C4-dicarboxylate transporter of Escherichia coli. J. Bacteriol. 180:4821-7 |
|---|---|
| Abstract |
Escherichia coli possesses three independent anaerobic C4-dicarboxylate transport systems encoded by the dcuA, dcuB, and dcuC genes. The dcuA and dcuB genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity. A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S. Six, S. C. Andrews, G. Unden, and J. R. Guest, J. Bacteriol. 176:6470-6478, 1994). These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a beta-lactamase protein lacking the N-terminal signal peptide. The resulting topological model differs from those previously predicted. It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6. The N and C termini are located in the periplasm and the predicted orientation is consistent with the "positive-inside rule." Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region. The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB. Overproduction of DcuA to 15% of inner-membrane protein was obtained with the lacUV5-promoter-based plasmid, pYZ4. |
| Links | |
| Keywords |
Amino Acid Sequence; Anaerobiosis; Bacterial Proteins/analysis; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Carrier Proteins/chemistry; Dicarboxylic Acid Transporters; Dicarboxylic Acids/metabolism; Escherichia coli/chemistry; Escherichia coli Proteins; Molecular Sequence Data; Recombinant Fusion Proteins/biosynthesis; Repressor Proteins; Transcription Factors/analysis; Transcription Factors/chemistry; Transcription Factors/genetics |
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