Topological analysis of DcuA, an anaerobic C4-dicarboxylate transporter of Escherichia coli.
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.
Golby
P
P
School of Animal and Microbial Sciences, University of Reading, Reading RG6 6AJ, United Kingdom.
Kelly
D J
DJ
Guest
J R
JR
Andrews
S C
SC
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry*; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins; D014157:Transcription Factors / Q000032:analysis / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; C414535:DcuA dicarboxylate transporter, bacteria; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids; D029968:Escherichia coli Proteins; C084955:PdhR protein, E coli; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors