TOPOLOGICAL ANALYSIS OF DCUA, AN ANAEROBIC C-4-DICARBOXYLATE TRANSPORTER OF ESCHERICHIA-COLI

Escherichia coil possesses three independent anaerobic C-4-dicarboxyla te transport systems encoded by the dcuA, dcuB, and dcuC genes. The dc uA and dcuB genes encode related integral inner-membrane proteins, Dcu A and DcuB (433 and 446 amino acid residues), which have 36% amino aci d sequence identity. A previous amino acid sequence-based analysis pre dicted that DcuA and DcuB contain either 12 or 14 transmembrane helice s, 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 analyzi ng 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a beta-lactamase protein lacking the N-terminal signal pe ptide. The resulting topological model differs from those previously p redicted. It has just 10 transmembrane helices and a central, 80-resid ue cytoplasmic loop between helices 5 and 6. The N and C termini are l ocated in the periplasm and the predicted orientation is consistent wi th the ''positive-inside rule.'' Two highly hydrophobic segments are n ot 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 obt ained with the lacUV5-promoter-based plasmid, pYZ4.