Topological analysis of DctQ, the small integral membrane protein of the C4-dicarboxylate TRAP transporter of Rhodobacter capsulatus.
Tripartite ATP-independent periplasmic ('TRAP') transporters are a novel group of bacterial and archaeal secondary solute uptake systems which possess a periplasmic binding protein, but which are unrelated to ATP-binding cassette (ABC) systems. In addition to the binding protein, TRAP transporters contain two integral membrane proteins or domains, one of which is 40-50 kDa with 12 predicted transmembrane (TM) helices, thought to be the solute import protein, while the other is 20-30 kDa and of unknown function. Using a series of plasmid-encoded beta-lactamase fusions, we have determined the topology of DctQ, the smaller integral membrane protein from the high-affinity C4-dicarboxylate transporter of Rhodobacter capsulatus, which to date is the most extensively characterised TRAP transporter. DctQ was predicted by several topology prediction programmes to have four TM helices with the N- and C-termini located in the cytoplasm. The levels of ampicillin resistance conferred by the fusions when expressed in Escherichia coli were found to correlate with this predicted topology. The data have provided a topological model which can be used to test hypotheses concerning the function of the different regions of DctQ and which can be applied to other members of the DctQ family.
Wyborn
N R
NR
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, S10 2TN, UK.
Alderson
J
J
Andrews
S C
SC
Kelly
D J
DJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003998:Dicarboxylic Acids / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; C108092:DctQ protein, Rhodobacter capsulatus; D003998:Dicarboxylic Acids; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases