Oligomeric structure and functional characterization of the urea transporter from Actinobacillus pleuropneumoniae.
Urea transporters (UTs) facilitate urea permeation across cell membranes in prokaryotes and eukaryotes. Bacteria use urea as a means to survive in acidic environments and/or as a nitrogen source. The UT from Actinobacillus pleuropneumoniae, ApUT, the pathogen that causes porcine pleurisy and pneumonia, was expressed in Escherichia coli and purified. Analysis of the recombinant protein using cross-linking and blue-native gel electrophoresis established that ApUT is a dimer in detergent solution. Purified protein was reconstituted into proteoliposomes and urea efflux was measured by stopped-flow fluorometry to determine the urea transport kinetics of ApUT. The measured urea flux was saturable, could be inhibited by phloretin, and was not affected by pH. Two-dimensional crystals of the biologically active ApUT show that it is also dimeric in a lipid membrane and provide the first structural information on a member of the UT family.
Raunser
Stefan
S
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Mathai
John C
JC
Abeyrathne
Priyanka D
PD
Rice
Amanda J
AJ
Zeidel
Mark L
ML
Walz
Thomas
T
D016977:Actinobacillus pleuropneumoniae / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003460:Crystallization; D003902:Detergents / Q000737:chemistry; D019281:Dimerization; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010539:Permeability; D020836:Protein Structure, Quaternary; D013552:Swine; D014508:Urea / Q000378:metabolism; D000096982:Urea Transporters
D001426:Bacterial Proteins; D003902:Detergents; D026901:Membrane Transport Proteins; D014508:Urea; D000096982:Urea Transporters