Oligomeric Structure and Functional Characterization of the Urea Transporter from Actinobacillus pleuropneunomiae

摘要

Urea transporters facilitate urea permeation across cell membranes in prokaryotes and eukaryotes. Bacteria use urea either as a means to survive in acidic environments and/or as a nitrogen source. The urea transporter ApUT from Actinobacillus pleuropneumoniae, the pathogen that causes porcine pleurisy and pneumonia, was expressed in E. 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. To determine the urea transport kinetics of ApUT, purified protein was reconstituted into proteoliposomes, and urea efflux was measured by stopped-flow fluorometry. 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 urea transporter family.