Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli

A large family of membrane channel proteins selective for transport of wate r (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms. Escherichia coli has two members of this family-a wate r channel, AqpZ, and a glycerol facilitator, GlpF. Despite having similar p rimary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed. Here we report bioche mical and functional characterizations of affinity-purified GlpF and compar e it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF ) polypeptides encoded by a bicistronic construct were expressed in bacteri a. HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetat e affinity purification. Sucrose gradient sedimentation analyses showed tha t the oligomeric state of octyl glucoside-solubilized GlpF varies: low ioni c strength favors subunit dissociation, whereas Mg2+ stabilizes tetrameric assembly. Reconstitution of affinity-purified GlpF into proteoliposomes inc reases glycerol permeability more than 100-fold and water permeability up t o 10-fold compared with control liposomes. Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibl y inhibited by HgCl2. Our studies demonstrate that, unlike AqpZ, a water-se lective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.