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

Abstract

A large family of membrane channel proteins selective for transport of water (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms. Escherichia coli has two members of this family-a water channel, AqpZ, and a glycerol facilitator, GlpF. Despite having similar primary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed. Here we report biochemical and functional characterizations of affinity-purified GlpF and compare it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF) polypeptides encoded by a bicistronic construct were expressed in bacteria. HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetate affinity purification. Sucrose gradient sedimentation analyses showed that the oligomeric state of octyl glucoside-solubilized GlpF varies: low ionic strength favors subunit dissociation, whereas Mg 2؉ stabilizes tetrameric assembly. Reconstitution of affinity-purified GlpF into proteoliposomes increases glycerol permeability more than 100-fold and water permeability up to 10-fold compared with control liposomes. Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibly inhibited by HgCl2. Our studies demonstrate that, unlike AqpZ, a waterselective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB

“…Arrhenius activation energies (E a ) for the three systems were also very comparable (Fig. S7) and in accordance with other studies (27). We conclude that water is conducted neither through the pore of wild type nor through the pore of F107A/F215A AmtB at a rate sufficient to increase measurably the permeability of proteoliposomes.…”

Structural basis of water-specific transport through the AQP1 water channel

“…Both glycerol and water molecules were visualized within the GlpF selectivity filter indicating that at high concentrations of glycerol there should be almost one-to-one transport of glycerol and water. Although GlpF permeability to water is significant, it is substantially less than that measured for its bacterial aquaporin counterpart and AQP1 homologue, AqpZ 22 . Since the constriction region of GlpF is wider than that of AQP1 it would appear that the more hydrophobic nature of GlpF is the primary factor responsible for its relatively reduced ability to transport water.…”

Identity of a Plasmodium lactate/H+ symporter structurally unrelated to human transporters

“…PfFNT was bound to Ni-NTA and eluted using increasing concentrations of imidazole (20-500 mM) in purification buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Brij 78). For proteoliposome preparation 36 E. coli polar lipids (Avanti Polar Lipids) were dissolved in chloroform to 25 mg ml À 1 , evaporated in a nitrogen stream and hydrated in 2 mM 2-mercaptoethanol to a final concentration of 50 mg ml À 1 for 1 h at room temperature. Hundred microlitres of lipid solution plus 10 ml of preparation buffer (20 mM HEPES, pH 6.8) were sonicated for 45 min in a nitrogen atmosphere.…”