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 HgCl(2). Our studies demonstrate that, unlike AqpZ, a water-selective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.

Figure 1

SDS/PAGE and velocity sedimentation analyses of His-GlpF and His-AqpZ. (A) Protein samples (≈1 μg) in 1% SDS and 140 mM 2-mercaptoethanol were analyzed with 10–20% acrylamide gradient gel slabs and stained with Coomassie brilliant blue (34). AqpZ samples were previously acidified with HCl and neutralized to disrupt the SDS-stable tetramer (1). (B) Membranes from transformed bacteria were solubilized in OG, layered over a 5–20% continuous sucrose gradient, and sedimented at 140,000 × g for 18 hr at 4°C. Twenty fractions were collected; top of gradient is on the left. Mobility of His-GlpF or His-AqpZ was determined by immunoblotting.

Figure 2

Interaction between His-GlpF and HA-GlpF. Membranes from bacteria expressing His-GlpF, HA-GlpF, or both were extracted in 3% OG and incubated for 1 hr at 4°C. The solutions were then mixed with Ni-NTA magnetic beads and eluted with imidazole. (Note that only His-GlpF or proteins associated with His-GlpF will adsorb to Ni-NTA beads.) The eluates were analyzed by SDS/PAGE gels stained with Coomassie brilliant blue (CBB) or probed with monoclonal antibodies to His (α-His) or HA (α-HA). (A) Membranes from a bacterial culture coexpressing His-GlpF and HA-GlpF from a bicistronic operon were adsorbed to Ni-NTA and eluted. (B) Membranes from a bacterial culture expressing His-GlpF and from a second culture expressing HA-GlpF were mixed before adsorption to Ni-NTA and elution.

Figure 3

Velocity sedimentation analysis of purified His-GlpF. OG-solubilized, affinity-purified His-GlpF was sedimented at 140,000 × g, for 18 hr at 20°C on a continuous gradient containing 5–20% sucrose. The gradient contained 20 mM Tris⋅HCl (pH 8.0), 5 mM EDTA, and 3% OG. (A) The sedimentation coefficient of GlpF was determined by comparison with multiple water-soluble standards. (B) Mobility of purified GlpF was determined in gradients also containing no additional salt, 300 mM NaCl, or 300 mM MgCl₂, or in 0.1% SDS. Twenty fractions were collected; top of gradient is on the left. Mobility of His-GlpF was determined by immunoblotting. (C) Fractions 6 and 11 from a gradient containing OG and 300 mM NaCl were analyzed by immunoblotting with anti-His or anti-HA monoclonal antibodies before (a) and after (b) a second affinity purification using Ni-NTA-agarose magnetic beads.

Figure 4

Functional reconstitution of glycerol transport activity in GlpF and AqpZ + GlpF proteoliposomes. GlpF (100 μg), AqpZ + GlpF (100 μg each), or no protein was mixed with pure phospholipid, and proteoliposomes were formed and equilibrated with glycerol (855 mosM). A concentration gradient for glycerol was then imposed by rapidly mixing 100 μl of the equilibrated proteoliposome suspension (1 μg of protein) with an equal volume of isoosmotic solution containing sucrose as a nonpermeant osmolyte in a stopped-flow apparatus. Release of glycerol from the proteoliposomes creates an osmotic gradient producing water efflux, and vesicle shrinkage was measured by light scattering. The average kinetics of 5–10 measurements were normalized and fitted to a characteristic single-order exponential equation dependent on the time course of glycerol efflux. Notice the different time scales in plots.

Figure 5

Functional reconstitution of water transport activity in GlpF and AqpZ proteoliposomes. GlpF, AqpZ, or no protein was mixed with pure phospholipid and proteoliposomes were formed. A 2-fold osmotic gradient was imposed by rapidly mixing 100 μl of proteoliposome suspension (1 μg of protein) with an equal volume of hyperosmotic solution containing sucrose as a nonpermeant osmolyte in a stopped-flow apparatus. Water efflux causes vesicle shrinkage, which was measured by light scattering. The average kinetics of 5–10 measurements was normalized and fitted to a characteristic single-order exponential equation dependent on the time course of water efflux. Notice the different time scales in plots.