of the galactose-H+ transport protein [GalP-(Hk)gl of Escherichia coli. Neil M. Sanderson. Giles E.M. Martin, Nicholas G. Rutherford and Peter J.F. Henderson.
Biochemical Society Transactions (1997) 25 471S
Purification, reconstitution and drcular dichroism of the galactose-H+ transport protein [GalP-(Hk)gl of Escherichia coli
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Department of Biochemistry and Molecular Biology, University of Leeds.L.eeds Ls2 9JT, U.K. GalP is an integral membrane protein found in Escherichia coli that catalyzes sugar-H+ symport across the cytoplasmic membrane. GalP shows strong homology to membrane proteins from bacteria, algae, fungi, yeasts, protozoa, plants and mammals. that transport various substrates by both active and facilitative mechanisms [ 11. Structural analysis of this superfamily suggests that these proteins contain 12 hydrophobic membrane-spanning ahelices. The N- and C-termini are predicted to be located on the cytoplasmic side of the membrane, together with a central region of 60-80 amino acids which is also thought to be mostly a-helical. To facilitate purification, GalP has been overexpressed and tagged with hexahistidine in E. coli. The bacterial inner membranes were isolated by explosive decompression of the cells in a French press followed by sucrose density gradient centrifugation. Following solubilization in buffer (pH 8) containing 1 % (wlv) dodecyl maltoside (DDM), the membrane fraction was applied to a nickel-affinity column that was subsequently resuspended in a buffer containing 0.05 % DDM. Bound GalP-(His),5was eluted at pH 4. E. coli lipid extract (Avanti Polar Lipids) was purified by acetondether-washing [2]. Lipid (10 mg) in chloroform was dried under N2 and rehydrated on addition of 1 ml 50 mM potassium phosphate (pH 7.6). 20 mM D-galactose, 1 mM dithiothreitol (Kpi/galactose/DlT) at 40°C. followed by vortexing. This led to the formation of a homogenous population of multilamellar liposomes. These were passed three times through an extruder (Lipex Biomembranes) containing two polycarbonate filters (100 nm pores) at 4OoC to produce unilamellar vesicles with a narrow size distribution. The following steps were all performed at 4OC. The liposomes (1 ml) were treated with 100 pl octyl glucoside (13.6 96) and mixed with 1 ml (0.6 mg) of purified GalP (15 min). The mixture was then diluted with 130 ml KPi/galactose/DTT, centrifuged at 100 OOO x R for 1 h and the supernatant discarded. The peilets were resuspended in a totai volume of 1 ml KPilgalactose/DlT. -Entrance-counterflow assays (Fig. 1) were carried out at 4°C. At zero time, the galactose-loaded proteoliposomes (20 p1 containing 12.6 pg protein) were diluted into a mixture of 940 4 5 0 mM KPi (pH 7.6). 1 mM D l T with 40 pl[3H]-galactose (50 phi, 4 pCi). At each time point, a 90 pl sample was filtered (Millipore GSW filters, pore size 0.22 mm) and washed with 4 ml ice-cold KP,iDlT. Radioactivity appearing in the proteoliposomes retained on the filter was determined by liquid scintillation counting. In the absence of added sugar, uptake of D-galactose by the
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Neil M. Sanderson. Giles E.M.Martin,Nicholas G. Rutherford and Peter J.F. Henderson
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Effect of D- and L-glucose on the CD spectrum of GalP proteoliposomes.
liposomes was rapid, reaching a peak after 5 min. This was followed by slow efflux of the labelled substrate for the remainder of the experiment (90 min). In the presence of 20 mM L-glucose, which is not a GalP substrate, there was no significant difference in the uptake. However, the addition of 20 mM D-glucose, a GalP substrate, significantly reduced the uptake. The counterflow activity was also inhibited by the antibiotics cytochalasin B and forskolin (not shown). The circular dichroism spectrum of GalP proteoliposomes was obtained using a Jasco model J-715 spectropolarimeter at 25OC with constant nitrogen flushing. The samples were analysed in Hellma quartz glass cells of 1 mm path length. The spectrum of GalP proteoliposomes (in 10 mM Kpi. 1 mM DlT) suggests that the protein is predominantly (65+ 5%) helical (Fig. 2). This is indicated by the strong positive band at 193 nm and the negative band at 222 nm. Addition of D-glucose (50 mM) leads to a shift in the spectrum that corresponds to a drop in helical content of about 20 8.This change is not seen when a similar concentration of Lglucose (not a substrate for GalP) is present instead. In both cases, the CD spectra generated by the relevant sugar alone was subtracted from the spectra of the sugar with the proteoliposomes. This specificity perhaps gives an indication of a structural change that GalP undergoes during substrate binding andor transport. These CD results are similar to those found by Chin et af. [3] with the human erythrocyte glucose transporter in liposomes. This work demonstrates that the GalP protein can be purified in a native form that retains activity and secondary structure when purified and reconstituted. Future work with the proteoliposomes will involve electrophysiological methods to characterise the transmembrane electrical gradients generated by GalP when transporting sugar.
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Supported by grants from BBSRC and the Wellcome Trust. We thank Mr. W.J. OReilly for isolation of inner membranes.
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Henderson, P.J.F. (1993) Curr. Opinion. Cell. Biol. 5,70872 1 Newman, M.J. and Wilson, T.H. (1980) J. Biol. Chem.
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Effect of addition of D- or L-glucose on galactosecounterflow activity in GalP proteoliposomes.
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Chin, J.J., Jung. E.K.Y, Chen, V. and Jung. C.Y. (1987) Proc. Natl. Acad. Sci. USA 84.41 13-4116
