Unidirectional Reconstitution into Detergent-destabilized Liposomes of the Purified Lactose Transport System of

Abstract

The lactose transport protein (LacS) of Streptococcus thermophilus was amplified to levels as high as 8 and 30% of total membrane protein in Escherichia coli and S. thermophilus, respectively. In both organisms the protein was functional and the expression levels were highest with the streptococcal lacS promoter. Also a LacS deletion mutant, lacking the carboxyl-terminal regulatory domain, could be amplified to levels >20% of membrane protein. Membranes from S. thermophilus proved to be superior in terms of efficient solubilization and ease and extent of purification of LacS; >95% of LacS was solubilized with relatively low concentrations of Triton X-100, n-octyl-beta-D-glucoside, n-dodecyl-beta-D-maltoside, or C12E8. The LacS protein carrying a poly-histidine tag was purified in large quantities (approximately 5 mg/liter of culture) and with a purity >98% in a two-step process involving nickel chelate affinity and anion exchange chromatography. The membrane reconstitution of LacS was studied systematically by stepwise solubilization of preformed liposomes, prepared from E. coli phospholipid and phosphatidylcholine, and protein incorporation at the different stages of liposome solubilization. The detergents were removed by adsorption onto polystyrene beads and H+-lactose symport and lactose counterflow were measured. Highest transport activities were obtained when Triton X-100 was used throughout the solubilization/purification procedure, whereas activity was lost irreversibly with n-octyl-beta-D-glucoside. For reconstitutions mediated by n-dodecyl-beta-D-maltoside, C12E8, and to a lesser extent Triton X-100, the highest transport activities were obtained when the liposomes were titrated with low amounts of detergent (onset of liposome solubilization). Importantly, under these conditions proteoliposomes were obtained in which LacS was reconstituted in an inside-out orientation, as suggested by the outside labeling of a single cysteine mutant with a membrane impermeable biotin-maleimide. The results are consistent with a mechanism of reconstitution in which the hydrophilic regions of LacS prevent a random insertion of the protein into the membrane. Consistent with the in vivo lactose/galactose exchange catalyzed by the LacS protein, the maximal rate of lactose counterflow was almost 2 orders of magnitude higher than that of H+-lactose symport.

Studies of Cytochrome c Oxidase-Driven H+-Coupled Phosphate Transport Catalyzed by the Saccharomyces cerevisiae Pho84 Permease in Coreconstituted Vesicles

“…Although previous characterization of phosphate transport activity of the purified protein was accomplished by reconstitution into liposomes with the same lipid composition as used in this work, the driving force for Pho84-catalyzed phosphate accumulation was provided by an artificial pH gradient (interior alkaline). Reconstitution of Pho84 into liposomes composed of E. coli phospholipids and egg phosphatidylcholine as previously described for reconstitution of lactose transporter of St. thermophilus (13) and prepared according to the procedure described by Berhe et al (10) resulted in an inactive membrane-bound Pho84 permease. In the study presented here, we have shown that purified cytochome c oxidase from beef heart can be functionally reconstituted into artificial lipid membranes with the lipid composition previously successfully employed for coreconstitution of purified nicotinamide nucleotide transhydrogenase and ATPase from beef heart mitochondria (26), purified beef heart nicotinamide nucleotide transhydrogenase and bacteriorhodopsin from Halobacterium halobium (27), and the Pho84 phosphate permease of S. cereVisiae (10).…”

“…The appropriate amounts of lipids were dried under N 2 (g), redissolved in diethyl ether, washed with ethanol, dried, resuspended to a final concentration of 20 mg/mL in 25 mM K + /Hepes (pH 7.0) and 0.1% Triton X-100, sonicated to clarity, and mixed with purified Pho84 protein at a concentration of 0.14 mg/mL. The protein/lipid mixture was incubated for 30 min at 4°C, after which the detergent was removed by a sequential Biobeads treatment essentially according to the method of Knol et al (13). Fresh Biobeads (80 mg/mL of liposome mixture), extensively washed with methanol and ethanol and finally rinsed in water, were added and incubated at 4°C under slow agitation.…”

“…Reconstitution of active H + -pumping cytochrome c oxidase purified from beef heart mitochondria in liposomes composed of acetone/ether-washed E. coli lipids and L-Rphosphatidylcholine from egg yolk in a ratio of 3:1 (w/w) has previously been shown (15). The same lipid composition was also used in the functional unidirectional reconstitution of the purified lactose transport system (LacS) of Streptococcus thermophilus (13). Although the purified Pho84p could be integrated into the liposomes composed of E. coli lipids and L-R-phosphatidylcholine, the protein was not active in this lipid environment (not shown).…”

Static light scattering to characterize membrane proteins in detergent solution

“…Examples of SEC-LS for three membrane proteins will be given: the glutamate transporter GltT [16], the lactose transporter LacS [17] and the riboflavin transporter RibU [18]. Other examples can be found in references [2,3,7,[19][20][21][22].…”

“…Histidine tagged LacS (A 280 = 1.01 ml mg À1 cm À1 ), GltT (A 280 = 0.62 ml mg À1 cm À1 ) and RibU (A 445 = 0.45 ml mg À1 cm À1 ) were purified as described using Ni-affinity chromatography [16][17][18]. The proteins were further purified using size exclusion chromatography in SEC buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.04% DDM, filtered through 0.1 lm pore size VVLP filters (Millipore)) as described for RibU [18].…”