Biochemistry zyxwvu 1984, 23, zyxwvu 6901-691 1 6901 zyxwvutsrqp Human Erythrocyte Hexose Transporter Activity Is Governed by Bilayer Lipid Composition in Reconstituted Vesicles? A. Carruthers* and D. L. Melchior* ABSTRACT: Purified membrane protein band 4.5 (a sugar transport protein) from human erythrocytes was reconstituted by reverse-phase evaporation into a variety of bilayers formed from the synthetic lecithins. The number of proteins recon- stituted was estimated by determination of D-glucose-sensitive cytochalasin B binding sites. D-Glucose transport activity in reconstituted vesicles was assessed by monitoring cytochalasin B sensitive D-glucose fluxes using microturbidimetric analysis. The major points are as follows: (1) The sugar transport activity of the reconstituted system is directly proportional to the number of cytochalasin B binding sites reconstituted. The ratio of cytochalasin B binding sites per band 4.5 protein is 0.8. These data suggest that the functional protein unit is a monomer. (2) Inhibitor studies of reconstituted hexose transfer support the notion that the kinetics of reconstituted transport are intrinsically symmetric. (3) The turnover number for transport is not consistent with transport proceeding via water-filled pores. (4) The absolute activity of the reconsti- tuted system (V,,,,, per reconstituted cytochalasin B binding protein) is governed by the bulk lipid composition of the synthetic membrane. (5) At temperatures where bilayers formed from dimyristoyl- or dipalmitoyllecithin (DML and DPL, respectively) are “crystalline”, hexose transport activity is not observed. Over the same temperature range, however, A m o n g the integral membrane proteins, many serve as vectorial enzymes catalyzing the transbilayer flux of specific molecules. Kinetic analysis of protein-mediated transport indicates two general classes of transport systems. The pore or channel mechanisms describe substrate flux via presumed membrane-spanning hydrophilic channels. The carrier-type mechanisms describe flux via proteins that undergo confor- mational or positional change during transport (Lieb zyxwvuts & Stein, 1974; Stein & Lieb, 1974). These kinetic schema, for the most part, employ conceptual changes in the protein state, not necessarily the actual physical transformations of the pro- tein(s); hence, their utility in describing the molecular events underlying protein-mediated transport is limited. Such events might be studied more directly by employing physical tech- niques that address the molecular requirements of both the native and reconstituted transport systems. Reconstituting transport systems into bilayers in which variables such as lipid composition and physical state are strictly controlled is an approach that should permit a more complete understanding of both the intrinsic and extrinsic molecular factors involved in protein-mediated transport. At the same time, this approach allows a direct investigation of how the different membrane lipids and their physical states From the Department of Biochemistry, University of Massachusetts Medical School, Worcester, Massachusetts 01605. Receiued April 16, 1984. This work was funded in part by National Institutes of Health Grant P30 AM32520. A.C. gratefully acknowledges the receipt of a Wellcome Trust Travel Award and assistance from N.A.T.O. and the zyxwvut S.E.R.C. of Great Britain. 0006-2960/84/0423-6901$01.50/0 crystalline bilayers formed from the longer chain lecithins, distearoyl-, diarachidonoyl-, and dielaidoyllecithin (DSL, DAL, and DEL, respectively), support significant protein-mediated transport activity. (6) In a given synthetic membrane, the bilayer transition from the liquid-crystalline to the fluid state results in increased protein-mediated sugar transport activity. In the one synthetic membrane (DEL) in which the activation energy (E,) for transport could be measured both above and below the bilayer phase transition, E, was unaffected by the phase change. (7) E, and the Arrhenius constant (A) for transport are dependent on lecithin acyl chain length and saturation. For b9th parameters, the order of increase is DML < DPL = dipalmitoleoyllecithin (DPOL) < DSL < DAL < DEL = dioleoyllecithin (DOL). This means that at 59-60 OC, the order of catalytic activity follows the lipid sequence DML < DPOL < DAL < DOL << DPL = DEL < DSL. (8) Cholesterol (48 mol 9%) restores protein-mediated transport activity to crystalline DPL bilayers and reduces the activity supported by fluid DPL bilayers. This effect is zyx not simply related to the effects of cholesterol on the bilayer partial specific volume. (9) The intuitive parameter “bilayer fluidity” appears to be a relatively unimportant determinant of pro- tein-mediated hexose flux. influence transport processes. For most biomembranes, bilayer lipid may be considered a solvent of integral membrane pro- teins. By analogy with water-soluble enzymes, it would be expected that bilayer protein activity is governed in part by various parameters of its solvent (the bilayer lipid). The membrane transport system chosen for study was the human erythrocyte hexose transporter. This system is par- ticularly well suited to these studies for the following reasons: (1) The transport system is passive and thus is uncomplicated by additional reactions typical of active transport systems such as ATP hydrolysis or coupled cation transport. (2) The ki- netics of the transport system are well-defined. (3) The lipid composition and organization of the red cell membrane have been extensively studied. (4) The transporters are abundant in the red cell membrane and may be readily purified without the additional complications of preexposure of tissue to en- zymes or contamination by proteins from intracellular orga- nelles. (5) The red blood cell is an ideal simple system for studying lipid effects on transport for there is no de novo synthesis of transporter nor any possibility of recruitment of transporter from cytosolic sites. Two red cell membrane proteins (band 4.5 and a component of band 3) appear to catalyze the saturable, selective transbilayer flux of D-glucose in synthetic membranes (Kasahara & Hinkle, 1977; Jones & Nickson, 1981; Shelton & Langdon, 1983; Carruthers & Melchior, 1984). Of these, band 4.5 proteins offer advantages of ease of purification and direct quantitation (Kasahara & Hinkle, 1977; Baldwin et al., 1980). Previous studies on the rat adipocyte hexose transport system reconstituted into syn- zyxwvutsrqp 0 1984 American Chemical Society