Effects of bilayer cholesterol on human erythrocyte hexose transport protein activity in synthetic lecithin bilayers

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Abstract
This study describes the effects of altered bilayer cholesterol content on reconstituted, protein-mediated sugar transport. The system used was the human erythrocyte sugar transporter (band 4.5) reconstituted into the bilayers of large unilamellar vesicles. Vesicle preparations were formed from synthetic lecithins whose bilayer cholesterol content ranged from 0-50 mol%. Transport was measured by microturbidimetric analysis over the temperature range of 0-65.degree. C while bilayer physical state was characterized by differential scanning calorimetry. Reconstituted transport activity was irreversibly lost between 62 and 65.degree. C. The Km for reconstituted transport was found to increase only slightly with increasing temperature and was not systematically affected by bilayer cholesterol content. The most striking observation of this study is that over certain critical cholesterol concentrations, as little as a 2.5% change in bilayer cholesterol can result in as much as a 100-fold change in Vmax per reconstituted protein. These findings run counter to the view that increasing bilayer cholesterol content monotonically transforms a membrane into a state of intermediate fluidity. Abrupt, cholesterol-induced bilayer reorganizations occurring at 15-20 and 30 mol% bilayer cholesterol are markedly reflected in altered sugar transport rates. Increasing the cholesterol content of crystalline distearoyllecithin bilayers inhibits the activity of the reconstituted transporter. It is apparent from these studies that bilayer fluidity is neither the sole nor a major determinant of the transport activity is independent of its ability to fluidize membranes. This is in agreement with a previous study in which bilayer lipid composition was shown to be a more important determinant of reconstituted transport rates than bilayer fluidity. As in the previous study, the activity of the sugar transporter is governed by the bulk properties of the membrane and not by features of a physically or compositionally distinct hypothetical boundary or annular lipid. In accord with other recent findings, these studies illustrate the potential for control of membrane transport protein activity by small changes in membrane lipid composition.