Modulation of dolichyl-phosphomannose synthase activity by changes in the lipid environment of the enzyme

Rat liver dolichyl-phosphomannose synthase (GDP mannose-dolicholphosphate mannosyltransferase; EC 2.4.1.83) was previously shown to catalyze optimal rates of mannosyl transfer to dolichyl-P when the polyprenol acceptor was incorporated into a phosphatidylethanolamine (PE) matrix that has a tendency to adopt a nonbilayer (hexagonal HII) phase [Jensen, J. W., & Schutzbach, J. S. (1985) Eur. J. Biochem. 153, 41-48]. The present investigations now further define the properties of the lipid environment that are essential for mannosyltransferase activity. Monogalactosyl diglyceride (MGDG), a glycoglycerolipid that prefers a nonbilayer-phase organization in isolation, was shown to provide a suitable lipid matrix for synthase activity. By comparison, the enzyme was not activated by digalactosyl diglyceride (DGDG), which forms stable bilayer structures upon hydration. Enzyme activity in MGDG/DGDG mixtures decreased as the proportion of DGDG in the dispersion was increased. Although bilayer-forming phospholipids supported low rates of mannosyl transfer, enzyme activity was stimulated by the addition of MGDG to either phosphatidylcholine (PC) or PE/PC (1:1) membranes. The incorporation of agents known to destabilize bilayer structures including dolichols, ubiquinone, dodecane, and cholesterol into PE/PC (1:1) membranes also increased the rate of mannosyl transfer. Enzyme activity in PC membranes was stimulated by the presence of gramicidin and also by greatly increased concentrations of the substrate, dolichyl-P. The results demonstrate that the enzyme does not have a requirement for PE and suggest that the physical state of the lipid matrix is an important determinant for reconstitution of the synthase and polyprenol phosphate substrate in a productive complex. The formation of an enzyme/lipid complex was demonstrated by sucrose density gradient centrifugation and could be correlated with the lipid requirements for enzyme activity.