Key Role of the Diglucosyldiacylglycerol Synthase for the Nonbilayer−Bilayer Lipid Balance of Acholeplasma laidlawii Membranes

Abstract

In the single membrane of Acholeplasma laidlawii, a specific glucosyltransferase (DGlcDAG synthase) synthesizes the major, bilayer-forming lipid diglucosyldiacylglycerol (DGlcDAG) from the preceding major, nonbilayer-prone monoglucosyldiacylglycerol (MGlcDAG). This is crucial for the maintenance of phase equilibria close to a potential bilayer-nonbilayer transition and a nearly constant spontaneous curvature for the membrane bilayer lipid mixture. The glucolipid pathway is also balanced against the phosphatidylglycerol (PG) pathway to maintain a certain lipid surface charge density. The DGlcDAG synthase was purified approximately 5000-fold by three chromatographic techniques and identified as a minor 40 kDa membrane protein. In CHAPS mixed micelles, a cooperative dependence on anionic lipid activators was confirmed, with PG as the best. The dependence of the enzyme on the soluble UDP-glucose substrate followed Michaelis-Menten kinetics, while the kinetics for the other (lipid) substrate MGlcDAG exhibited cooperativity, with Hill coefficients in the range of 3-5. Vmax and the Hill coefficient, but not Km, for the MGlcDAG substrate were increased by increased PG concentrations, but above 3 mol % MGlcDAG, the rate of synthesis was constant. Hence, the DGlcDAG synthase is more affected by the lipid activator than by the lipid substrate at physiological lipid concentrations. The enzyme was shown to be sensitive to curvature "stress" changes, i.e., was stimulated by various nonbilayer lipids but inhibited by certain others. Certain phosphates were also stimulatory. With the two purified MGlcDAG and DGlcDAG synthases reconstituted together in the presence of a potent nonbilayer lipid, the strong responses in the amounts of MGlcDAG and DGlcDAG synthesized mimicked the responses in vivo. This supports the important regulatory functions of these enzymes.

The Nonbilayer/Bilayer Lipid Balance in Membranes

“…Assay for Glucosyltransferase Activity-Assays in mixed micelles were technically performed as described before (11,14). The CHAPS assay buffer contained 18.8 mM CHAPS, 100 mM Tris maleate, pH 8, and 5-20 mM MgCl 2 .…”

“…PG is particularly important because the consecutively acting DGlcDAG synthase is only active, in a cooperative fashion, in the presence of substantial amounts of an activator lipid, especially PG. The nonbilayer/bilayer lipid balance and curvature are more likely regulated by the synthesis of DGlcDAG because this step is strongly stimulated by certain nonbilayer-prone additives in vitro (13,14) but also by "patching" of the activator PG (15). 1 The abbreviations used are: PA, phosphatidic acid; CHAPS, 3-…”

“…the transmembrane electrical potential, the cellular K ϩ and ATP content, and glucose consumption, respectively (16,17). Nucleotide phosphates also affect the two glucosyltransferases differently in vitro (18), and certain soluble phosphates stimulated the purified DGlcDAG synthase (14). Important phosphate molecules in Mycoplasmas are, in addition to RNA, DNA, polyphosphates, and phospholipids (19), also fructose 1,6-bisphosphate (FBP) and ATP (20).…”

“…The presence of substantial amounts of activator PG for DGlcDAG synthesis is crucial in vitro, both with crude cellular proteins and the purified enzyme (13,14). In vivo there is a correlation between these two lipids; (14), and (filled circles) liposome models (13).…”

“…In vivo there is a correlation between these two lipids; (14), and (filled circles) liposome models (13). caused by nonbilayer-prone molecules, like 1,3-DOG (Fig.…”