Abstract
1,2-Diacylglycerol 3-glucosyltransferase synthesizes the major nonbilayer-prone lipid monoglucosyldiacylglycerol (MGlcDAG) in the membrane of Acholeplasma laidlawii, which is important for the spontaneous curvature, and is a regulatory site for the lipid surface charge density. A potential connection between activity and a conformational change of this enzyme, governed by essential lipid activators, was studied with purified MGlcDAG synthase in different lipid aggregates. Critical fractions of anionic phospholipids 1, 2-dioleoyl-phosphatidylglycerol (DOPG) and 1,2-dioleoyl-phosphatidylserine (DOPS) were essential for the restoration of enzyme activity, while the zwitterionic 1,2-dioleoyl-phosphatidylcholine (DOPC) and the uncharged diglucosyldiacylglycerol (DGlcDAG) were not. Proteolytic resistance had a very good correlation with the enzyme activity in various lipid-CHAPS mixed micelles. Anionic lipids DOPG and DOPS could protect the exposed MGlcDAG synthase from digestion, whereas DOPC and DGlcDAG could not. Similar features were observed in liposome bilayers. Likewise, the detergent dodecylphosphoglycerol (PGD), with a phosphatidylglycerol-like headgroup, could also stimulate the MGlcDAG synthase activity efficiently with a concomitant protection toward proteolytic digestion. Neither proteolytic resistance nor restored enzyme activity was observed using soluble glycerol 3-phosphate. It is concluded that in addition to critical amounts, both the negatively charged headgroup and hydrophobic chains of the activator amphiphiles, but not a certain aggregate curvature, seem necessary for a proper conformation and the resulting active state of the MGlcDAG synthase.
Highlights
The roles of native lipids are more and more evident in the function of membrane proteins, especially the influence of such lipids on protein conformation and folding state (e.g. 1, 2)
In Acholeplasma laidlawii membranes, the major phospholipids and glucolipids are extensively regulated under a variety of conditions to maintain certain properties like (i) a constant surface charge density [3], (ii) a nearly constant spontaneous curvature [4], and (iii) phase equilibria close to a potential bilayer to non-bilayer transition [5]
MGlcDAG, a major nonbilayer-forming lipid synthesized from DAG, is the first glucolipid in the glucolipid pathway; this is catalyzed by 1,2-diacylglycerol 3-glucosyltransferase (MGlcDAG synthase)
Summary
The roles of native lipids are more and more evident in the function of membrane proteins, especially the influence of such lipids on protein conformation and folding state (e.g. 1, 2). Proteolytic resistance had a very good correlation with the enzyme activity in various lipid-CHAPS mixed micelles. Anionic lipids DOPG and DOPS could protect the exposed MGlcDAG synthase from digestion, whereas DOPC and DGlcDAG could not.
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