Chloroplast membranes contain a substantial excess of the nonbilayer-prone monogalactosyldiacylglycerol (GalDAG) over the biosynthetically consecutive, bilayer-forming digalactosyldiacylglycerol (GalGalDAG), yielding a high membrane curvature stress. During phosphate shortage, plants replace phospholipids with GalGalDAG to rescue phosphate while maintaining membrane homeostasis. Here we investigate how the activity of the corresponding glycosyltransferase (GT) in Arabidopsis thaliana (atDGD2) depends on local bilayer properties by analyzing structural and activity features of recombinant protein. Fold recognition and sequence analyses revealed a two-domain GT-B monotopic structure, present in other plant and bacterial glycolipid GTs, such as the major chloroplast GalGalDAG GT atDGD1. Modeling led to the identification of catalytically important residues in the active site of at-DGD2 by site-directed mutagenesis. The DGD synthases share unique bilayer interface segments containing conserved tryptophan residues that are crucial for activity and for membrane association. More detailed localization studies and liposome binding analyses indicate differentiated anchor and substratebinding functions for these separated enzyme interface regions. Anionic phospholipids, but not curvature-increasing nonbilayer lipids, strongly stimulate enzyme activity. From our studies, we propose a model for bilayer "control" of enzyme activity, where two tryptophan segments act as interface anchor points to keep the substrate region close to the membrane surface. Binding of the acceptor substrate is achieved by interaction of positive charges in a surface cluster of lysines, arginines, and histidines with the surrounding anionic phospholipids. The diminishing phospholipid fraction during phosphate shortage stress will then set the new GalGalDAG/ phospholipid balance by decreasing stimulation of atDGD2.At least 30% of the genes in most cells encode proteins that are associated with the surface or embedded in the lipid matrix of the membranes, either as peripheral or as integral membrane proteins (1). Lipids form a bulk bilayer with certain properties and can interact specifically with sites in certain membrane proteins or complexes, potentially as structural cofactors. Hence, lipids exert a substantial control over the activity of many proteins or assist protein-protein interactions. A key characteristic of plants and photosynthetic bacteria is the high abundance of galactolipids in their membranes harboring the photosynthetic complexes. Analogous glycolipids are major components in many Gram-positive bacteria. Most are uncharged, consisting of a glycerol backbone esterified with two acyl chains and carrying one (monoglycosyldiacylglycerol) or two (diglycosyldiacylglycerol) sugar moieties in the headgroup. They are prone to interact by hydrogen bonding, and pure monoglycosyl-DAGs 3 tend to form nonbilayer aggregates due to their small headgroups, whereas diglycosylDAGs are always bilayer-forming. Their relative amounts will affect sponta...