Advanced Method for the In Vivo Measurements of Lysophospholipid Translocation Across the Inner (Cytoplasmic) Membrane of Escherichia coli.

Phospholipid translocation occurs ubiquitously in biological membranes and primarily is protein catalyzed. Lipid flippases mediate the net translocation of specific phospholipids from one leaflet of a membrane to the other. In the inner (cytoplasmic) membrane (IM) of Gram-negative bacteria, lysophospholipid translocase (LplT) and cytosolic bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase (Aas) form a glycerophospholipid regeneration system, which is capable of facilitating rapid retrograde translocation of lyso forms of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) but not exogenous (host-derived) phosphatidylcholine (PC) across the IM of Gram-negative diderm (two-membraned) bacteria in consequential order lyso-PE = lyso-PG > > lysophosphatidic acid (lyso-PA) >> lyso-PC. Although several flippases that bind and move non-glycerophosphatidyl lipids across the IM are characterized in Gram-negative bacteria, LplT appears to be the first example of a bacterial protein capable of facilitating the rapid translocation of monoacylated glycerophospholipids. On the cytoplasmic surface, Aas restores the lysophospholipids to their diacyl forms with comparable efficiency but excludes any exogenous monoacylated lipid species. This coupled remodeling enzyme tandem provides an effective means to examine substrate specificity of lipid regeneration and lysophospholipid transport per se across the membrane. The current chapter describes two distinct but complementary methods for the measurement of lysophospholipid transport across membranes using Escherichia coli spheroplasts.