RAPID TRANSMEMBRANE MOVEMENT OF NEWLY SYNTHESIZED PHOSPHATIDYLETHANOLAMINE ACROSS THE INNER MEMBRANE OF ESCHERICHIA-COLI

For the first time the transmembrane movement of an endogenously synth esized phospholipid across the inner membrane of E. coli is reported. [C-14]phosphatidylethanolamine (PE) was biosynthetically introduced in to inner membrane vesicles from the PE deficient strain AD93, by recon stitution with the enzyme phosphatidylserine (PS) synthetase, Upon add ition of wild type cell lysate containing PS synthetase, and the metab olic substrates CTP and [C-14]serine to inside-out vesicles from AD93, [C-14]PS was synthesized, which was for the most part converted into [C-14]PE. [C-14]PE was introduced in right-side out vesicles by enclos ing PS synthetase and CTP in the vesicle lumen and adding [C-14]serine . The newly synthesized [C-14]PE immediately equilibrated over both me mbrane leaflets (t(1/2) less than one min), as determined by its acces sibility toward the amino-reactive chemical fluorescamine. In both ins ide-out and right-side out vesicles, a 35-65% distribution was found o f the newly synthesized PE over the cytoplasmic and periplasmic leafle t, respectively, The transport process of PE was not influenced by the presence of ATP or the proton motive force in inside out vesicles. Pr etreatment of both types of vesicles with sulfhydryl reagents, or of r ight-side out vesicles with proteinase K, did not affect the rate and extent of the transmembrane distribution of the newly synthesized PE.