Phospholipid Synthesis and Exchange in Castor Bean Endosperm Homogenates

Abstract

Crude organelle preparations from castor bean (Ricinus communis L.) endosperm rapidly incorporate CDP-((14)C)choline and CDP-((14)C)-ethanolamine into phosphatidylcholine and phosphatidylethanolamine, respectively. Separation of organelles by sucrose density gradient centrifugation following incubation with these substrates demonstrated that most of the (14)C phospholipids thus formed were present in the endoplasmic reticulum membranes, although label was also found in mitochondria, proplastids, and glyoxysomes. The phospholipid-synthesizing enzymes, cholinephosphotransferase and ethanolaminephosphotransferase, are exclusively confined to the endoplasmic reticulum membrane fraction, suggesting that the appearance of (14)C-phospholipid in other organelles was due to phospholipid exchange. Phospholipid synthesis was inhibited by the cytoplasmic supernatant fraction. The active inhibitor in this fraction was not identified, but the inhibition was not significantly relieved by either dialyzing or boiling the supernatant. Phosphatidylcholine synthesis showed an absolute requirement for Mg(2+); the Michaelis constant was 1 mm. Ca(2+) was a potent inhibitor of Mg(2+)-stimulated phospholipid synthesis and enhanced the decay of (14)C-phospholipids from pre-labeled membranes, particularly when the membranes were resuspended in the cytoplasmic supernatant.The data are consistent with the concept that the endoplasmic reticulum is a major site of membrane proliferation where structural lipids, and possibly proteins, are inserted into, and thus expand, a pre-existing membrane fraction. Other organelle and cellular membranes could therefore originate from the proliferating endoplasmic reticulum by a process of membrane flow and differentiation.