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The synthesis of Phosphatidylinositol (PI) is limited to the Endoplasmatic Reticulum (ER), which is the largest membrane componement of the cell. This site also contributes the synthesis to the majority of phospholipids, namely phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and triacylglycerol (TG). The synthesis involves a series of enzymatic reactions.

The biosynthesis and phosphorylation of PI is mainly confined to the cytosolic facing surface of organelles by already residential kinases, but not at the ER spesifically. De novo PI synthesis of PI starts with an enzymatic process where phosphatidic acid (PA) is converted into the intermediate CDP- diacylglycerol (CDP-DG) by a process called CDP-diaglycerol synthase. This synthesis is catalyzed by the use of CDS1 and CDS2, CDS- enzymes. In the final enzymatic process, CDP-DG and inositol are catalyzed by the enzyme PI synthase (PIS) and synthesised into PI.