Molecular Species of Phosphatidylinositol-Cycle Intermediates in the Endoplasmic Reticulum and Plasma Membrane

摘要

Phosphatidylinositol (PI) turnover is a process requiring both the plasma and ER membranes.Wenhave determined the distribution of phosphatidic acid (PA) and PI and their acyl chain compositions in thesentwo subcellular membranes using mass spectrometry. We assessed the role of PI cycling in determining thenmolecular species and quantity of these lipids by comparing the compositions of the two membranes isolatednfrom embryonic fibroblasts obtained from diacylglycerol kinase ε (DGKε) knockout (KO) and wild-typen(WT) mice. In the KO cells, the conversion of arachidonoyl-rich DAG to PA is blocked by the absence ofnDGKε, resulting in a reduction in the rate of PI cycling. The acyl chain composition is very similar for PI andnPA in the endoplasmic reticulum (ER) versus plasma membrane (PM) and forWT versus KO. However, thenacyl chain profile for PI is very different from that for PA. This indicates that DGKε is not facilitating thendirect transfer of a specific species of PAbetween the PMand the ER.Approximately 20%of the PAin the ERnmembrane has one short acyl chain of 14 or fewer carbons. These species of PA are not converted into PI butnmay play a role in stabilizing regions of high positive curvature in the ER. There are also PI species in both thenER and PM for which there is no detectable PA precursor, indicating that these species of PI are unlikely tonarise via the PI cycle. We find that in the PM of KO cells the levels of PI and of PA are decreased ∼3-fold inncomparison with those in either the PM of WT cells or the ER of KO cells. The PI cycle is slowed in the KOncells; hence, the lipid intermediates of the PI cycle can no longer be interconverted and are depleted fromthe PIncycle by conversion to other species. There is less of an effect of the depletion in the ER where de novonsynthesis of PA occurs in comparison with the PM.