Reconstitution of ORP-mediated lipid exchange coupled to PI4P metabolism.

Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.

Fatty acid isoprenoid alcohol ester synthesis in fruits of the African Oil Palm (Elaeis guineensis).

The African Oil Palm (Elaeis guineensis; family Arecaceae) represents the most important oil crop for food and feed production and for biotechnological applications. Two types of oil can be extracted from palm fruits, the mesocarp oil which is rich in palmitic acid and in carotenoids (provitamin A) and tocochromanols (vitamin E), and the kernel oil with high amounts of lauric and myristic acid. We identified fatty acid phytyl esters (FAPEs) in the mesocarp and kernel tissues of mature fruits, mostly esterified with oleic acid and very long chain fatty acids. In addition, fatty acid geranylgeranyl esters (FAGGEs) accumulated in mesocarp and kernels to even larger amounts. In contrast, FAPEs and FAGGEs amounts and fatty acid composition in leaves were very similar. Analysis of wild accessions of African Oil Palm from Cameroon revealed a considerable variation in the amounts and composition of FAPEs and FAGGEs in mesocarp and kernel tissues. Exogenous supplementation of phytol or geranylgeraniol to mesocarp slices resulted in the incorporation of these alcohols into FAPEs and FAGGEs, respectively, indicating that they are synthesized via enzymatic reactions. Three candidate genes of the esterase/lipase/thioesterase (ELT) family were identified in the Oil Palm genome. The genes are differentially expressed in mesocarp tissue with EgELT1 showing the highest expression. Geranylgeraniol from FAGGE might be recycled and used as a substrate for the synthesis of carotenoids and tocotrienols during fruit development. Thus, FAPEs and FAGGEs in the mesocarp and kernel of Oil Palm provide an additional metabolic source for fatty acids and phytol or geranylgeraniol, respectively.