A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples

摘要

PtdIns3 P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3 P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3 P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3 P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells.Abbreviations: EEA1, early endosome antigen 1; GFP, green fluorescent protein; GST, glutathione transferase; MTM, myotubularin; MTMR, MTM-related; PE, phosphatidylethanolamine; PI3K, phosphoinositide 3-kinase; PtdInsP, phosphatidylinositol monophosphate; PtdInsP2, phosphatidylinositol diphosphate; PX, Phox homology; TRAP, thrombin-receptor-agonist peptide