Probing the Central Role of Phosphatidylinositol Synthesis in Lipid Metabolism of Eukaryotic Cells

Phosphatidylinositol (PI) is the precursor of all phosphoinositides (PPIns), a class of regulatory phospholipids that play key roles in cellular physiology. PI is synthesized in the ER by a single enzyme, phosphatidylinositol synthase (PIS). PI may have important roles of its own in membranes where PPIns are not produced such as in the ER and mitochondria. Disruption of PIS in yeast and zebrafish is lethal and PIS has been postulated to be an essential gene in studies of haploid cells. Therefore, it is difficult to evaluate the processes where this enzyme, or its lipid product, PI plays a critical role. To overcome this problem, we generated stable HEK293 cell lines in which a doxycycline (Dox)-inducible PIS-GFP transgene was stably integrated in a safe-harbor site of the genome and the endogenous PIS locus was inactivated via CRISPR/Cas9 genome editing. We verified the knock-out of the endogenous PIS and the inducible expression of PIS-GFP by western blot analysis and sequencing of genomic DNA. Using these cells, we found that PIS enzymatic activity assayed in ER membranes prepared from cells after Dox deprivation was indistinguishable from the assay blank. Dox treatment of cells elevated PIS activity in the isolated ER membrane although it was still lower than that found in the membranes prepared from parental HEK293. Surprisingly, in spite of the undetectable low PIS activity, Dox-deprived cells still contained about 50% of PI as assessed by equilibrium labeling with 3 H-inositol and retained their PPIns distribution as shown by confocal microscopy using GFP-tagged PPIns binding modules. This disparity between PIS activity and PI availability was observed even under culture conditions that deprived cells of any external PI source. Such cells were subjected to lipidomic analysis to assess the overall lipid changes associated with the reduction of PI synthesis and to identify processes that require PI. This analysis confirmed the ~50% PI loss and also revealed reprogramming of specific lipid metabolic pathways. Current work focuses on the possibility that alternate source(s) of PI exist and on the question of what lipid synthetic pathways show adaptation to the greatly reduced or missing PIS activity. These experiments have the promise of a better understanding of how PI lipids determine the overall lipid landscape and homeostasis of eukaryotic cells and may unravel new molecular targets that can be exploited for therapeutic interventions in human metabolic and lipid disorders.