The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids’ role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.

Zewe et al develop approaches to map the subcellular distribution of the major 14 phospholipid, phosphatidylinositol (PI), revealing that the lipid is present in most membranes 15 except for plasma membrane, where it is mainly found as PI4P and PI(4,5)P2. 16Abstract 1 The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in 2 eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these 3 lipids' role in health and disease. PPIn are derived from the major structural lipid, 4 phosphatidylinositol (PI). However, although the distribution of most PPIn have been 5 characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, 6 we have used several orthogonal approaches to map the subcellular distribution of PI, including 7 localizing exogenous fluorescent PI, as well as detecting lipid conversion products of 8 endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. 9We report that PI is broadly distributed throughout intracellular membrane compartments. 10However, there is a surprising lack of PI in the plasma membrane compared to the PPIn. These 11 experiments implicate regulation of PI supply to the plasma membrane, as opposed to 12 regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.