Defining the subcellular distribution and metabolic channeling of phosphatidylinositol

Abstract

Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.

MapPIng PI inside cells brings new light to polyphosphoinositide biology

“…Made in trace amounts (1% of total lipids) in a highly accurate manner, these lipids regulate processes—signaling pathways, vesicular trafficking, cytoskeletal dynamics, ion transport, and lipid exchange—in different cellular regions. Two studies, by Pemberton et al (2) and Zewe et al (3), present innovative approaches to detect PI in living cells, shedding new light on PI distribution and availability for PPIn production.…”

“…Pemberton et al (2) and Zewe et al (3) solved this issue by reengineering bacterial enzymes called PI-specific phospholipase C (PI-PLC), which bind to lipid membranes and convert PI into diacylglycerol (DAG), releasing inositol-1-phosphate. One strategy was to abolish the catalytic activity of the PI-PLC to get a mere PI-binding protein that, once fused to GFP, was amenable to intracellularly detect PI (Fig.…”

“…One strategy was to abolish the catalytic activity of the PI-PLC to get a mere PI-binding protein that, once fused to GFP, was amenable to intracellularly detect PI (Fig. 1 b; 2). A second approach, based on a previous work (7), was to use activatable PI-PLCs to produce DAG in the cytosolic side of a given organelle and, by measuring how much DAG is produced, to indirectly evaluate PI levels (2).…”

“…1 b; 2). A second approach, based on a previous work (7), was to use activatable PI-PLCs to produce DAG in the cytosolic side of a given organelle and, by measuring how much DAG is produced, to indirectly evaluate PI levels (2). For this, a PI-PLC variant, deficient in binding lipid surfaces, was fused to a FK506-binding protein (FKBP) and coexpressed in cells with a FKBP–rapamycin binding (FRP) domain anchored to the organelle.…”

“…One of the lobes can be anchored to a specific organelle for local analysis. An additional way to quantify PI was to detect its conversion into PI4P by recruiting on organelles a catalytic fragment of PI 4-kinase via the rapamycin-based strategy (2,3; Fig. 1 e).…”