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.

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Osh6, a member of the oxysterol-binding protein–related protein (ORP) family, is a lipid transport protein that is involved in the transport of phosphatidylserine (PS) between the endoplasmic reticulum (ER) and the plasma membrane (PM). We used a biophysical approach to characterize its transport mechanism in detail. We examined the transport of all potential ligands of Osh6. PI4P and PS are the best described lipid cargo molecules; in addition, we showed that PIP2 can be transported by Osh6 as well. So far, it was the exchange between the two cargo molecules, PS and PI4P, in the lipid-binding pocket of Osh6 that was considered an essential driving force for the PS transport. However, we showed that Osh6 can efficiently transport PS along the gradient without the help of PI4P and that PI4P inhibits the PS transport along its gradient. This observation highlights that the exchange between PS and PI4P is indeed crucial, but PI4P bound to the protein rather than intensifying the PS transport suppresses it. We considered this to be important for the transport directionality as it prevents PS from returning back from the PM where its concentration is high to the ER where it is synthesized. Our results also highlighted the importance of the ER resident Sac1 phosphatase that enables the PS transport and ensures its directionality by PI4P consumption. Furthermore, we showed that the Sac1 activity is regulated by the negative charge of the membrane that can be provided by PS or PI anions in the case of the ER membrane.

1 Pemberton et al. characterize a molecular toolbox for the visualization and manipulation of 2 phosphatidylinositol (PtdIns) within intact cells. Results using these approaches define the steady-state 3 distribution of PtdIns across subcellular membrane compartments as well as provide new insights into the 4 relationship between PtdIns availability and polyphosphoinositide turnover. 5 6 7 Abstract 8Phosphatidylinositol (PtdIns) is an essential structural component of eukaryotic membranes that also 9 serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance 10 of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of 11 how the dynamic regulation of PtdIns synthesis and transport contributes to the turnover of PPIn pools. To 12 address these shortcomings, we capitalized on the substrate selectivity of a bacterial enzyme, PtdIns-specific 13 PLC, to establish a molecular toolbox for investigations of PtdIns distribution and availability within intact cells. 14 In addition to its presence within the ER, our results reveal low steady-state levels of PtdIns within the plasma 15 membrane (PM) and endosomes as well as a relative enrichment of PtdIns within the cytosolic leaflets of the 16 Golgi complex, peroxisomes, and outer mitochondrial membranes. Kinetic studies also demonstrate the 17 requirement for sustained PtdIns supply from the ER for the maintenance of monophosphorylated PPIn 18 species within the PM, Golgi complex, and endosomal compartments. 19