Gradients of PtdIns4P between organelle membranes and the endoplasmic reticulum (ER) are thought to drive counter-transport of other lipids via non-vesicular traffic. This novel pathway requires the SAC1 phosphatase to degrade PtdIns4P in a ‘cis’ configuration at the ER to maintain the gradient. However, SAC1 has also been proposed to act in ‘trans’ at membrane contact sites, which could oppose lipid traffic. It is therefore crucial to determine which mode SAC1 uses in living cells. We report that acute inhibition of SAC1 causes accumulation of PtdIns4P in the ER, that SAC1 does not enrich at membrane contact sites, and that SAC1 has little activity in ‘trans’, unless a linker is added between its ER-anchored and catalytic domains. The data reveal an obligate ‘cis’ activity of SAC1, supporting its role in non-vesicular lipid traffic and implicating lipid traffic more broadly in inositol lipid homeostasis and function.

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.

8Gradients of PtdIns4P between organelle membranes and the endoplasmic reticulum (ER) 9 are thought to drive counter-transport of other lipids via non-vesicular traffic. This novel 10 pathway requires the SAC1 phosphatase to degrade PtdIns4P in a "cis" configuration at the 11 ER to maintain the gradient. However, SAC1 has also been proposed to act in "trans" at 12 membrane contact sites, which could oppose lipid traffic. It is therefore crucial to determine 13 which mode SAC1 uses in living cells. We report that acute inhibition of SAC1 causes 14 accumulation of PtdIns4P in the ER, that SAC1 does not enrich at membrane contact sites, 15and that SAC1 has little activity in "trans", unless a linker is added between its ER-anchored 16 and catalytic domains. The data reveal an obligate "cis" activity of SAC1, supporting its role 17 in non-vesicular lipid traffic and implicating lipid traffic more broadly in inositol lipid 18 homeostasis and function.

Phosphatidylinositol 4‐phosphate (PtdIns4P) is one of the most functionally diverse molecules utilized by eukaryotic cells. It is both a metabolic hub for other crucial signaling lipids such as PtdIns(4,5)P2 and PtdInsP3, and a key molecule for recruitment of membrane and lipid transport proteins in its own right. Most recently, it has been proposed to form a “phosphoinositide‐motive force”, whereby transport of PtdIns4P molecules down their concentration gradient from the plasma membrane (PM) or Golgi to the ER drives counter transport of other lipids up their own concentration gradients. A critical requirement for this model is that the main PtdIns4P degrading enzyme, the integral ER phosphatase SAC1, hydrolyzes PtdIns4P in the ER in a “cis” configuration. Alternatively, it has been suggested that other functions of PtdIns4P are regulated by SAC1 hydrolyzing PtdIns4P directly in the PM in a “trans” configuration at membrane contact sites (MCS). However, such activity would surely disrupt lipid counter transport. Therefore, we sought to determine whether SAC1 acts in “cis”, “trans”, or both in mammalian cells. Acute chemical ablation of SAC1 activity drives ectopic accumulation of PtdIns4P in the ER, revealing “cis” activity. Furthermore, endogenous or ectopically expressed SAC1 localizes to the ER and Golgi, but does not constitutively or dynamically enrich at ER‐PM MCS. Forced recruitment of SAC1 to experimentally induced MCS does not produce robust “trans” activity on PM PtdIns4P. However, “trans” activity can be induced by adding an approximately 6 nm long helical linker between the ER anchor and the catalytic domain. Together, our results reveal that SAC1 operates in a “cis” configuration. This ensures a “phosphoinositide‐motive force” for lipid transport is effective, and also implies regulation of PM phosphoinositide signaling is tightly linked to non‐vesicular traffic of PtdIns4P at ER‐PM MCS.Support or Funding InformationThis work was supported by National Institutes of Health grant 1R35GM119412‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.