1 SAC 1 Degrades its Lipid Substrate PtdIns 4 P in the 1 Endoplasmic Reticulum to Maintain a Steep 2 Chemical Gradient with Donor Membranes

8 Gradients 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 11 the ER to maintain the gradient. However, SAC1 has also been proposed to act in “trans” 12 at membrane contact sites, which could oppose lipid traffic. It is therefore crucial to 13 determine which mode SAC1 uses in living cells. We report that acute inhibition of SAC1 14 causes accumulation of PtdIns4P in the ER, that SAC1 does not enrich at membrane 15 contact sites, and that SAC1 has little activity in “trans”, unless a linker is added between 16 its ER-anchored and catalytic domains. The data reveal an obligate “cis” activity of SAC1, 17 supporting its role in non-vesicular lipid traffic and implicating lipid traffic more broadly in 18 inositol lipid homeostasis and function. 19