SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes.

James P Zewe,Rachel C Wills,Gerald Rv Hammond,Sahana Sangappa,Brady D Goulden

doi:10.7554/elife.35588

Abstract

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.

Highlights

Phosphatidylinositol-4-phosphate (PtdIns4P) is arguably the most functionally diverse lipid molecule in eukaryotic cells
We present evidence for ‘cis’ activity in mammalian cells, show that SAC1 fails to enrich at endoplasmic reticulum (ER)-plasma membrane (PM) membrane contact sites (MCS) and, we show that SAC1 does not possess a conformation that allows it to traverse ER-PM MCS and act in ‘trans’
We demonstrated that: (i) agents that acutely inhibit SAC1 activity in cells produce the accumulation of PtdIns4P substrate in the ER; (ii) under either resting or MCS forming conditions, SAC1 fails to enrich at ER-PM MCS, the would-be sites of ‘trans’ activity; (iii) SAC1 has robust ‘cis’ activity but very poor ‘trans’ activity in living cells; (iv) for appreciable ‘trans’ activity, an additional ~6 nm linker between the ER localized TMD and the catalytic domain must be introduced

Summary

Introduction

Phosphatidylinositol-4-phosphate (PtdIns4P) is arguably the most functionally diverse lipid molecule in eukaryotic cells. PtdIns4P binds to and thereby recruits and/or activates many proteins involved in cellular traffic (Tan et al, 2014) These include proteins regulating vesicular traffic at the endoplasmic reticulum (ER), late endosomes/lysosomes (LEL) and Golgi (Wang et al, 2007; Wang et al, 2003; Jovicet al., 2012; 2014; Klinkenberg et al, 2014), as well as non-vesicular lipid transport at the plasma membrane (PM), LEL and Golgi (Mesmin et al, 2013; Chung et al, 2015; Moser von Filseck et al, 2015a; Zhao and Ridgway, 2017). SAC1 is an integral membrane protein with two C-terminal transmembrane helices (Whitters et al, 1993; Konrad et al, 2002; Nemoto et al, 2000), which localizes primarily to the ER but is able to traffic to the Golgi depending on the growth status of the cell

Methods

Results

Conclusion