Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation

Abstract

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

Osh6 requires Ist2 for localization to the ER-PM contacts and efficient phosphatidylserine transport

“…We first evaluated the effect of ist2 mutations on general lipid homeostasis in the cell. Lipidomic analysis of whole cell extracts showed that both osh6D osh7D and ist2D cells had a dramatic (2.5-fold) decrease in PS levels compared to WT, similar to the decrease in PS observed in a strain lacking all ER-PM tethers (Quon et al, 2018). In contrast, the levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE) or phosphatidylinositol (PI) were not significantly affected ( Fig.…”

Sterol flow between the plasma membrane and the endosome is regulated by the LAM family protein Ltc1

“…While we have focused here on the role of Ltc1 in retrograde sterol flow from the PM, our observations suggest that Osh41 may promote PM sterol levels during steady-state growth and Osh42 may control sterol flow away from vacuoles. Osh41 and Osh42 are related to S. cerevisiae Osh4, which binds sterols in vitro (de Saint-Jean et al, 2011;Im et al, 2005;Schulz et al, 2009) and is proposed to promote ER to PM sterol transport by using the counter-gradient of PI4P (Moser von Filseck et al, 2015), although in vivo data suggest a role in membrane sterol organization rather than sterol transport (Georgiev et al, 2011;Quon et al, 2018;Raychaudhuri et al, 2006). These observations demonstrate that D4H is a highly valuable tool to study the intracellular trafficking of sterols.…”

“…Intriguingly, while most LTPs localize to dedicated organelle contact sites, S. cerevisiae mutants lacking all six known ER-PM tethering proteins show normal anterograde, and only partially diminished retrograde sterol transport (Quon et al, 2018).…”

Non-Canonical Regulation of Phosphatidylserine Metabolism by a Phosphatidylinositol Transfer Protein and a Phosphatidylinositol 4-OH Kinase

“…The metabolic phenotypes of stt4 G1782D and sac1 mutants suggested derangements in how PtdSer synthesis is regulated or otherwise organized. The role of ER-plasma membrane (PM) contact sites as regulatory/organizing nodes for lipid synthesis has been noted (Quon, Sere et al, 2018).…”

“…To determine whether simple tethering of ER to the PM was sufficient to rescue PtdSer homeostasis in tether cells, an artificial ER-PM protein tether was expressed in the double mutant (Quon, Sere et al, 2018). Expression of this artificial staple failed to rescue [ 3 H]-PtdSer levels in tetherΔ yeast and failed to restore growth of tetherΔpsd1Δ cells in Etn-free liquid media ( Figure 7D).…”