Effectors of yeast SAC1 and ER-PM membrane contact sites

In budding yeast cells, ~40-50% of the inner surface of the plasma membrane (PM) is covered with the endoplasmic reticulum (ER). This association is achieved by 7 proteins that together join cortical ER and the PM membranes at membrane contact sites (MCSs). Several of these membrane "tether" proteins physically interact with the phosphoinositide phosphatase Sac1p. In the ER, Sac1p hydrolyses phosphoinositide-4-phosphate (PI4P), which is a major precursor of other phosphoinositide lipids, and physically interacts with several tether proteins. Yeast cells that lack SAC1 exhibit only minor growth defects, and in ∆-super-tether (∆-s-tether) cells, in which the 7 proteins involved in ER-PM contact have been deleted, growth is only modestly impacted. However, the deletion of SAC1 in the ∆-s-tether cells results in lethality. I hypothesized that this genetic interaction reflected overlapping functions of SAC1 and the ER-PM tether genes. To determine the genetic pathways involved in this genetic interaction, I conducted genetic selections to identify high-copy suppressors that rescue the lethality of sac1Δ Δ–s-tether cells. Five suppressors were identified, four of which are involved in the phospholipid biosynthesis pathway. Based on these suppressors, as well as lipidomic analysis and determinations of intracellular lipid distributions, I propose a model in which SAC1 and ER-PM tethering together maintain the normal distribution of PI4P and phosphatidylserine (PS) between the ER and PM, respectively. The counter-exchange of PI4P for PS occurs at membrane contact sites between the ER and PM, where Sac1p in the ER turns-over the transferred PI4P from the PM. Mutations in SAC1 or ER-PM contacts sites disrupt normal PI4P distribution and increase PI4P in the PM. The intermembrane balance of the lipids can be restored by increased PS and phosphatidyl choline (PC) synthesis. In addition, ER membrane stress in Δ-s-tether cells activates the unfolded protein response (UPR), which is exacerbated when combined with a temperature conditional sac1 mutation. Through transcriptomic analysis, I show that sac1ts Δ-s-tether cells exhibit broader changes in the global environmental stress response (ESR) pathway. High-copy suppressors of sac1∆ ∆-s-tether lethality reduce this ER stress and curtail ESRs. These findings support a model where SAC1 and ER-PM MCSs collectively act as a nexus for regulating lipid biosynthesis and membrane stress.