Mechanisms and regulation of plasma membrane-endoplasmic reticulum Membrane Contact Sites in Saccharomyces cerevisiae

Membrane tether proteins staple the endoplasmic reticulum (ER) to other cellular membranes at Membrane Contact Sites (MCSs), as possible conduits to coordinately regulate lipid metabolism and membrane exchange. To determine the role of ER-PM MCSs in membrane regulation, I generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tether proteins (the yeast E-Syts Tcb1p-3p; VAP homologs Scs2p and Scs22p; and the TMEM16 homolog Ist2p), as well as the presumptive tether Ice2p. Although Δ-s-tether cells lack direct ER-PM MCSs they were viable, albeit with severe defects in lipid homeostasis and membrane organization. To determine the role of MCSs in non-vesicular sterol transport, the bi-directional exchange of sterols between the ER-PM in Δ-s-tether cells was directly assayed in vivo. No significant defects in non-vesicular sterol transport were detected though lipidomic analysis revealed major dysfunction in phospholipid and sphingolipid synthesis. Based on these findings I hypothesized that Δ-s-tether growth defects might be rescued either metabolically, by increasing phosphatidylcholine synthesis through choline addition, or by overexpressing the phospholipid methyltransferase OPI3. These modifications did restore growth, but not by re-establishing ER-PM MCSs, suggesting that these membrane attachments are not physically required for lipid transport but rather as regulators of lipid metabolism. Phosphatidylinositol-4-phosphate (PI4P) accumulated in the PM of Δ-s-tether cells, consistent with the observed synthetic lethality between Δ-s-tether mutations and mutations in either the SAC1 PI4P phosphatase gene or OSH4, which encodes a PI4P binding protein. Even though transport assays indicated that MCSs do not mediate ER-PM sterol exchange, we tested if sterols played a role in contact site generation. Cells depleted of sterols by repressing ERG9, which encode the sterol specific biosynthetic enzyme squalene synthase, exhibited nearly complete coverage of the inner cytoplasmic PM surface with cortical ER. These results support a model for ER-PM MCSs in which they collectively act as a regulatory nexus for coordinating multiple lipid metabolic pathways to control cell membrane composition.