Orthogonal Targeting of SAC1 to Mitochondria Implicates ORP2 as a Major Player in PM PI4P Turnover

Oxysterol-binding protein (OSBP)-related proteins (ORPs) 5 and 8 have been shown to deplete the lipid phosphatidylinositol 4-phosphate (PI4P) at sites of membrane contact between the endoplasmic reticulum (ER) and plasma membrane (PM). This is believed to be caused by transport of PI4P from the PM to the ER, where PI4P is degraded by an ER-localized SAC1 phosphatase. This is proposed to power the anti-port of phosphatidylserine (PS) lipids from ER to PM, up their concentration gradient. Alternatively, ORPs have been proposed to sequester PI4P, dependent on the concentration of their alternative lipid ligand. Here, we aimed to distinguish these possibilities in living cells by orthogonal targeting of PI4P transfer and degradation to PM-mitochondria contact sites. Surprisingly, we found that orthogonal targeting of SAC1 to mitochondria enhanced PM PI4P turnover independent of targeting to contact sites with the PM. This turnover could be slowed by knock-down of soluble ORP2, which also has a major impact on PM PI4P levels even without SAC1 over-expression. The data reveal a role for contact site-independent modulation of PM PI4P levels and lipid antiport.