Sac1 degrades its lipid substrate PI4P in the ER to maintain a steep electrochemical gradient on donor membranes

Abstract

PI4P metabolism has been proposed to form the basis of an “inositide motive force”, whereby a steep electrochemical gradient of this lipid that is high in a target membrane and low in the endoplasmic reticulum (ER) drives counter transport of other lipids. This mechanism assumes that the ER‐resident Sac1 phosphatase degrades PI4P in the ER (i.e. acting in cis). However, several studies have proposed that the enzyme instead operates in trans at contact sites between the ER and other PI4P rich membranes. We therefore sought to establish whether Sac1 truly acts in cis or trans in living cells. Compared with bona fide plasma membrane (PM)‐ER contact site proteins and broadly distributed ER proteins, Sac1 exhibits a localization consistent with the latter. Strategies to test activity of the enzyme at the PM in living cells revealed far more robust activity when the enzyme met its substrate in cis than at contact sites in trans. Finally, we used chemical and genetic strategies to inactivate Sac1 in yeast and mammalian cells, revealing dramatic accumulation of PI4P in the ER. This result is consistent with a failure to degrade the lipid only after its delivery to the ER. Together, the data show that Sac1 acts in cis, establishing the plausibility of an inositide motive force, and implicating PI4P transfer proteins as essential to phosphoinositide and lipid homeostasis.Support or Funding Information1R35GM119412‐01: NIH(NIGMS) R35