Osh6 Revisited: Control of PS Transport by the Concerted Actions of PI4P and Sac1 Phosphatase

Abstract

Osh6, a member of the oxysterol-binding protein–related protein (ORP) family, is a lipid transport protein that is involved in the transport of phosphatidylserine (PS) between the endoplasmic reticulum (ER) and the plasma membrane (PM). We used a biophysical approach to characterize its transport mechanism in detail. We examined the transport of all potential ligands of Osh6. PI4P and PS are the best described lipid cargo molecules; in addition, we showed that PIP2 can be transported by Osh6 as well. So far, it was the exchange between the two cargo molecules, PS and PI4P, in the lipid-binding pocket of Osh6 that was considered an essential driving force for the PS transport. However, we showed that Osh6 can efficiently transport PS along the gradient without the help of PI4P and that PI4P inhibits the PS transport along its gradient. This observation highlights that the exchange between PS and PI4P is indeed crucial, but PI4P bound to the protein rather than intensifying the PS transport suppresses it. We considered this to be important for the transport directionality as it prevents PS from returning back from the PM where its concentration is high to the ER where it is synthesized. Our results also highlighted the importance of the ER resident Sac1 phosphatase that enables the PS transport and ensures its directionality by PI4P consumption. Furthermore, we showed that the Sac1 activity is regulated by the negative charge of the membrane that can be provided by PS or PI anions in the case of the ER membrane.

Depletion of Plasma Membrane PI4P by ORP5 Requires Hydrolysis by SAC1 in Acceptor Membranes

“…Our results make perfect sense in the light of recent, elegant in vitro experiments with Osh6p in artificial membranes (Eisenreichova et al, 2021). Here, the ORD domain was shown to have exceptionally high affinity for PI4P, such that it actually inhibited binding and transport of the counter lipid, PS.…”

“…A number of scenarios could be envisioned that may preclude productive transfer of PM PI4P to mitochondria by ORP5 ∆TMD -FKBP. One possibility is suggested by recent studies of the homologous yeast protein, Osh6p (Eisenreichova et al, 2021). That study reported that binding of PI4P by Osh6p is so tight that lipid transfer is actually inhibited.…”

Orthogonal targeting of SAC1 to mitochondria implicates ORP2 as a major player in PM PI4P turnover

“…Several scenarios could be envisioned that may preclude productive transfer of PM PI4P to mitochondria by ORP5 ∆TMD -FKBP. One possibility is suggested by recent studies of the homologous yeast protein, Osh6p (Eisenreichova et al, 2021). That study reported that binding of PI4P by Osh6p is so tight that lipid transfer is inhibited.…”

“…And if PI4P can be presented to and hydrolyzed by SAC1 mito , why isn’t PI4P transfer to the mitochondrial outer membrane not normally observed? A clue comes from elegant in vitro work on Osh6 and ORP8 (Eisenreichova et al, 2021; Ikhlef et al, 2021); here, the ORD domain was shown to have exceptionally high affinity for PI4P, such that it inhibited binding and transport of the counter lipid, PS. These experiments predicted and demonstrated a requirement for PI4P hydrolysis for PS transport to occur.…”

“…Why the discrepancy? Yet another clue comes from the work on Osh6 and ORP8 (Eisenreichova et al, 2021; Ikhlef et al, 2021): here, ORD domains could bind both PI4P and PI(4,5)P 2 , but to have a higher affinity for the former. Additionally, cells are known to preserve PI(4,5)P 2 levels in the face of declining PI4P (Hammond et al, 2009, 2012; Bojjireddy et al, 2014).…”