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.
Highlights
In the past few years, the emerging interest in lipid transport proteins shed some light on the biology of the oxysterol-binding protein (OSBP)-related protein (ORP) family (Olkkonen, 2013; Olkkonen and Li, 2013)
We examined the transport of PS first because Osh6 was reported to be a PS transporter in several studies (Maeda et al, 2013; von Filseck et al, 2015a)
We have utilized a FRET assay to monitor the amount of transported PS in the acceptor membrane by observing the energy transfer between CFP fused to a PS biosensor [C2 domain from Lactadherin C (LactC) ] and ATTO488-DOPE in the acceptor membrane (Figures 1A,B)
Summary
In the past few years, the emerging interest in lipid transport proteins shed some light on the biology of the oxysterol-binding protein (OSBP)-related protein (ORP) family (Olkkonen, 2013; Olkkonen and Li, 2013). The mechanism which allows such a selective lipid transport across the concentration gradient relies on phosphoinositides (PPIns), mostly phosphatidylinositol 4-phosphate (PI4P) (von Filseck et al, 2015b). It has been postulated that the main feature of the transport mechanism is the exchange of the cargo lipid molecule at the target membrane for the PPIn molecule which, upon delivery to the ER, is hydrolyzed, providing the PPIn gradient as a driving force (energy) for the transport against the concentration gradient. This has been first shown elegantly for the yeast ergosterol transporter Osh. To maintain the phosphoinositide gradient in live cells, three processes are required: i) PI transport to the PM, ii) its conversion to PI4P by phosphatidylinositol 4-kinases, and iii) PI4P hydrolysis in the ER membrane back to PI carried out by the Sac phosphatase (Cai et al, 2014; Chung et al, 2015) that has an essential role in the transport process (von Filseck et al, 2015b)
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