Osh Proteins Regulate Phosphoinositide Metabolism at ER-Plasma Membrane Contact Sites

Abstract

Sac1 phosphoinositide (PI) phosphatases are essential regulators of PI-signaling networks. Yeast Sac1, an integral endoplasmic reticulum (ER) membrane protein, controls PI4P levels at the ER, Golgi, and plasma membrane (PM). Whether Sac1 can act in trans and turn over PI4P at the Golgi and PM from the ER remains a paradox. We find that Sac1-mediated PI4P metabolism requires the oxysterol-binding homology (Osh) proteins. The PH domain-containing family member, Osh3, localizes to PM/ER membrane contact sites dependent upon PM PI4P levels. We reconstitute Osh protein-stimulated Sac1 PI phosphatase activity in vitro. We also show that the ER membrane VAP proteins, Scs2/Scs22, control PM PI4P levels and Sac1 activity in vitro. We propose that Osh3 functions at ER/PM contact sites as both a sensor of PM PI4P and an activator of the ER Sac1 phosphatase. Our findings further suggest that the conserved Osh proteins control PI metabolism at additional membrane contact sites.

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

“…Previous studies have identified the PM-associated ER as sites of phosphatidylinositol (PI) metabolism, nonvesicular transfer of sterols, and Ca 2þ level regulation (Li and Prinz 2004;Baumann et al 2005;Carrasco and Meyer 2011;Stefan et al 2011). EM analysis has revealed that 20% -45% of the cytoplasmic surface of the PM in budding yeast is within tethering distance of the ER and ribosome excluded (Fig.…”

“…Regulation of the essential lipid-signaling molecule phosphatidylinositol 4-phosphate (PI4P) is controlled by Osh and VAP mediated activation of Sac1 phosphatase (Roy and Levine 2004;Stefan et al 2011). Deletion of Osh proteins in yeast cells results in a six-to sevenfold increase in PI4P levels; furthermore, addition of recombinant Osh3 to a microsome fraction depleted of peripherally bound proteins (including endogenous Osh proteins) was able to stimulate Sac1 phosphatase activity, suggesting that Osh proteins control PI4P levels at ER-PM contact sites (Stefan et al 2011).…”

Cellular microcompartments constitute general suborganellar functional units in cells

“…A similar arrangement has been reported for the oxysterol-binding proteins Osh2 and Osh3, which contain FFAT and PH domains required for targeting them to ER-PM contact sites (Toulmay and Prinz , 2011 ). Osh proteins have previously been implicated in non-vesicular sterol trafficking between the ER and the PM, but recent work suggests an additional role of these proteins as phosphatidylinositol-4-phosphate (PI-4-P) sensors that modulate PI-4-P levels in the PM by regulating the ER-resident PI-4-P phosphatase Sac1 at the ER-PM contact sites (Stefan et al , 2011 ). A recent study of Drin, Antonny, and coworkers demonstrated that Osh4 binds sterols and PI-4-P in the same binding site and may, thus, generate a sterol gradient between the ER and the Golgi (de Saint -Jean et al, 2011 ).…”

“…Microcompartments can be subdomains on an organelle membrane such as subdomains on endosomes (de Renzis et al , 2002 ), the plasma membrane (Kusumi et al , 2012 ), the ER (Stefan et al , 2011 ), the Golgi (Bankaitis , 2009 ), or protein-lipid-based supercomplexes of the mitochondrial respiratory chain (Schagger and Pfeiffer , 2000 ). They also include contact zones between distinct membranes (Elbaz and Schuldiner , 2011 ) or defined regions within the bacterial plasma membrane (Shapiro et al , 2009 ).…”

Nonvesicular Lipid Transfer from the Endoplasmic Reticulum

“…Osh2p and Osh3p, which both contain a FFAT motif and a PH domain, might stabilize ER-PM MCSs, through interaction with Scs2p, the yeast homolog of VAP-A/B on the ER, and PI4P on the PM (Stefan et al 2011). Similarly, OSBP might stabilize ER-Golgi MCSs, thereby facilitating ERto-Golgi ceramide transport in the presence of 25-hydroxycholesterol (25OH) (Perry and Ridgway 2006), whereas ORPL1 may stabilize ERlate endosome MCSs under low cholesterol conditions (Rocha et al 2009).…”