Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

Abstract

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large gene family that differentially localize to organellar membranes, reflecting a functional role in sterol signaling and/or transport. OSBP partitions between the endoplasmic reticulum (ER) and Golgi apparatus where it imparts sterol-dependent regulation of ceramide transport and sphingomyelin synthesis. ORP9L also is localized to the ER-Golgi, but its role in secretion and lipid transport is unknown. Here we demonstrate that ORP9L partitioning between the trans-Golgi/trans-Golgi network (TGN), and the ER is mediated by a phosphatidylinositol 4-phosphate (PI-4P)-specific PH domain and VAMP-associated protein (VAP), respectively. In vitro, both OSBP and ORP9L mediated PI-4P-dependent cholesterol transport between liposomes, suggesting their primary in vivo function is sterol transfer between the Golgi and ER. Depletion of ORP9L by RNAi caused Golgi fragmentation, inhibition of vesicular somatitus virus glycoprotein transport from the ER and accumulation of cholesterol in endosomes/lysosomes. Complete cessation of protein transport and cell growth inhibition was achieved by inducible overexpression of ORP9S, a dominant negative variant lacking the PH domain. We conclude that ORP9 maintains the integrity of the early secretory pathway by mediating transport of sterols between the ER and trans-Golgi/TGN. INTRODUCTIONThe Golgi apparatus is a highly compartmentalized organelle that serves as a nexus for the processing, sorting, and vesicular transport of protein and lipid cargo between organelles. Regulation of vesicular transport is dependent on coat proteins and factors that control coat assembly/disassembly, as well as membrane dynamics and individual lipid regulators. A continuing challenge is the identification of the proteins responsible for spatial generation of Golgi lipid regulators by localized synthesis and/or transport (Huijbregts et al., 2000;De Matteis et al., 2007). In this study we investigated one such lipid-binding protein, oxysterol-binding protein (OSBP)-related protein 9 (ORP9), and its role in endoplasmic reticulum (ER)-to-Golgi transport.Lipid synthesis and transport to the Golgi apparatus regulates local and distal cellular functions. For example, synthesis of sphingomyelin (SM) and glycosphingolipids (Huitema et al., 2004) in combination with cholesterol is required for raft assembly in the Golgi and cargo delivery to the apical surface of polarized cells (Hoekstra et al., 2003). SM synthesis in the Golgi apparatus is dependent on delivery of ceramide from the ER by the ceramide transfer protein (CERT; Hanada et al., 2003). Ceramide transport by CERT involves binding of a phosphatidylinositol 4-phosphate (PI-4P)-specific pleckstrin homology (PH) domain to the Golgi apparatus and interaction with vesicle-associated membrane protein (VAMP)-associated-protein (VAP) by the "two phenylalanines in an acidic tract" (FFAT) motif (Hanada et al., 2003). In addition, CERT activates protein kinase D activity and secretory t...

Nonvesicular Lipid Transfer from the Endoplasmic Reticulum

“…PI(4,5)P2 may, therefore, enhance the directional transport of sterol from PI(4,5)P2-poor to PI(4,5)P2-rich membranes. Likewise, in vitro cholesterol transport by OSBP and ORP9, which bind PI4P via their pleckstrin homology (PH) domain (Levine and Munro 1998), is markedly stimulated by the presence of PI4P (Ngo and Ridgway 2009). These examples show how the membrane environment of different LTPs (PI[4,5]P2 content for Osh4 and PI4P for OSBP and ORP9) influences their lipid (sterol) transfer activity.…”

OSBP- and FAN-mediated sterol requirement for spermatogenesis inDrosophila

“…Upon binding 25-hydroxycholesterol, OSBP translocates from the cytosol to the Golgi, but the consequence of this is unknown (Ridgway et al, 1992). Knockdown of ORP9 in cells results in Golgi fragmentation, accumulation of sterols in endosomes or lysosomes and, subsequently, ER-to-Golgi transport defects, indicating that the partitioning of sterols in the Golgi and other organelles affects intracellular transport (Ngo and Ridgway, 2009). Furthermore, in CHO-K1 cells, OSBP synergizes with ceramide transport protein (CERT) in ER-to-Golgi ceramide transport, and the interaction of OSBP with the ER and Golgi apparatus is required for Golgi translocation of CERT (Perry and Ridgway, 2006).…”

Membrane Phospholipid Asymmetry Counters the Adverse Effects of Sterol Overloading in the Golgi Membrane of Drosophila

“…Osbp; apparatus with its pleckstrin homolog domain and to VAP in the ER with its FFAT motif. Disturbing the level or the activity of several OSBPs leads to Golgi fragmentation and inefficient protein transport from the Golgi apparatus to the plasma membrane (Ngo and Ridgway 2009;Nhek et al 2010). Interestingly, although PKD inhibits OSBP, it enhances the activity of PI4KIII by phosphorylation and stimulates PI4P production, which might result in the recruitment of more OSBP to the Golgi apparatus (Hausser et al 2005).…”

“…Genetic manipulation of ORP levels alters cholesterol distribution and homeostasis in cells. For example, RNA interference (RNAi) depletion of ORP9L leads to profound cholesterol accumulation in lysosomal membranes (Ngo and Ridgway 2009). Similarly, knocking down ORP5 causes cholesterol accumulation in late endosomes and lysosomes in culture cells (Du et al 2011).…”