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...

Protein kinase D (PKD) is a critical regulator of Golgi structure and function. Biochemical evidence is presented that demonstrates the oxysterol-binding protein OSBP as a novel PKD substrate. Phosphorylation inhibits OSBP Golgi localization, impairs CERT Golgi localization, and promotes Golgi fragmentation.

The study identifies a sterol- and oxysterol binding protein (OSBP)-regulated phosphatidylinositol 4-kinase that regulates ceramide transport protein (CERT) activity and sphingomyelin (SM) synthesis. RNA interference silencing experiments identify PI4KIIα; as the mediator of Golgi recruitment of CERT, providing a potential mechanism for coordinating assembly of SM and cholesterol in the Golgi or more distal compartments.

Cholesterol and its numerous oxygenated derivatives (oxysterols) profoundly affect the biophysical properties of membranes, and positively and negatively regulate sterol homoeostasis through interaction with effector proteins. As the bulk of cellular sterols are segregated from the sensory machinery that controls homoeostatic responses, an important regulatory step involves sterol transport or signalling between membrane compartments. Evidence for rapid, energy-independent transport between organelles has implicated transport proteins, such as the eukaryotic family of OSBP (oxysterol-binding protein)/ORPs (OSBP-related proteins). Since the founding member of this family was identified more than 25 years ago, accumulated evidence has implicated OSBP/ORPs in sterol signalling and/or sterol transport functions. However, recent evidence of sterol transfer activity by OSBP/ORPs suggests that other seemingly disparate functions could be the result of alterations in membrane sterol distribution or ancillary to this primary activity.

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