Elucidating the role of oxysterol binding protein-related protein 9 (ORP9) in cholesterol transport and Golgi function

Oxysterol-binding protein (OSBP)-related protein 9 (ORP9) is one of twelve members of an OSBP gene family implicated in sterol transport and signaling, vesicular trafficking and lipid regulation. Two variants of ORP9 are produced from alternate promoter start sites: full length ORP9 long (ORP9L) and ORP9 short (ORP9S), which lacks the N-terminal pleckstrin homology (PH) domain. ORP9L localizes to the trans-Golgi apparatus via PH domain interaction with phosphatidylinositol-4-phosphate (PI-4P), and both ORP9L and ORP9S localize to the endoplasmic reticulum by interaction between t&barbelow;wo p&barbelow;henylalanines in an a&barbelow;cid t&barbelow;ract (FFAT) domains and vesicle-associated membrane protein (VAMP)-associated protein (VAP). The purpose of this study was to test whether ORP9 can bind and transport sterols between the ER and Golgi apparatus, and to identify whether this affects vesicular trafficking, organelle integrity and cell signaling pathways. We demonstrate that ORP9L did not bind sterols dispersed in solution or Triton-X100 micelles. However, ORP9L and OSBP bound and extracted cholesterol presented in liposomes. Furthermore, ORP9L and OSBP transfer cholesterol between liposomes in a PI-4P-dependent mechanism. RNAi-dependent knockdown of ORP9L by RNAi and overexpression of ORP9S in CHO cells resulted in Golgi fragmentation and defective ER-Golgi transport. In addition, ORP9L knockdown in CHO cells elevated cholesterol mass in endosomes/lysosomes. Overexpression of ORP9S decreased cholesterol mass suggesting that ORP9S is a dominant negative inhibitor of ORP9L function. ORP9L and ORP9S each contain a phosphorylation site for phosphoinositide-dependent kinase-2 (PDK-2), which phosphorylates AGC kinases, such as protein kinase B (PKB)/Akt, and promotes cell growth and viability. Interestingly,ORP9 knockdown increased phosphorylation of the Akt PDK-2 site (Ser473). Protein kinase D (PKD) also phosphorylates ORP9L and ORP9S and is involved in regulation of vesicular trafficking from the trans-Golgi network (TGN). PKD phosphorylation and interaction with ORP9L could regulate vesicular transport activity and Golgi secretion. Our data suggests that ORP9L and OSBP localize and transfer sterols to the ER and Golgi, however they have separate distinct functions. ORP9L regulates cholesterol trafficking between ER and Golgi compartments to maintain Golgi structure, organization and secretion.