| Oxysterol binding protein (OSBP) and eleven OSBP-related proteins (ORPs) constitute a highly conserved protein family characterized by N-terminal pleckstrin homology (PH) and C-terminal OSBP homology domains (OHD)s. OSBP and ORPs are implicated in the regulation of vesicle trafficking, cell signalling and lipid metabolism, but their precise roles are poorly understood. To gain insight into the function of this gene family, yeast two-hybrid analysis was used to clone the OSBP-interacting protein vesicle-associated membrane protein (VAMP)-associated protein (VAP)-A, an ER-localized protein implicated in vesicle transport. Further yeast two-hybrid analysis determined that VAP-A interacted with both ORP9 variants, ORP9S and ORP9L. Furthermore, GST-VAP-A pulldown assays showed that ORPIL, ORP3(Id) and ORP4L also interacted with VAP-A, defining it as a central and functional regulator of the ORP family. The role of VAP-A/OSBP and VAP-A/ORP9 complex(es) in ER structure and export was characterized by inducible overexpression in CHO cells. Overexpressed OSBP was localized in a Golgi/vesicular compartment similar to endogenous OSBP. Overexpressed OSBP W174A, in which the conserved PH domain tryptophan was mutated, displayed enhanced interaction with VAP-A and caused the formation of irregular ER-derived vesicular structures to which VAP and OSBP W174A localized. We demonstrated that these ER-derived structures are involved at an early stage of ceramide and protein export from the ER. Similar to OSBP W174A, overexpression of both ORP9 variants caused dramatic changes in the structure of the ER, ERGIC (ER/Golgi intermediate compartment) and nucleus. This was accompanied by reduced cell growth, and in the case of ORP9S, cell death by a non-apoptotic mechanism. In summary, we have identified protein-protein interactions between VAP-A, OSBP and several ORPs, and implicated this network in an early stage of protein and ceramide export from the ER, as well as the regulation of ER membrane structure. |
