Abstract

We recently showed that oxysterol-binding protein (OSBP), one of twelve related PH domain containing proteins with lipid and sterol binding activity, interacts with VAMP-associated protein (VAP)-A on the endoplasmic reticulum (ER). In addition to OSBP, seven OSBP-related proteins (ORPs) bind VAP-A via a conserved E-F/Y-F/Y-DA 'FFAT' motif. We focused on this interaction for ORP9, which is expressed as a full-length (ORP9L) or truncated version missing the PH domain (ORP9S). Mutation analysis showed that the interaction required the ORP9 FFAT motif and the N-terminal conserved domain of VAP. Endogenous ORP9L displayed Golgi localization, which was partially mediated by the PH domain based on limited localization of OPR9-PH-GFP with the Golgi apparatus. When inducibly overexpressed, ORP9S and ORP9L colocalized with VAP-A and caused vacuolation of the ER as well as retention of the ER-Golgi intermediate compartment marker ERGIC-53/p58 in the ER. ORP9L mutated in the VAP-A binding domain (ORP9L-FY-->AA) did not localize to the ER but appeared with giantin and Sec31 on large vesicular structures, suggesting the presence of a hybrid Golgi-COPII compartment. Normal Golgi localization was also observed for ORP9L-FY-->AA. Results show that VAP binding and PH domains target ORP9 to the ER and a Golgi-COPII compartment, respectively, and that ORP9L overexpression in these compartments severely perturbed their organization.