Ligand-dependent localization and function of ORP–VAP complexes at membrane contact sites

Abstract

Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP-VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP-VAP association, alters the subcellular targeting of ORP-VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP-VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER-lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP-VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling.

Sterol liganding of OSBP-related proteins (ORPs) regulates the subcellular distribution of ORP–VAPA complexes and their impacts on organelle structure

“…Consistent with this idea, four-aa deletion mutants interfering with the sterol binding were reported to alter the localization of OSBP [41,42], ORP1L [16,42], and ORP9L [17,42]. However, no one has thus far studied how the cellular sterol status and high-affinity oxysterol ligands of ORPs affect the subcellular localization of ORP-VAP complexes, which according to the current view represent the functionally active form of these proteins.…”

“…Eight out of 12 ORPs contain a FFAT motif, which binds specifically to the type II integral ER proteins VAPA and VAPB [23,24,42]. In order to investigate the effects of ORP sterol ligands on the subcellular targeting of ORP-VAPA complexes, we employed the Bimolecular Fluorescence Complementation (BiFC) technique, which allows the visualization of protein proximity in living cells, with the fluorescence intensity varying according to the protein interaction strength [45].…”

“…Plasmids for Bimolecular Fluorescence Complementation (BiFC) of ORP and VAP proteins in mammalian cells have been published previously [22,42]. Plasmid for expression of mCherry-galactosyltransferase 1 (GalT1) fragment aa 1-81 was from Prof.…”

A loss-of-function variant in OSBPL1A predisposes to low plasma HDL cholesterol levels and impaired cholesterol efflux capacity

“…Moreover, the protein co-localized in some endosomes with Alexa568-labeled apoA-I internalized into the cells (Fig 4E). The apoA-I-containing organelles were occasionally encircled by the OSBPL1A signal, demonstrating that they represent apoA-I internalized in endosomes decorated by OSBPL1A or by ER cisternae with associated OSBPL1A [37]. OSBPL1A-39X, the short truncated N-terminal fragment, was only present in the cytosolic compartment and in the nucleus (Fig.…”

Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans

“…Osh1 localization was observed in the cytoplasm, Golgi and the nucleus-vacuole junction (45)(46)(47)(48). Osh2, Osh3, Osh6, and Osh7 were enriched in regions of the ER that are closely apposed to the plasma membrane (PM) (36).…”