ORP/Osh mediate cross-talk between ER-plasma membrane contact site components and plasma membrane SNAREs

OSBP-homologous proteins (ORPs, Oshp) are lipid binding/transfer proteins. Several ORP/Oshp localize to membrane contacts between the endoplasmic reticulum (ER) and the plasma membrane, where they mediate lipid transfer or regulate lipid-modifying enzymes. A common way in which they target contacts is by binding to the ER proteins, VAP/Scs2p, while the second membrane is targeted by other interactions with lipids or proteins. We have studied the cross-talk of secretory SNARE proteins and their regulators with ORP/Oshp and VAPA/Scs2p at ER-plasma membrane contact sites in yeast and murine primary neurons. We show that Oshp-Scs2p interactions depend on intact secretory SNARE proteins, especially Sec9p. SNAP-25/Sec9p directly interact with ORP/Osh proteins and their disruption destabilized the ORP/Osh proteins, associated with dysfunction of VAPA/Scs2p. DeletingOSH1-3in yeast or knocking down ORP2 in primary neurons reduced the oligomerization of VAPA/Scs2p and affected their multiple interactions with SNAREs. These observations reveal a novel cross-talk between the machineries of ER-plasma membrane contact sites and those driving exocytosis.Peer reviewe

OSBP-related protein 4L (ORP4L) interacts with the phosphoinositide 5-phosphatase OCRL1

Tiivistelmä – Referat – Abstract OCRL1 is a phosphatase that cleaves phosphatidylinositol 4,5-bisphosphate (PI4,5P) to phosphatidylinositol-4-phosphate (PI4P). ORP4 is a lipid binding/transport protein implicated in G-protein coupled signaling, cellular calcium homeostasis, proliferation and viability. OCRL1 and ORP4 are found in the endoplasmic reticulum and membrane contact sites throughout the endosomal system and the Golgi complex. OCRL1 is also present in the plasma membrane and vesicular structures. ORP4 has high affinity for binding sterols through the OSBP related domain (ORD). ORP4 also interacts with vimentin intermediate filaments via the ORD and influences the localization and organization of vimentin. The membrane lipid phosphatidylinositol (PI) can be phosphorylated either singly or in combination at three different positions on its inositol ring (D-3, D-4, and D-5)—yielding 8 possible phosphoinositides; the interconversion between the species is regulated by kinases and phosphatases that either add or remove phosphate groups from the various positions on the ring. Phosphoinositide metabolism is heavily involved in signal transduction pathways and cytoskeletal organization. Interestingly, it has also been found to be spatially regulated with distinct phosphoinositides being enriched in particular membrane compartments. BiFC assays can provide an important tool for visualizing protein-protein interactions. Not only is BiFC able to determine protein-protein proximity but it is also able to localize the interaction with regard to compartment membranes and structures. This study examined the interaction of ORP4 with OCRL1 by using BiFC analysis. We were able to determine that the protein pair seems to be in close proximity in the endoplasmic reticulum near the Golgi. Interaction only took place when the OCRL1 was tagged with VB at the C-terminal

ORP/Osh mediate cross-talk between ER-plasma membrane contact site components and plasma membrane SNAREs

OSBP-homologous proteins (ORPs, Oshp) are lipid binding/transfer proteins. Several ORP/Oshp localize to membrane contacts between the endoplasmic reticulum (ER) and the plasma membrane, where they mediate lipid transfer or regulate lipid-modifying enzymes. A common way in which they target contacts is by binding to the ER proteins, VAP/Scs2p, while the second membrane is targeted by other interactions with lipids or proteins. We have studied the cross-talk of secretory SNARE proteins and their regulators with ORP/Oshp and VAPA/Scs2p at ER-plasma membrane contact sites in yeast and murine primary neurons. We show that Oshp-Scs2p interactions depend on intact secretory SNARE proteins, especially Sec9p. SNAP-25/Sec9p directly interact with ORP/Osh proteins and their disruption destabilized the ORP/Osh proteins, associated with dysfunction of VAPA/Scs2p. Deleting OSH1-3 in yeast or knocking down ORP2 in primary neurons reduced the oligomerization of VAPA/Scs2p and affected their multiple interactions with SNAREs. These observations reveal a novel cross-talk between the machineries of ER-plasma membrane contact sites and those driving exocytosis

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

Background and aims: Among subjects with high-density-lipoprotein cholesterol (HDL-C) below the 1st percentile in the general population, we identified a heterozygous variant OSBPL1A p.C39X encoding a short truncated protein fragment that co-segregated with low plasma HDL-C. Methods: We investigated the composition and function of HDL from the carriers and non-carriers and studied the properties of the mutant protein in cultured hepatocytes. Results: Plasma HDL-C and apolipoprotein (apo) A-I were lower in carriers versus non-carriers, whereas the other analyzed plasma components or HDL parameters did not differ. Sera of the carriers displayed a reduced capacity to act as cholesterol efflux acceptors (p <0.01), whereas the cholesterol acceptor capacity of their isolated HDL was normal. Fibroblasts from a p.C39X carrier showed reduced cholesterol efflux to lipid-free apoA-I but not to mature HDL particles, suggesting a specific defect in ABCA1-mediated efflux pathway. In hepatic cells, GFP-OSBPL1A partially co-localized in endosomes containing fluorescent apoA-I, suggesting that OSBPL1A may regulate the intracellular handling of apoA-I. The GFP-OSBPL1A-39X mutant protein remained in the cytosol and failed to interact with Rab7, which normally recruits OSBPL1A to late endosomes/lysosomes, suggesting that this mutation represents a loss-of-function. Conclusions: The present work represents the first characterization of a human OSBPL1A mutation. Our observations provide evidence that a familial loss-of-function mutation in OSBPL1A affects the first step of the reverse cholesterol transport process and associates with a low HDL-C phenotype. This suggests that rare mutations in OSBPL genes may contribute to dyslipidemias. (C) 2016 Elsevier Ireland Ltd. All rights reserved.Peer reviewe