Coordinated lipid transfer between the endoplasmic reticulum and the Golgi complex requires the VAP proteins and is essential for Golgi-mediated transport.
ABSTRACT: Lipid transport between intracellular organelles is mediated by vesicular and nonvesicular transport mechanisms and is critical for maintaining the identities of different cellular membranes. Nonvesicular lipid transport between the endoplasmic reticulum (ER) and the Golgi complex has been proposed to affect the lipid composition of the Golgi membranes. Here, we show that the integral ER-membrane proteins VAP-A and VAP-B affect the structural and functional integrity of the Golgi complex. Depletion of VAPs by RNA interference reduces the levels of phosphatidylinositol-4-phosphate (PI4P), diacylglycerol, and sphingomyelin in the Golgi membranes, and it leads to substantial inhibition of Golgi-mediated transport events. These effects are coordinately mediated by the lipid-transfer/binding proteins Nir2, oxysterol-binding protein (OSBP), and ceramide-transfer protein (CERT), which interact with VAPs via their FFAT motif. The effect of VAPs on PI4P levels is mediated by the phosphatidylinositol/phosphatidylcholine transfer protein Nir2, which is required for Golgi targeting of OSBP and CERT and the subsequent production of diacylglycerol and sphingomyelin. We propose that Nir2, OSBP, and CERT function coordinately at the ER-Golgi membrane contact sites, thereby affecting the lipid composition of the Golgi membranes and consequently their structural and functional identities.
Project description:Sphingolipids such as ceramide are important constituents of cell membranes. The ceramide transfer protein (CERT) moves ceramide from the endoplasmic reticulum to the Golgi apparatus in a nonvesicular manner. Hyperphosphorylation of the serine-repeat motif (SRM) adjacent to the pleckstrin homology (PH) domain of CERT down-regulates the inter-organelle ceramide transport function of CERT. However, the mechanistic details of this down-regulation remain elusive. Using solution NMR and binding assays, we herein show that a hyperphosphorylation-mimetic CERT variant in which 10 serine/threonine residues of SRM had been replaced with glutamate residues (the 10E variant) displays an intramolecular interaction between SRM and positively charged regions of the PH domain, which are involved in the binding of this domain to phosphatidylinositol 4-monophosphate (PI4P). Of note, the binding of the PH domain to PI4P-embedded membranes was attenuated by the SRM 10E substitutions in cell-free assays. Moreover, the 10E substitutions reduced the Golgi-targeting activity of the PH-SRM construct in living cells. These results indicate that hyperphosphorylated SRM directly interacts with the surface of the PH domain in an intramolecular manner, thereby decreasing the PI4P-binding activity of the PH domain. In light of these findings, we propose that the hyperphosphorylation of SRM may trigger the dissociation of CERT from the Golgi apparatus, resulting in a functionally less active conformation of CERT.
Project description:The endoplasmic reticulum (ER)-resident proteins vesicle-associated membrane protein (VAMP)-associated protein A and B (VAPA and VAPB) have been reported to be necessary for efficient hepatitis C virus (HCV) replication, but the specific mechanisms are not well understood. VAPs are known to recruit lipid transfer proteins to the ER, including oxysterol binding protein (OSBP), which has been previously shown to be necessary for cholesterol delivery to the HCV replication organelle in exchange for phosphatidylinositol 4-phosphate [PI(4)P]. Here, we show that VAPA and VAPB are redundant for HCV infection and that dimerization is not required for their function. In addition, we identify the phosphatidylinositol transfer protein Nir2 as an effector of VAPs to support HCV replication. We propose that Nir2 functions to replenish phosphoinositides at the HCV replication organelle to maintain elevated steady-state levels of PI(4)P, which is removed by OSBP. Thus, Nir2, along with VAPs, OSBP, and the phosphatidylinositol 4-kinase, completes a cycle of phosphoinositide flow between the ER and viral replication organelles to drive ongoing viral replication.IMPORTANCE Hepatitis C virus (HCV) is known for its ability to modulate phosphoinositide signaling pathways for its replication. Elevated levels of phosphatidylinositol 4-phosphate [PI(4)P] in HCV replication organelles (ROs) recruits lipid transfer proteins (LTPs), like oxysterol-binding protein (OSBP). OSBP exchanges PI(4)P with cholesterol, thus removing PI(4)P from the HCV RO. Here, we found that the phosphatidylinositol transfer protein Nir2 acts as an LTP and may replenish PI at the HCV RO by interacting with VAMP-associated proteins (VAPs), enabling continuous viral replication during chronic infection. Therefore, the coordination of OSBP, Nir2, and VAPs completes our understanding of the phosphoinositide cycle between the ER and HCV ROs.
Project description:Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport.
Project description:The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol-binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi network (TGN). Using the inhibitor OSW-1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4-kinase PI4KIII? triggers large periodic traveling waves of PI4P across the TGN These waves are cadenced by long-range PI4P production by PI4KII? and PI4P consumption by OSBP Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4-kinases to control the lipid composition of subcellular membranes.
Project description:Sphingomyelin (SM) and cholesterol are coregulated metabolically and associate physically in membrane microdomains involved in cargo sorting and signaling. One mechanism for regulation of this metabolic interface involves oxysterol binding protein (OSBP) via high-affinity binding to oxysterol regulators of cholesterol homeostasis and activation of SM synthesis at the Golgi apparatus. Here, we show that OSBP regulation of SM synthesis involves the endoplasmic reticulum (ER)-to-Golgi ceramide transport protein (CERT). RNA interference (RNAi) experiments in Chinese hamster ovary (CHO)-K1 cells revealed that OSBP and vesicle-associated membrane protein-associated protein (VAP) were required for stimulation of CERT-dependent ceramide transport and SM synthesis by 25-hydroxycholesterol and cholesterol depletion in response to cyclodextrin. Additional RNAi experiments in human embryonic kidney 293 cells supported OSBP involvement in oxysterol-activated SM synthesis and also revealed a role for OSBP in basal SM synthesis. Activation of ER-to-Golgi ceramide transport in CHO-K1 cells required interaction of OSBP with the ER and Golgi apparatus, OSBP-dependent Golgi translocation of CERT, and enhanced CERT-VAP interaction. Regulation of CERT by OSBP, sterols, and VAP reveals a novel mechanism for integrating sterol regulatory signals with ceramide transport and SM synthesis in the Golgi apparatus.
Project description:Hepatitis C virus (HCV) RNA replicates its genome on specialized endoplasmic reticulum modified membranes termed membranous web and utilizes lipid droplets for initiating the viral nucleocapsid assembly. HCV maturation and/or the egress pathway requires host sphingolipid synthesis, which occur in the Golgi. Ceramide transfer protein (CERT) and oxysterol-binding protein (OSBP) play a crucial role in sphingolipid biosynthesis. Protein kinase D (PKD), a serine/threonine kinase, is recruited to the trans-Golgi network where it influences vesicular trafficking to the plasma membrane by regulation of several important mediators via phosphorylation. PKD attenuates the function of both CERT and OSBP by phosphorylation at their respective Ser(132) and Ser(240) residues (phosphorylation inhibition). Here, we investigated the functional role of PKD in HCV secretion. Our studies show that HCV gene expression down-regulated PKD activation. PKD depletion by shRNA or inhibition by pharmacological inhibitor Gö6976 enhanced HCV secretion. Overexpression of a constitutively active form of PKD suppressed HCV secretion. The suppression by PKD was subverted by the ectopic expression of nonphosphorylatable serine mutant CERT S132A or OSBP S240A. These observations imply that PKD negatively regulates HCV secretion/release by attenuating OSBP and CERT functions by phosphorylation inhibition. This study identifies the key role of the Golgi components in the HCV maturation process.
Project description:Vesicle-associated membrane protein-associated protein (VAP) is an endoplasmic reticulum (ER)-resident integral membrane protein that controls a nonvesicular mode of ceramide and cholesterol transfer from the ER to the Golgi complex by interacting with ceramide transfer protein and oxysterol-binding protein (OSBP), respectively. We report that VAP and its interacting proteins are required for the processing and secretion of pancreatic adenocarcinoma up-regulated factor, whose transport from the trans-Golgi network (TGN) to the cell surface is mediated by transport carriers called "carriers of the trans-Golgi network to the cell surface" (CARTS). In VAP-depleted cells, diacylglycerol level at the TGN was decreased and CARTS formation was impaired. We found that VAP forms a complex with not only OSBP but also Sac1 phosphoinositide phosphatase at specialized ER subdomains that are closely apposed to the trans-Golgi/TGN, most likely reflecting membrane contact sites. Immobilization of ER-Golgi contacts dramatically reduced CARTS production, indicating that association-dissociation dynamics of the two membranes are important. On the basis of these findings, we propose that the ER-Golgi contacts play a pivotal role in lipid metabolism to control the biogenesis of transport carriers from the TGN.
Project description:Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIbeta and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.
Project description:Mutations in the ER-associated VAPB/ALS8 protein cause amyotrophic lateral sclerosis and spinal muscular atrophy. Previous studies have argued that ER stress may underlie the demise of neurons. We find that loss of VAP proteins (VAPs) leads to an accumulation of aberrant lysosomes and impairs lysosomal degradation. VAPs mediate ER to Golgi tethering and their loss may affect phosphatidylinositol-4-phosphate (PtdIns4P) transfer between these organelles. We found that loss of VAPs elevates PtdIns4P levels in the Golgi, leading to an expansion of the endosomal pool derived from the Golgi. Fusion of these endosomes with lysosomes leads to an increase in lysosomes with aberrant acidity, contents, and shape. Importantly, reducing PtdIns4P levels with a PtdIns4-kinase (PtdIns4K) inhibitor, or removing a single copy of Rab7, suppress macroautophagic/autophagic degradation defects as well as behavioral defects observed in Drosophila Vap33 mutant larvae. We propose that a failure to tether the ER to the Golgi when VAPs are lost leads to an increase in Golgi PtdIns4P levels, and an expansion of endosomes resulting in an accumulation of dysfunctional lysosomes and a failure in proper autophagic lysosomal degradation. Abbreviations: ALS: amyotrophic lateral sclerosis; CSF: cerebrospinal fluid; CERT: ceramide transfer protein; FFAT: two phenylalanines in an acidic tract; MSP: major sperm proteins; OSBP: oxysterol binding protein; PH: pleckstrin homology; PtdIns4P: phosphatidylinositol-4-phosphate; PtdIns4K: phosphatidylinositol 4-kinase; UPR: unfolded protein response; VAMP: vesicle-associated membrane protein; VAPA/B: mammalian VAPA and VAPB proteins; VAPs: VAMP-associated proteins (referring to Drosophila Vap33, and human VAPA and VAPB).
Project description:Cholesterol, which is endocytosed to the late endosome (LE)/lysosome, is delivered to other organelles through vesicular and nonvesicular transport mechanisms. In this study, we discuss a novel mechanism of cholesterol transport from recycling endosomes (REs) to the trans-Golgi network (TGN) through RELCH/KIAA1468, which is newly identified in this study as a Rab11-GTP- and OSBP-binding protein. After treating cells with 25-hydroxycholesterol to induce OSBP relocation from the cytoplasm to the TGN, REs accumulated around the TGN area, but this accumulation was diminished in RELCH- or OSBP-depleted cells. Cholesterol content in the TGN was decreased in Rab11-, RELCH-, and OSBP-depleted cells and increased in the LE/lysosome. According to in vitro reconstitution experiments, RELCH tethers Rab11-bound RE-like and OSBP-bound TGN-like liposomes and promotes OSBP-dependent cholesterol transfer from RE-like to TGN-like liposomes. These data suggest that RELCH promotes nonvesicular cholesterol transport from REs to the TGN through membrane tethering.