Vesicle-associated Membrane Protein-associated Protein Research Articles

ALS-Linked VapB P56S Mutation Alters Neuronal Mitochondrial Turnover at the Synapse.

Mitochondrial population maintenance in neurons is essential for neuron function and survival. Contact sites between mitochondria and the endoplasmic reticulum (ER) are poised to regulate mitochondrial homeostasis in neurons. These contact sites can facilitate transfer of calcium and lipids between the organelles and have been shown to regulate aspects of mitochondrial dynamics. Vesicle-associated membrane protein-associated protein B (VapB) is an ER membrane protein present at a subset of ER-mitochondrial contact sites. A proline-to-serine mutation in VapB at amino acid 56 (P56S) correlates with susceptibility to amyotrophic lateral sclerosis (ALS) type 8. Given the relationship between failed mitochondrial health and neurodegenerative disease, we investigated the function of VapB in mitochondrial population maintenance. We demonstrated that transgenic expression of VapBP56S in zebrafish larvae (sex undetermined) increased mitochondrial biogenesis, causing increased mitochondrial population size in the axon terminal. Expression of wild-type VapB did not alter biogenesis but, instead, increased mitophagy in the axon terminal. Using genetic manipulations to independently increase mitochondrial biogenesis, we show that biogenesis is normally balanced by mitophagy to maintain a constant mitochondrial population size. VapBP56S transgenics fail to increase mitophagy to compensate for the increase in mitochondrial biogenesis, suggesting an impaired mitophagic response. Finally, using a synthetic ER-mitochondrial tether, we show that VapB's function in mitochondrial turnover is likely independent of ER-mitochondrial tethering by contact sites. Our findings demonstrate that VapB can control mitochondrial turnover in the axon terminal, and this function is altered by the P56S ALS-linked mutation.

The Ribosome Assembly Factor LSG1 Interacts with Vesicle-Associated Membrane Protein-Associated Proteins (VAPs).

LSG1 is a conserved GTPase involved in ribosome assembly. It is required for the eviction of the nuclear export adapter NMD3 from the pre-60S subunit in the cytoplasm. In human cells, LSG1 has also been shown to interact with vesicle-associated membrane protein-associated proteins (VAPs) that are found primarily on the endoplasmic reticulum. VAPs interact with a large host of proteins which contain FFAT motifs (two phenylalanines (FF) in an acidic tract) and are involved in many cellular functions including membrane traffic and regulation of lipid transport. Here, we show that human LSG1 binds to VAPs via a noncanonical FFAT-like motif. Deletion of this motif specifically disrupts the localization of LSG1 to the ER, without perturbing LSG1-dependent recycling of NMD3 in cells or modulation of LSG1 GTPase activity invitro.

RILP Induces Cholesterol Accumulation in Lysosomes by Inhibiting Endoplasmic Reticulum-Endolysosome Interactions.

Endoplasmic reticulum (ER)-endolysosome interactions regulate cholesterol exchange between the ER and the endolysosome. ER-endolysosome membrane contact sites mediate the ER-endolysosome interaction. VAP-ORP1L (vesicle-associated membrane protein-associated protein- OSBP-related protein 1L) interaction forms the major contact site between the ER and the lysosome, which is regulated by Rab7. RILP (Rab7-interacting lysosomal protein) is the downstream effector of Rab7, but its role in the organelle interaction between the ER and the lysosome is not clear. In this study, we found RILP interacts with ORP1L to competitively inhibit the formation of the VAP-ORP1L contact site. Immunofluorescence microscopy revealed that RILP induces late endosome/lysosome clustering, which reduces the contact of endolysosomes with the ER, interfering with the ER-endolysosome interaction. Further examination demonstrated that over-expression of RILP results in the accumulation of cholesterol in the clustered endolysosomes, which triggers cellular autophagy depending on RILP. Our results suggest that RILP interferes with the ER-endolysosome interaction to inhibit cholesterol flow from the endolysosome to the ER, which feedbacks to trigger autophagy.

Coordination of oxysterol binding protein 1 and VAP-A/B modulates the generation of cholesterol and viral inclusion bodies to promote grass carp reovirus replication.

Similar to other RNA viruses, grass carp reovirus, the causative agent of the hemorrhagic disease, replicates in cytoplasmic viral inclusion bodies (VIBs), orchestrated by host proteins and lipids. The host pathways that facilitate the formation and function of GCRV VIBs are poorly understood. This work demonstrates that GCRV manipulates grass carp oxysterol binding protein 1 (named as gcOSBP1) and vesicle-associated membrane protein-associated protein A/B (named as gcVAP-A/B), 3 components of cholesterol transport pathway, to generate VIBs. By siRNA-mediated knockdown, we demonstrate that gcOSBP1 is an essential host factor for GCRV replication. We reveal that the nonstructural proteins NS80 and NS38 of GCRV interact with gcOSBP1, and that the gcOSBP1 is recruited by NS38 and NS80 for promoting the generation of VIBs. gcOSBP1 increases the expression of gcVAP-A/B and promotes the accumulation of intracellular cholesterol. gcOSBP1 also interacts with gcVAP-A/B for forming gcOSBP1-gcVAP-A/B complexes, which contribute to enhance the accumulation of intracellular cholesterol and gcOSBP1-mediated generation of VIBs. Inhibiting cholesterol accumulation by lovastatin can completely abolish the effects of gcOSBP1 and/or gcVAP-A/B in promoting GCRV infection, suggesting that cholesterol accumulation is vital for gcOSBP1- and/or gcVAP-A/B-mediated GCRV replication. Thus, our results, which highlight that gcOSBP1 functions in the replication of GCRV via its interaction with essential viral proteins for forming VIBs and with host gcVAP-A/B, provide key molecular targets for obtaining anti-hemorrhagic disease grass carp via gene editing technology.

Triazole derivatives inhibit the VOR complex-mediated nuclear transport of extracellular particles: Potential application in cancer and HIV-1 infection

Extracellular vesicles (EVs) appear to play an important role in intercellular communication in various physiological processes and pathological conditions such as cancer. Like enveloped viruses, EVs can transport their contents into the nucleus of recipient cells, and a new intracellular pathway has been described to explain the nuclear shuttling of EV cargoes. It involves a tripartite protein complex consisting of vesicle-associated membrane protein-associated protein A (VAP-A), oxysterol-binding protein (OSBP)-related protein-3 (ORP3) and late endosome-associated Rab7 allowing late endosome entry into the nucleoplasmic reticulum. Rab7 binding to ORP3-VAP-A complex can be blocked by the FDA-approved antifungal drug itraconazole. Here, we design a new series of smaller triazole derivatives, which lack the dioxolane moiety responsible for the antifungal function, acting on the hydrophobic sterol-binding pocket of ORP3 and evaluate their structure–activity relationship through inhibition of VOR interactions and nuclear transfer of EV and HIV-1 cargoes. Our investigation reveals that the most effective compounds that prevent nuclear transfer of EV cargo and productive infection by VSV-G-pseudotyped HIV-1 are those with a side chain between 1 and 4 carbons, linear or branched (methyl) on the triazolone region. These potent chemical drugs could find clinical applications either for nuclear transfer of cancer-derived EVs that impact metastasis or viral infection.

The VAMP-associated protein VAP27-1 plays a crucial role in plant resistance to ER stress by modulating ER–PM contact architecture in Arabidopsis

The endoplasmic reticulum (ER) and the plasma membrane (PM) form ER–PM contact sites (EPCSs) that allow the ER and PM to exchange materials and information. Stress-induced disruption of protein folding triggers ER stress, and the cell initiates the unfolded protein response (UPR) to resist the stress. However, whether EPCSs play a role in ER stress in plants remains unclear. VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP)-ASSOCIATED PROTEIN 27-1 (VAP27-1) functions in EPCS tethering and is encoded by a family of 10 genes (VAP27-1–10) in Arabidopsis thaliana. Here, we used CRISPR-Cas9-mediated genome editing to obtain a homozygous vap27-1 vap27-3 vap27-4 (vap27-1/3/4) triple mutant lacking three of the key VAP27 family members in Arabidopsis. The vap27-1/3/4 mutant exhibits defects in ER–PM connectivity and EPCS architecture, as well as excessive UPR signaling. We further showed that relocation of VAP27-1 to the PM mediates specific VAP27-1-related EPCS remodeling and expansion under ER stress. Moreover, the spatiotemporal dynamics of VAP27-1 at the PM increase ER–PM connectivity and enhance Arabidopsis resistance to ER stress. In addition, we revealed an important role for intracellular calcium homeostasis in the regulation of UPR signaling. Taken together, these results broaden our understanding of the molecular and cellular mechanisms of ER stress and UPR signaling in plants, providing additional clues for improving plant broad-spectrum resistance to different stresses.

Mitochondria-associated endoplasmic reticulum membranes tethering protein VAPB-PTPIP51 protects against ischemic stroke through inhibiting the activation of autophagy.

Mitochondria-associated endoplasmic reticulum membranes (MAMs) serve as a crucial bridge connecting the endoplasmic reticulum (ER) and mitochondria within cells. Vesicle-associated membrane protein-associated protein B (VAPB) and protein tyrosine phosphatase interacting protein 51 (PTPIP51) are responsible for the formation and stability of MAMs, which have been implicated in the pathogenesis of various diseases. However, the role of MAMs in ischemic stroke (IS) remains unclear. We aimed to investigate the role of MAMs tethering protein VAPB-PTPIP51 in experimental cerebral ischemia. We simulated cerebral ischemia-reperfusion injury (CIRI) by using a mouse middle cerebral artery occlusion (MCAO) model. We observed a decrease in VAPB-PTPIP51 expression in the brain tissue. Our findings suggested compromised MAMs after MCAO, as a decreased mitochondria-ER contact (MERC) coverage and an increased distance were observed through the transmission electron microscope (TEM). Upon VAPB or PTPIP51 knockdown, the damage to MAMs was exacerbated, accompanied by excessive autophagy activation and increased reactive oxygen species (ROS) production, resulting in an enlarged infarct area and exacerbated neurological deficits. Notably, we observed that this damage was concomitant with the inhibition of the PI3K/AKT/mTOR pathway and was successfully mitigated by the treatment with the PI3K activator. Our findings suggest that the downregulation of VAPB-PTPIP51 expression after IS mediates structural damage to MAMs. This may exacerbate CIRI by inhibiting the PI3K pathway and activating autophagy, thus providing new therapeutic targets for IS.

VAP spatially stabilizes dendritic mitochondria to locally support synaptic plasticity

Synapses are pivotal sites of plasticity and memory formation. Consequently, synapses are energy consumption hotspots susceptible to dysfunction when their energy supplies are perturbed. Mitochondria are stabilized near synapses via the cytoskeleton and provide the local energy required for synaptic plasticity. However, the mechanisms that tether and stabilize mitochondria to support synaptic plasticity are unknown. We identified proteins exclusively tethering mitochondria to actin near postsynaptic spines. We find that VAP, the vesicle-associated membrane protein-associated protein implicated in amyotrophic lateral sclerosis, stabilizes mitochondria via actin near the spines. To test if the VAP-dependent stable mitochondrial compartments can locally support synaptic plasticity, we used two-photon glutamate uncaging for spine plasticity induction and investigated the induced and adjacent uninduced spines. We find VAP functions as a spatial stabilizer of mitochondrial compartments for up to ~60 min and as a spatial ruler determining the ~30 μm dendritic segment supported during synaptic plasticity.

CircTP63 promotes prostate cancer progression via miR-421/VAMP associated protein A axis.

Background: Circular RNAs (circRNA) have a vital role in the progression of cancers. For instance, circTP63 is upregulated in prostate cancer (PCa) tissues compared with adjacent normal tissues. However, the role of circTP63 in prostate cancer is still unclear. Methods: qRT-PCR assays were applied to detected the expression of circTP63 and miR-421 in PCa samples. Functionally, CCK-8, apoptosis assay, and transwell migration and invasion assays were used to explore the role of circTP63 in PCa progression. Mechanistically, the interaction between circTP63 and miR-421 were verified using qRT-PCR and dual-luciferase report assay. Western blot, qRT-PCR, and dual-luciferase report assay were applied to detect the interaction between miR-421 and VAMP associated protein A (VAPA). And xenograft animal model was used to detect the role of circTP63 in vivo. Results: circTP63 was upregulated and miR-421 was downregulated in PCa tissues. Functional assays revealed that circTP63 promoted the proliferation and metastasis of PCa cells in vitro. In addition, the inhibition effect of circTP63 knockdown could be rescued by miR-421 inhibition or VAPA overexpression. Mechanistically, circTP63-mediated PCa progression through directly binding to miR-421, and subsequently releasing the VAPA. In vivo, silencing of circTP63 significantly impaired PCa progression. Conclusion: In summary, our study identified circTP63 as an oncogenic circRNA, which could be a promising diagnostic and therapeutic target for PCa treatment.

Serine-129 Phosphorylated α-Synuclein Regulates Endoplasmic Reticulum-Mitochondria Calcium Homeostasis via Interaction with VAPB and PTPIP51 in an α-Synuclein-induced Parkinson Disease Model

Serine-129 phosphorylated α-synuclein (p-α-syn), accounting for nearly 90% of α-synuclein (α-syn) found in Lewy bodies (LBs), is linked to the pathogenesis of Parkinson's disease (PD). The molecular targets for the cytotoxic effect of p-α-syn are not fully understood; however, so we sought to determine the role of p-α-syn in cell injury and describe the underlying molecular mechanism. Mitochondrial dysfunction was observed in primary neurons from Thy1-SNCA transgenic mice. Using co-immunoprecipitation coupled with mass spectrometry, we screened the p-α-syn interacting proteins in the midbrains of TG mice and validated the interaction with vesicle-associated membrane protein-associated protein (VAPB) and protein tyrosine phosphatase interacting protein 51 (PTPIP51), which are both located in the mitochondrion-associated endoplasmic reticulum (ER) membrane. VAPB binds to PTPIP51 tethering ER and mitochondria and has an important role in the transport of calcium. We showed that inhibition of α-syn phosphorylation at ser 129 increased the interaction between VAPB and PTPIP51. Moreover, we also demonstrated that inhibition of α-syn phosphorylation at ser 129 alleviated ER and mitochondrial calcium overload. These findings suggest that p-α-syn is involved in regulation of the ER and mitochondrial calcium, which provides new insight into the mechanism by which p-α-syn induces cellular toxicity and neurodegeneration.

ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.

Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between theER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3β decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.

Differential roles for ACBD4 and ACBD5 in peroxisome–ER interactions and lipid metabolism

Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. While collaborating in lipid metabolism, for example, of very long-chain fatty acids (VLCFAs) and plasmalogens, the ER also plays a role in peroxisome biogenesis. Recent work identified tethering complexes on the ER and peroxisome membranes that connect the organelles. These include membrane contacts formed via interactions between the ER protein VAPB (vesicle-associated membrane protein-associated protein B) and the peroxisomal proteins ACBD4 and ACBD5 (acyl-coenzyme A-binding domain protein). Loss of ACBD5 has been shown to cause a significant reduction in peroxisome-ER contacts and accumulation of VLCFAs. However, the role of ACBD4 and the relative contribution these two proteins make to contact site formation and recruitment of VLCFAs to peroxisomes remain unclear. Here, we address these questions using a combination of molecular cell biology, biochemical, and lipidomics analyses following loss of ACBD4 or ACBD5 in HEK293cells. We show that the tethering function of ACBD5 is not absolutely required for efficient peroxisomal β-oxidation of VLCFAs. We demonstrate that loss of ACBD4 does not reduce peroxisome-ER connections or result in the accumulation of VLCFAs. Instead, the loss of ACBD4 resulted in an increase in the rate of β-oxidation of VLCFAs. Finally, we observe an interaction between ACBD5 and ACBD4, independent of VAPB binding. Overall, our findings suggest that ACBD5 may act as a primary tether and VLCFA recruitment factor, whereas ACBD4 may have regulatory functions in peroxisomal lipid metabolism at the peroxisome-ER interface.

ARTC1-mediated VAPB ADP-ribosylation regulates calcium homeostasis.

Mono-ADP-ribosylation (MARylation) is a post-translational modification that regulates a variety of biological processes, including DNA damage repair, cell proliferation, metabolism, and stress and immune responses. In mammals, MARylation is mainly catalyzed by ADP-ribosyltransferases (ARTs), which consist of two groups: ART cholera toxin-like (ARTCs) and ART diphtheria toxin-like (ARTDs, also known as PARPs). The human ARTC (hARTC) family is composed of four members: two active mono-ADP-ARTs (hARTC1 and hARTC5) and two enzymatically inactive enzymes (hARTC3 and hARTC4). In this study, we systematically examined the homology, expression, and localization pattern of the hARTC family, with a particular focus on hARTC1. Our results showed that hARTC3 interacted with hARTC1 and promoted the enzymatic activity of hARTC1 by stabilizing hARTC1. We also identified vesicle-associated membrane protein-associated protein B (VAPB) as a new target of hARTC1 and pinpointed Arg50 of VAPB as the ADP-ribosylation site. Furthermore, we demonstrated that knockdown of hARTC1 impaired intracellular calcium homeostasis, highlighting the functional importance of hARTC1-mediated VAPB Arg50 ADP-ribosylation in regulating calcium homeostasis. In summary, our study identified a new target of hARTC1 in the endoplasmic reticulum and suggested that ARTC1 plays a role in regulating calcium signaling.

Dynamic interactome of the MHC I peptide loading complex in human dendritic cells

Dendritic cells (DCs) orchestrate immune responses by presenting antigenic peptides on major histocompatibility complex (MHC) molecules to T cells. Antigen processing and presentation via MHC I rely on the peptide-loading complex (PLC), a supramolecular machinery assembled around the transporter associated with antigen processing (TAP), which is the peptide transporter in the endoplasmic reticulum (ER) membrane. We studied antigen presentation in human DCs by isolating monocytes from blood and differentiating them into immature and mature DCs. We uncovered that during DC differentiation and maturation, additional proteins are recruited to the PLC, including B-cell receptor-associated protein 31 (BAP31), vesicle-associated membrane protein-associated protein A (VAPA), and extended synaptotagmin-1 (ESYT1). We demonstrated that these ER cargo export and contact site-tethering proteins colocalize with TAP and are within 40 nm proximity of the PLC, suggesting that the antigen processing machinery is located near ER exit- and membrane contact sites. While CRISPR/Cas9-mediated deletion of TAP and tapasin significantly reduced MHC I surface expression, single-gene deletions of the identified PLC interaction partners revealed a redundant role of BAP31, VAPA, and ESYT1 in MHC I antigen processing in DCs. These data highlight the dynamics and plasticity of PLC composition in DCs that previously was not recognized by the analysis of cell lines.

Lipid transport protein ORP2A promotes glucose signaling by facilitating RGS1 degradation.

Heterotrimeric GTP-binding proteins (G proteins) are a group of regulators essential for signal transmission into cells. Regulator of G protein signaling 1 (AtRGS1) possesses intrinsic GTPase-accelerating protein (GAP) activity and could suppress G protein and glucose signal transduction in Arabidopsis (Arabidopsis thaliana). However, how AtRGS1 activity is regulated is poorly understood. Here, we identified a knockout mutant of oxysterol binding protein-related protein 2A, orp2a-1, which exhibits similar phenotypes to the arabidopsis g-protein beta 1-2 (agb1-2) mutant. Transgenic lines overexpressing ORP2A displayed short hypocotyls, a hypersensitive response to sugar, and lower intracellular AtRGS1 levels than the control. Consistently, ORP2A interacted with AtRGS1 in vitro and in vivo. Tissue-specific expression of 2 ORP2A alternative splicing isoforms implied functions in controlling organ size and shape. Bioinformatic data and phenotypes of orp2a-1, agb1-2, and the orp2a-1 agb1-2 double mutant revealed the genetic interactions between ORP2A and Gβ in the regulation of G protein signaling and sugar response. Both alternative protein isoforms of ORP2A localized in the endoplasmic reticulum (ER), plasma membrane (PM), and ER-PM contact sites and interacted with vesicle-associated membrane protein-associated protein 27-1 (VAP27-1) in vivo and in vitro through their two phenylalanines in an acidic track-like motif. ORP2A also displayed differential phosphatidyl phosphoinositide binding activity mediated by the pleckstrin homology domain in vitro. Taken together, the Arabidopsis membrane protein ORP2A interacts with AtRGS1 and VAP27-1 to positively regulate G protein and sugar signaling by facilitating AtRGS1 degradation.

DNA Methylation Analysis Identifies Novel Epigenetic Loci in Dilated Murine Heart upon Exposure to Volume Overload

Left ventricular (LV) dilatation, a prominent risk factor for heart failure (HF), precedes functional deterioration and is used to stratify patients at risk for arrhythmias and cardiac mortality. Aberrant DNA methylation contributes to maladaptive cardiac remodeling and HF progression following pressure overload and ischemic cardiac insults. However, no study has examined cardiac DNA methylation upon exposure to volume overload (VO) despite being relatively common among HF patients. We carried out global methylome analysis of LV harvested at a decompensated HF stage following exposure to VO induced by aortocaval shunt. VO resulted in pathological cardiac remodeling, characterized by massive LV dilatation and contractile dysfunction at 16 weeks after shunt. Although methylated DNA was not markedly altered globally, 25 differentially methylated promoter regions (DMRs) were identified in shunt vs. sham hearts (20 hypermethylated and 5 hypomethylated regions). The validated hypermethylated loci in Junctophilin-2 (Jph2), Signal peptidase complex subunit 3 (Spcs3), Vesicle-associated membrane protein-associated protein B (Vapb), and Inositol polyphosphate multikinase (Ipmk) were associated with the respective downregulated expression and were consistently observed in dilated LV early after shunt at 1 week after shunt, before functional deterioration starts to manifest. These hypermethylated loci were also detected peripherally in the blood of the shunt mice. Altogether, we have identified conserved DMRs that could be novel epigenetic biomarkers in dilated LV upon VO exposure.

Glycolipid transfer protein knockout disrupts vesicle trafficking to the plasma membrane

The glycolipid transfer protein (GLTP) has been linked to many cellular processes aside from its best-known invitro function as a lipid transport protein. It has been proposed to act as a sensor and regulator of glycosphingolipid homeostasis in cells. Furthermore, through its previously determined interaction with the endoplasmic reticulum membrane protein VAP-A (vesicle-associated membrane protein-associated protein A), GLTP may also be involved in facilitating vesicular transport in cells. In this study, we characterized the phenotype of CRISPR/Cas9-mediated GLTP KO HeLa cells. We showed that motility, three-dimensional growth, and cellular metabolism were all altered by GLTP knockout. Expression of a GLTP mutant incapable of binding VAP disrupted cell spheroid formation, indicating that the GLTP-VAP interaction is linked to cellular adhesion, cohesion, and three-dimensional growth. Most notably, we found evidence that GLTP, through its interaction with VAP-A, affects vesicular trafficking, marking the first cellular process discovered to be directly impacted by a change in GLTP expression.

Legionella- and host-driven lipid flux at LCV-ER membrane contact sites promotes vacuole remodeling.

Legionella pneumophila replicates in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with ER-derived vesicles and a tight association with the ER. Proteomics of purifiedLCVs indicate the presence of membrane contact sites (MCS)proteins possibly implicated in lipid exchange. Using duallyfluorescence-labeled Dictyostelium discoideum amoeba, we revealthat VAMP-associated protein (Vap) and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER, and Vap also localizes to the LCV membrane. Furthermore, Vap as well as Sac1 promote intracellular replication of L.pneumophila and LCV remodeling. Oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and restrict (OSBP8) or promote (OSBP11) bacterial replication and LCV expansion. The sterol probes GFP-D4H* and filipin indicate that sterols are rapidly depleted from LCVs, while PtdIns(4)P accumulates. In addition to Sac1, the PtdIns(4)P-subverting L.pneumophila effector proteins LepB and SidC also support LCV remodeling. Taken together, the Legionella- and host cell-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole maturation.

Stranger Twins: A Tale of Resemblance and Contrast Between VAP Proteins.

When considering the vesicle-associated membrane protein-associated protein (VAP) family, major receptors at the surface of the endoplasmic reticulum (ER), it appears that VAP-A and VAP-B paralogs largely overlap in structure and function, and that specific features to distinguish these two proteins hardly exist or are poorly documented. Here, we question the degree of redundancy between VAP-A and VAP-B: is one simply a backup plan, in case of loss of function of one of the two genes, or are there molecular and functional divergences that would explain their maintenance during evolution?

Arabidopsis ORP2A mediates ER–autophagosomal membrane contact sites and regulates PI3P in plant autophagy

Autophagy is an intracellular degradation system for cytoplasmic constituents which is mediated by the formation of a double-membrane organelle termed the autophagosome and its subsequent fusion with the lysosome/vacuole. The formation of the autophagosome requires membrane from the endoplasmic reticulum (ER) and is tightly regulated by a series of autophagy-related (ATG) proteins and lipids. However, how the ER contacts autophagosomes and regulates autophagy remain elusive in plants. In this study, we identified and demonstrated the roles of Arabidopsis oxysterol-binding protein-related protein 2A (ORP2A) in mediating ER-autophagosomal membrane contacts and autophagosome biogenesis. We showed that ORP2A localizes to both ER-plasma membrane contact sites (EPCSs) and autophagosomes, and that ORP2A interacts with both the ER-localized VAMP-associated protein (VAP) 27-1 and ATG8e on the autophagosomes to mediate the membrane contact sites (MCSs). In ORP2A artificial microRNA knockdown (KD) plants, seedlings display retarded growth and impaired autophagy levels. Both ATG1a and ATG8e accumulated and associated with the ER membrane in ORP2A KD lines. Moreover, ORP2A binds multiple phospholipids and shows colocalization with phosphatidylinositol 3-phosphate (PI3P) invivo. Taken together, ORP2A mediates ER-autophagosomal MCSs and regulates autophagy through PI3P redistribution.