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VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites

ABSTRACT: VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites

PROVIDER: PRJNA777941 | ENA |

REPOSITORIES: ENA

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VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites

Project description:RNA transfer via extracellular vesicles (EVs) influences cell phenotypes; however, lack of information regarding biogenesis of RNA-containing EVs has limited progress in the field. Here, we identify endoplasmic reticulum membrane contact sites (ER MCS) as platforms for generation of RNA-containing EVs. We identify a subpopulation of small EVs that is highly enriched in RNA and regulated by the ER MCS linker protein VAP-A. Functionally, VAP-A-regulated EVs are critical for miR-100 transfer between cells and in vivo tumor formation. Lipid analysis of VAP-A-knockdown EVs revealed reductions in the EV biogenesis lipid ceramide. Knockdown of the VAP-A-binding ceramide transfer protein CERT led to similar defects in EV RNA content. Imaging experiments revealed that VAP-A promotes lumenal filling of multivesicular bodies (MVBs). CERT localizes to MVBs, and the ceramide-generating enzyme neutral sphingomyelinase 2 colocalizes with VAP-A-positive ER. We propose that ceramide transfer via VAP-A-CERT linkages drives biogenesis of a select RNA-containing population.

2022-04-05 | GSE188223 | GEO

Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites

Project description:Membrane contact sites are cellular structures that mediate inter-organelle exchange and communication. The endoplasmic reticulum (ER), which forms a network extending throughout the cytoplasm, possesses two known major tether proteins, named VAP-A and VAP-B, that allow its interaction with other organelles. VAP-A and VAP-B interact with proteins from other organelles that possess a small VAP-interacting motif, named FFAT (two phenylalanines in an acidic track), and consequently scaffold contact sites implicating the ER. In this study, by using an unbiased proteomic approach, we identified a novel ER tether named MOtile SPerm Domain-containing protein 2 (MOSPD2). We showed that MOSPD2 possesses a Motile Sperm Protein (MSP) domain which binds FFAT motifs and consequently allows membrane tethering in vitro. Accordingly, MOSPD2, which is an ER-anchored protein, interacts with several FFAT-containing proteins bound to diverse organelles,such as endosomes, mitochondria or Golgi. Consequently, the specific interaction between MOSPD2 and these organelle-bound proteins results in the formation of contact sites between the ER and these organelles. Thus, MOSPD2 is a novel tether for membrane contact sites between the ER and the other cellular organelles.

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The Legionella-driven PtdIns(4)P gradient at LCV-ER membrane contact sites promotes Vap-, OSBP and Sac1-dependent pathogen vacuole remodeling

Project description:The causative agent of Legionnaires’ disease, Legionella pneumophila, governs interactions with host cells by secreting ca. 330 different “effector” proteins. The facultative intracellular bacteria replicate in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide (PI) lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with endoplasmic reticulum (ER)-derived vesicles and a tight association with the ER. Proteomics of purified LCVs revealed the presence of membrane contact sites (MCS) proteins implicated in lipid exchange. Using dually fluorescence-labeled Dictyostelium discoideum amoeba, we reveal that the VAMP-associated protein (Vap), the PtdIns(4)P 4-phosphatase Sac1, and the large fusion GTPase Sey1/atlastin-3 localize to the ER, but not to the LCV membrane, and these ER-resident proteins promote intracellular replication of L. pneumophila and LCV remodeling. Moreover, oxysterol binding proteins (OSBPs) exclusively localize to the ER (OsbH) or the LCV membrane (OsbK), respectively, and promote (OsbH) or restrict (OsbK) intracellular replication of L. pneumophila and LCV expansion. Furthermore, the PtdIns(4)P-subverting L. pneumophila effectors LepB and SidC also promote LCV remodeling. Taken together, the Legionella-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole remodeling.

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Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites

Project description:Vesicular traffic and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of membrane tethers to contact sites between the endoplasmic reticulum (ER) and the plasma membrane is often triggered by calcium. In contrast, we reveal here a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or sustained receptor signaling triggers the depletion of cholesterol and associated complex glycosphingolipids from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of bacterial Shiga toxin. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites.

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APEX2 proximity labeling of PI(3)P dependent ER and early endosome membrane contact tether proteins

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An ER translocon for multi-pass membrane protein biogenesis.

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