Via Proteins and Lipids – Versatility of VAPs at Dynamic Membrane Contact Sites

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

Unesterified cholesterol accumulates in late endosomes in cells expressing the misfolded cystic fibrosis transmembrane conductance regulator (CFTR). CFTR misfolding in the endoplasmic reticulum (ER) or general activation of ER stress led to dynein-mediated clustering of cholesterol-loaded late endosomes at the Golgi region, a process regulated by ER-localized VAMP-associated proteins (VAPs). We hypothesized that VAPs serve as intracellular receptors that couple lipid homeostasis through interactions with two phenylalanines in an acidic track (FFAT) binding signals (found in lipid sorting and sensing proteins, LSS) with proteostasis regulation. VAPB inhibited the degradation of ΔF508-CFTR. The activity was mapped to the ligand-binding major sperm protein (MSP) domain, which was sufficient in regulating CFTR biogenesis. We identified mutations in an unstructured loop within the MSP that uncoupled VAPB-regulated CFTR biogenesis from basic interactions with FFAT. Using this information, we defined functional and physical interactions between VAPB and proteostasis regulators (ligands), including the unfolded protein response sensor ATF6 and the ER degradation cluster that included FAF1, VCP, BAP31, and Derlin-1. VAPB inhibited the degradation of ΔF508-CFTR in the ER through interactions with the RMA1-Derlin-BAP31-VCP pathway. Analysis of pseudoligands containing tandem FFAT signals supports a competitive model for VAP interactions that direct CFTR biogenesis. The results suggest a model in which VAP-ligand binding couples proteostasis and lipid homeostasis leading to observed phenotypes of lipid abnormalities in protein folding diseases.

VAP (VAMP-associated protein) is a type II integral membrane protein of the endoplasmic reticulum (ER), and its N-terminal major sperm protein (MSP) domain faces the cytoplasmic side. VAP functions as a tethering molecule at the membrane contact sites between the ER and intracellular organelles and regulates a wide variety of cellular functions, including lipid transport, membrane trafficking, microtubule reorganization and unfolded protein response. VAP-point mutations in human vapb are strongly associated with amyotrophic lateral sclerosis. Importantly, the MSP domain of VAP is cleaved, secreted and interacts with the axon growth cone guidance receptors (Eph, Robo, Lar), suggesting that VAP could function as a circulating hormone similar to the Caenorhabditis elegans MSP protein. In this review, we discuss not only the intracellular functions of VAP but also the recently discovered extracellular functions and their implications for neurodegenerative disease.

Membrane contact sites (MCS) are sites of close apposition of two organelles that help in lipid transport and synthesis, calcium homeostasis and several other biological processes. The VAMP-associated proteins (VAPs) VAPA, VAPB, MOSPD2 and the recently described MOSPD1 and MOSPD3 are tether proteins of MCSs that are mainly found at the endoplasmic reticulum (ER). VAPs interact with various proteins with a motif called FFAT (two phenylalanines in an acidic tract), recruiting the associated organelle to the ER. In addition to the conventional FFAT motif, the recently described FFNT (two phenylalanines in a neutral tract) and phospho-FFAT motifs contribute to the interaction with VAPs. In this review, we summarize and compare the recent interactome studies described for VAPs, including in silico and proximity labeling methods. Collectively, the interaction repertoire of VAPs is very diverse and highlights the complexity of interactions mediated by the different FFAT motifs to the VAPs.

A point mutation (P56S) in VAPB (VAMP-associated membrane protein B) is associated with certain forms of familial amyotrophic lateral sclerosis (ALS), which is characterized by progressive loss of motor neuron function. VAPB is a member of the VAP family of proteins, which are typically associated with the endoplasmic reticulum (ER) and thought to function in lipid metabolism. They have an N-terminal major sperm protein (MSP) domain, a coiled-coil motif, and a transmembrane domain. Tsuda et al . found with immunofluorescent analysis of either endogenous proteins or versions tagged on the N and C termini with different epitope tags that the Drosophila homolog of human VAPB, which they call dVAP, was localized both intracellularly and extracellularly in wing imaginal discs. Western blot analysis of fly extracts or human white blood cells suggested that a cleaved form of the protein was present in addition to the full-length protein, and detection of the short forms in human serum suggested that the cleavage product was secreted. Analysis of a version of dVAP in which the residue homologous to the one associated with ALS was mutated (P58S) showed that the protein was not present extracellularly but instead formed aggregates in the wing disc cells or neurons. These P58S aggregates also included wild-type dVAP and ER proteins and appeared to trigger the ER stress response in motor neurons. Antibodies against ubiquitin recognized the aggregated P58S protein on Western blots of immunoprecipitated P58S, and ubiquitin colocalized with the aggregates in Drosophila neurons. Although presynaptic overexpression of wild-type dVAP alters motor neuron morphology and electrophysiological properties and causes muscle defects, as well as impairs flight, overexpression of P58S did not cause such phenotypes. Furthermore, the P58S mutant rescued the lethality of null mutations in dVAP; thus, P58S retains some biological activity but not all properties of the wild-type protein. Because VAPs have a MSP domain and MSPs are ligands for Eph homologs in Caenorhabditis elegans , the authors performed genetic experiments that placed dVAP and Eph in common developmental pathways in flies and placed VPR-1, the C. elegans VAP homolog, and VAB-1, the C. elegans Eph receptor, in common pathways in worms. Fluorescein isothiocyanate (FITC)-conjugated versions of the MSP domain of human VAP, dVAP, or VPR-1 were incubated with isolated C. elegans gonads, and all three proteins bound in the same pattern as that of MSP-FITC. Additionally, the hVAP MSP domain competed with MSP-FITC, suggesting that they recognized the same binding sites. The MSP domain of hVAP also competed with EphrinB2 for binding to EphA4 in a dose-dependent manner, suggesting that VAPs may serve as Eph ligands in mammals as well. Ackerman and Cox provide insight into the importance of these results in understanding the mechanism underlying ALS. H. Tsuda, S. M. Han, Y. Yang, C. Tong, Y. Q. Lin, K. Mohan, C. Haueter, A. Zoghbi, Y. Harati, J. Kwan, M. A. Miller, H. J. Bellen, The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors. Cell 133 , 963-977 (2008). [PubMed]

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.

Decision letter: The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

Membrane contact sites are cellular structures that mediate interorganelle exchange and communication. The two major tether proteins of the endoplasmic reticulum (ER), VAP‐A and VAP‐B, interact with proteins from other organelles that possess a small VAP‐interacting motif, named FFAT [two phenylalanines (FF) in an acidic track (AT)]. In this study, using an unbiased proteomic approach, we identify a novel ER tether named motile sperm domain‐containing protein 2 (MOSPD2). We show that MOSPD2 possesses a Major Sperm Protein (MSP) domain which binds FFAT motifs and consequently allows membrane tethering in vitro. MOSPD2 is an ER‐anchored protein, and it interacts with several FFAT‐containing tether proteins from endosomes, mitochondria, or Golgi. Consequently, MOSPD2 and these organelle‐bound proteins mediate the formation of contact sites between the ER and endosomes, mitochondria, or Golgi. Thus, we characterized here MOSPD2, a novel tethering component related to VAP proteins, bridging the ER with a variety of distinct organelles.

Productive developmental cycle of the obligate intracellular bacterial pathogen Chlamydia trachomatis depends on the interaction of the replicative vacuole, named the inclusion, with cellular organelles. We have recently reported the formation of ER-Inclusion membrane contact sites (MCSs), where the endoplasmic reticulum (ER) is in apposition to the inclusion membrane. These platforms contain the C. trachomatis inclusion membrane protein IncD, the mammalian ceramide transfer protein CERT and the ER resident proteins VAPA/B and were proposed to play a role in the non-vesicular trafficking of lipids to the inclusion. Here, we identify STIM1 as a novel component of ER-Inclusion MCSs. STIM1, an ER calcium (Ca2+) sensor that relocate to ER-Plasma Membrane (PM) MCSs upon Ca2+ store depletion, associated with C. trachomatis inclusion. STIM1, but not the general ER markers Rtn3C and Sec61ß, was enriched at the inclusion membrane. Ultra-structural studies demonstrated that STIM1 localized to ER-Inclusion MCSs. Time-course experiments showed that STIM1, CERT and VAPB co-localized throughout the developmental cycle. By contrast, Orai1, the PM Ca2+ channel that interacts with STIM1 at ER-PM MCSs, did not associate with C. trachomatis inclusion. Upon ER Ca2+ store depletion, a pool of STIM1 relocated to ER-PM MCSs, while the existing ER-Inclusion MCSs remained enriched in STIM1. Finally, we have identified the CAD domain, which mediates STIM1-Orai1 interaction, as the minimal domain required for STIM1 enrichment at ER-Inclusion MCSs. Altogether this study identifies STIM1 as a novel component of ER-C. trachomatis inclusion MCSs. We discuss the potential role(s) of STIM1 during the infection process.

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.

In many non-excitable cells, the depletion of endoplasmic reticulum (ER) Ca2+ stores leads to the dynamic formation of membrane contact sites (MCSs) between the ER and the plasma membrane (PM), which activates the store-operated Ca2+ entry (SOCE) to refill the ER store. Two different Ca2+-sensitive proteins, STIM1 and extended synaptotagmin-1 (E-syt1), are activated during this process. Due to the lack of live cell super-resolution imaging, how MCSs are dynamically regulated by STIM1 and E-syt1 coordinately during ER Ca2+ store depletion and replenishment remain unknown. With home-built super-resolution microscopes that provide superior axial and lateral resolution in live cells, we revealed that extracellular Ca2+ influx via SOCE activated E-syt1s to move towards the PM by ~12 nm. Unexpectedly, activated E-syt1s did not constitute the MCSs per se, but re-arranged neighboring ER structures into ring-shaped MCSs (230~280 nm in diameter) enclosing E-syt1 puncta, which helped to stabilize MCSs and accelerate local ER Ca2+ replenishment. Overall, we have demonstrated different roles of STIM1 and E-syt1 in MCS formation regulation, SOCE activation and ER Ca2+ store replenishment.

Maintaining lipid composition diversity in membranes from different organelles is critical for numerous cellular processes. However, many lipids are synthesized in the endoplasmic reticulum (ER) and require delivery to other organelles. In this scenario, formation of membrane contact sites (MCS) between neighbouring organelles has emerged as a novel non-vesicular lipid transport mechanism. Dissecting the molecular composition of MCS identified phosphoinositides (PIs), cholesterol, scaffolding/tethering proteins as well as Ca2+ and Ca2+-binding proteins contributing to MCS functioning. Compelling evidence now exists for the shuttling of PIs and cholesterol across MCS, affecting their concentrations in distinct membrane domains and diverse roles in membrane trafficking. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the plasma membrane (PM) not only controls endo-/exocytic membrane dynamics but is also critical in autophagy. Cholesterol is highly concentrated at the PM and enriched in recycling endosomes and Golgi membranes. MCS-mediated cholesterol transfer is intensely researched, identifying MCS dysfunction or altered MCS partnerships to correlate with de-regulated cellular cholesterol homeostasis and pathologies. Annexins, a conserved family of Ca2+-dependent phospholipid binding proteins, contribute to tethering and untethering events at MCS. In this chapter, we will discuss how Ca2+ homeostasis and annexins in the endocytic compartment affect the sensing and transfer of cholesterol and PIs across MCS.

STARD3 [(StAR)‐related lipid transfer domain protein 3] belongs to the START domain protein family, a group of protein having lipid transfer properties. STARD3 is a sterol binding protein located on the limiting membrane of late endosomes, where it is involved in cholesterol transport. Indeed in mammalian cells, high levels of STARD3 are associated with the presence of enlarged endosomes enriched in cholesterol. Using a correlative electron and light microscopy approach, we showed that STARD3 remodels the subcellular architecture. Remarkably, STARD3 ties endosomes with the endoplasmic reticulum. The molecular mechanism of this attachment was solved and relies on the specific interaction between STARD3 and VAP proteins [VAMP‐Associated Proteins (VAP‐A and VAP‐B)]. Altogether our results show that STARD3 and VAP proteins form a novel molecular machinery creating inter‐organelle membrane contacts sites (MCSs), governing the formation of a specific subcellular territory between endosomes and the endoplasmic reticulum. Using in vitro reconstitution, we are able to address the lipid transfer potential of this novel machinery. How STARD3‐made endoplasmic reticulum‐endosomes MCSs control the dynamics of the endosomal compartment will be presented.

The endoplasmic reticulum transmembrane protein vesicle-associated membrane protein-associated protein (VAP) plays a central role in the formation and function of membrane contact sites (MCS) through its interactions with proteins. The major sperm protein (MSP) domain of VAP binds to a variety of sequences which are referred to as FFAT-like motifs. In this study, we investigated the interactions of eight peptides containing FFAT-like motifs with the VAP-A MSP domain (VAP-AMSP ) by solution NMR. Six of eight peptides are specifically bound to VAP-A. Furthermore, we found that the RNA-dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2 has an FFAT-like motif which specifically binds to VAP-AMSP as well as other FFAT-like motifs. Our results will contribute to the discovery of new VAP interactors.