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?

New insights into the OSBP‒VAP cycle.

VAP-A is a major endoplasmic reticulum (ER) receptor that allows this organelle to engage numerous membrane contact sites with other organelles. One highly studied example is the formation of contact sites through VAP-A interaction with Oxysterol-binding protein (OSBP). This lipid transfer protein transports cholesterol from the ER to the trans-Golgi network owing to the counter-exchange of the phosphoinositide PI(4)P. In this review, we highlight recent studies that advance our understanding of the OSBP cycle and extend the model of lipid exchange to other cellular contexts and other physiological and pathological conditions.

VAP-A intrinsically disordered regions enable versatile tethering at membrane contact sites.

Membrane contact sites (MCSs) are heterogeneous in shape, composition, and dynamics. Despite this diversity, VAP proteins act as receptors for multiple FFAT motif-containing proteins and drive the formation of most MCSs that involve the endoplasmic reticulum (ER). Although the VAP-FFAT interaction is well characterized, no model explains how VAP adapts to its partners in various MCSs. We report that VAP-A localization to different MCSs depends on its intrinsically disordered regions (IDRs) in human cells. VAP-A interaction with PTPIP51 and VPS13A at ER-mitochondria MCS conditions mitochondria fusion by promoting lipid transfer and cardiolipin buildup. VAP-A also enables lipid exchange at ER-Golgi MCS by interacting with oxysterol-binding protein (OSBP) and CERT. However, removing IDRs from VAP-A restricts its distribution and function to ER-mitochondria MCS. Our data suggest that IDRs do not modulate VAP-A preference toward specific partners but do adjust their geometry to MCS organization and lifetime constraints. Thus, IDR-mediated VAP-A conformational flexibility ensures membrane tethering plasticity and efficiency.

A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterisation in different cell types.

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.

Molecular and cellular dissection of the oxysterol-binding protein cycle through a fluorescent inhibitor.

ORPphilins are bioactive natural products that strongly and selectively inhibit the growth of some cancer cell lines and are proposed to target intracellular lipid-transfer proteins of the oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phosphatidylinositol 4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular OSBP levels. By taking advantage of the intrinsic fluorescence of SWG, we followed its fate in cell cultures and show that its incorporation at the trans-Golgi network depends on cellular abundance of OSBP. Using in vitro membrane reconstitution systems and cellular imaging approaches, we also report that SWG inhibits specifically the lipid transfer activity of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels, and PI(4)P turnover were affected. Finally, using intermolecular FRET analysis, we demonstrate that SWG directly binds to the lipid-binding cavity of OSBP. Collectively these results describe SWG as a specific and intrinsically fluorescent pharmacological tool for dissecting OSBP properties at the cellular and molecular levels. Our findings indicate that SWG binds OSBP with nanomolar affinity, that this binding is sensitive to the membrane environment, and that SWG inhibits the OSBP-catalyzed lipid exchange cycle.