The endoplasmic reticulum–mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 β-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the β-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and β-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.

A deregulation of ceramide biosynthesis in the endoplasmic reticulum (ER) is frequently linked to induction of mitochondrial apoptosis. Although in vitro studies suggest that ceramides might initiate cell death by acting directly on mitochondria, their actual contribution to the apoptotic response in living cells is unclear. Here, we have analyzed the consequences of targeting the biosynthetic flow of ceramides to mitochondria using a ceramide transfer protein (encoded by COL4A3BP) equipped with an OMM anchor, mitoCERT. Cells expressing mitoCERT import ceramides into mitochondria and undergo Bax-dependent apoptosis. Apoptosis induction by mitoCERT was abolished through (i) removal of its ceramide transfer domain, (ii) disruption of its interaction with VAMPassociated proteins (VAPs) in the ER, (iii) addition of antagonistic CERT inhibitor HPA12, (iv) blocking de novo ceramide synthesis and (v) targeting of a bacterial ceramidase to mitochondria. Our data provide the first demonstration that translocation of ER ceramides to mitochondria specifically commits cells to death and establish mitoCERT as a valuable new tool to unravel the molecular principles underlying ceramide-mediated apoptosis.

G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs.

PurposeThe purpose of this paper is to describe and compare the different ways in which the shareholder disclosure requirements under the European Union Transparency Directive have been implemented in the UK, France, and Germany. Belgium and Luxembourg have not yet implemented the Directive.Design/methodology/approachThe paper explains the categories of entities that must comply with reporting requirements, disclosure/reporting thresholds, categories of direct and indirect shareholdings to which these thresholds apply, and the reporting required methods of notification.FindingsThe paper finds that the new European rules broaden the categories of entities that must comply with the reporting requirements. In the UK, France and Germany, thresholds are calculated based on all shares or other financial instruments to which voting rights are attached and indirect holdings must be aggregated and separately identified. Additionally, Germany imposes further notification requirements with respect to options.Originality/valueThe paper provides a useful comparative analysis of new shareholder disclosure requirements in the UK, France, and Germany.