Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome.

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two sub-assemblies: Retriever composed of VPS35L, VPS26C, and VPS29; and the CCC complex which contains twelve subunits: COMMD1-COMMD10 and the coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy, and in silico predictions, we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilized by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a 16th subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

Specific subdomain localization of ER resident proteins and membrane contact sites resolved by electron microscopy.

The endoplasmic reticulum (ER) is a large, single-copy, membrane-bound organelle that comprises an elaborate 3D network of diverse structural subdomains, including highly curved tubules, flat sheets, and parts that form contacts with nearly every other organelle. The dynamic and complex organization of the ER poses a major challenge on understanding how its functioning - maintenance of the structure, distribution of its functions and communication with other organelles - is orchestrated. In this study, we resolved a unique localization profile within the ER network for several resident ER proteins representing a broad range of functions associated with the ER using immuno-electron microscopy and calculation of a relative labeling index (RLI). Our results demonstrated the effect of changing cellular environment on protein localization and highlighted the importance of correct protein expression level when analyzing its localization at subdomain resolution. We present new software tools for anonymization of images for blind analysis and for quantitative assessment of membrane contact sites (MCSs) from thin section transmission electron microscopy micrographs. The analysis of ER-mitochondria contacts suggested the presence of at least three different types of MCSs that responded differently to changes in cellular lipid loading status.