ABSTRACT Eukaryotic cells are characterized by their exquisite compartmentalization. Membrane-bound organelles form highly dynamic and interconnected networks. This complexity makes a permanent crosstalk between the organelles a necessity for the coordination of cellular functions. The tight juxtaposition of membranes from different types of organelles is essential to the controlled exchanges of matter and information within cells and is mediated by various organelle-tethering protein complexes. Small metabolites and messengers such as phospholipids (PLs) and Ca2+ are exchanged at these membrane contact sites (MCSs). Understanding the molecular mechanisms that regulate interactions between organelles will offer new insights into this fundamental aspect of eukaryotic cell biology. Our research focuses on the Endoplasmic Reticulum- Mitochondrion Encounter Structure (ERMES), a tether identified in the model eukaryote organism yeast, and functioning at ER-mitochondrial junctions also named Mitochondrion-Associated Membranes (MAMs). While many groups investigate MCSs, most of them use approaches based on genetic screens and cellular imaging methods combined with proteomics or metabolomics. We bring to bear biochemical, biophysical and structural methods to characterize ERMES at the level of molecular structure to understand its precise cellular functions and mechanism of action. ERMES is composed of five subunits, but besides its subunit composition nothing else is known about its architecture and mode of assembly at MAMs. Three of these subunits, the ER- anchored protein Mmm1, the soluble subunit Mdm12, and the mitochondrial membrane protein Mdm34, contain a SMP domain, a lipid-binding protein domain exclusively found in proteins located at MCSs from yeast to humans. Using mass-spectrometry, we showed that Mdm12 preferentially binds phosphatidylcholines while our 17-Å resolution negative-stain EM structure of the Mmm1/Mdm12 hetero-tetramer revealed that the soluble SMP domains not only bind phospholipids but also function as specific protein scaffolds to assemble the tether. This led us to propose a first and very rudimentary structural model for the ERMES-mediated exchange of PLs at MAMs. The lack of a biochemically tractable system reconstituted in vitro has hindered efforts to definitely establish the function(s) of ERMES. Here, we propose to complete the reconstitution of ERMES to characterize its subunit stoichiometry and identify its bona-fide lipid ligands. Using purified subunits we will reconstitute the SMP-core of ERMES on two distinctly labeled types of proteoliposomes and assess tethering and PL exchange using fluorescence-based biophysical methods in vitro. With this system, we will also dissect the mechanisms of ERMES regulation by the tail-anchored mitochondrial GTPase Gem1, an integral ERMES subunit that was shown to control tether assembly and lipid exchange. Last, we will determine the structure of ERMES by a `hybrid' approach comb...