Linking Architecture and Function of Endoplasmic Reticulum - Plasma Membrane Contact Sites

All organelles of eukaryotic cells form an intricate network of membrane contact sites (MCS) and thereby promote cellular functions such as lipid metabolism, signalling and organelle biogenesis. The formation of cortical endoplasmic reticulum (cER) - plasma membrane (PM) contacts in budding yeast cells is mediated by a set of six conserved proteins. By combining correlative light and electron microscopy (CLEM) and electron cryo-tomography (cryo-ET) with yeast genetic techniques, this work addresses unanswered questions about the cellular architecture of ER-PM contacts and how protein organisation in uences their functionality. CLEM on resin-embedded cells shows that protein composition at MCS modulates the cER membrane curvature rather than the membrane distance to the PM. Speci cally, the tricalbins (Tcbs), the yeast orthologs of Extended-Synaptotagmins, localise to cortical ER of high membrane curvature. A high-throughput genetic screen uncovers a new role for Tcbs in the maintenance of PM bilayer lipid asymmetry and redundancies in lipid routes through other cellular MCS, such as nucleus-vacuole junctions and ER-mitochondria contacts. In order to visualise protein organisation in cells at the highest level of preservation, cryo-ET acquisition and image analysis methods for this study were rst established on BAR domain- containing proteins at the PM. These methods, in combination with cryo-focussed ion beam milling, were subsequently used to visualise ER-PM contact site protein organisation in situ. The tricalbins are lipid transfer proteins integral to the ER, which contact the PM via C2 domains, potentially in a calcium dependent manner. The cryo-ET data shows an increase in protein layer bound to the PM at high cellular calcium, compatible with increased C2 binding to the PM. Cryo-ET of cells induced for expression of tricalbin-3, but devoid of all other ER-PM-forming proteins, reveals protein densities bridging the ER and PM. The range of structures observed strongly favours a model in which the lipid transfer domain functions as shuttle between the two contact site membranes in vertical orientation