Abstract: A defining feature of cells are membrane bilayers that separate them from their environment and, in eukaryotic cells, delineate intracellular organelles with specialized functions. For cells to live and replicate, they must be able to maintain and expand these membranes. In eukaryotes, the lipid building blocks of membranes are made mostly in the endoplasmic reticulum (ER) and then transported to other organelles from there. While the role of vesicle trafficking for lipid transfer between organelles has long been known, we have only in the last decade begun to appreciate the importance of non-vesicular protein-mediated lipid transfer at so-called membrane contact sites, where two organelles are closely apposed. Indeed, the ER, the main site of lipid synthesis, contacts every other cellular organelle at such sites. The main focus of the laboratory is to identify and characterize the protein residents of contact sites in order to better elucidate the still largely unknown lipid transfer processes that occur there, their molecular basis, and their role in physiology. Contact site residents include lipid “shuttles”, proteins that equilibrate lipids between organelles, typically one lipid at a time, in order to fine-tune the lipid compositions of apposed contact site membranes. In addition, just in this last grant period, the PIs group was central to the discovery of elongated lipid transfer proteins in the VPS13 family that span between organelles and serve as bridges, with long hydrophobic grooves that solubilize lipid fatty acid moieties, allowing for bulk lipid flow across the aqueous space between membranes. Paradigm-shifting studies from the PI’s group and collaborator suggest that these lipid transfer bridges work in partnership with integral membrane proteins, such as scramblases, to function in membrane expansion and organelle biogenesis, for example in the de novo formation of the autophagosome or the yeast prospore membrane. The role of protein-mediated lipid transfer in organelle biogenesis was unanticipated and previously thought to involve exclusively the fusion of hundreds of vesicles, as vesicles were until recently the only known mechanism for bulk lipid movement. The PI will continue the systematic characterization of contact site proteins and their physiological roles; emphasis going forward will be to dissect the mechanisms by which bridge-like lipid transporters collaborate with integral membrane proteins, including scramblases, to transfer lipids directionally from their site of synthesis to other organelles for membrane maintenance or de novo organelle formation.