New pathways for metabolic organization at organelle contact sites Hanaa Hariri, Wayne State University Mike Henne, UT Southwestern Medical Center Fatty acids (FA) are toxic to cells and thus sequestered in the form of triacylglycerides in cytoplasmic organelles called lipid droplets (LD) that bud from the endoplasmic reticulum (ER). We recently showed that, in response to nutrient stress, yeast LDs cluster adjacent to the vacuole/lysosome, but how this LD accumulation is spatially coordinated was unknown. We report that yeast ER-vacuole tether Mdm1 spatially regulates LD biogenesis at ER-vacuole contacts to maintain ER lipid homeostasis and protect cell from FA-induced lipotoxicity. Mdm1 binds to LDs and free FAs via its uncharacterized PX-Associated (PXA) domain and co-enriches with fatty acyl-CoA ligase Faa1 at LD bud sites. Consistent with this, loss of MDM1 perturbs free FA activation and subsequent TAG generation, elevating cellular FAs that perturbs ER morphology and sensitizes yeast to FA-induced lipotoxicity. Our data support a model where the molecular tether, Mdm1, organizes free FA activation adjacent to the vacuole to promote LD production in response to stress. This maintains ER lipid homeostasis by efficiently segregating toxic FAs into LDs. These findings establish a functional significance for organelle contacts in organizing FA metabolism and lipid flux. As an new investigator at WSU, I am currently investigating the mechanisms that spatially regulate the metabolic fates of free FAs at organelle contact sites using a combination of in vitro reconstitution, global lipidomics, and high-resolution microscopy.