Lipid droplet targeting drives substrate access during very long chain fatty acid activation by Fat1.

Fatty acids require conjugation with coenzyme A (CoA) for their incorporation into membrane lipids. Of these, very-long-chain fatty acids (VLCFAs), the precursors of yeast ceramides, are particularly hydrophobic. Yet, it remains unresolved where and how the sole VLCFA-CoA synthetase Fat1 activates them. Here, we show that lipid droplets (LDs) are both the site of free VLCFA storage and their place of activation. Free VLCFAs preferentially partition into LDs in molecular simulations and in vitro reconstitutions, whereas Fat1 targets the LD surface in cells via an N-terminal amphipathic helix. Structural predictions identify an essential hydrophobic cavity in Fat1 that connects the active site to the membrane, suggesting that VLCFAs shuttle directly from the LD core into the enzymatic pocket. Our model explains how the organelle targeting of a fatty acid activator evolved to match the biophysical properties, and consequently, the subcellular localization of its highly hydrophobic substrate.