PROJECT SUMMARY Lipid storage is a fundamental process for organisms to buffer fluctuations in the availability and need for metabolic energy. Lipids are predominantly stored as neutral lipids, such as triacylglycerols (TGs), in organelles called lipid droplets (LDs). These organelles are hubs of metabolism with most of their biochemical functions being executed by proteins targeting their surface. Consistent with the important function of LD proteins in metabolism, their aberrant accumulation on LDs can cause diseases, such as liver disease or lipodystrophy. Despite the fundamental importance of LD proteins and their intimate link to human physiology and pathology, little is known about the mechanisms how protein target LDs. In this proposal, we address this question for some of the most important lipid metabolism enzymes that target LDs after initial insertion into the endoplasmatic reticulum. Capitalizing on results from the previous funding period, we will define the machinery building bridges between the ER and LDs for protein targeting and decipher the molecular driving forces for transport to LDs. Completing these aims will reveal a fundamental aspect of evolutionarily conserved cell biology. Since accumulation of protein variants on LDs causes common metabolic diseases, our may also provide new therapeutic avenues for intervention.