Tld1 is a novel regulator of triglyceride lipolysis that demarcates a lipid droplet subpopulation.

Cells store lipids in the form of triglyceride (TG) and sterol-ester (SE) in lipid droplets (LDs). Distinct pools of LDs exist, but a pervasive question is how proteins localize to and convey functions to LD subsets. Here, we show the yeast protein YDR275W/Tld1 (for TG-associated LD protein 1) localizes to a subset of TG-containing LDs, and reveal it negatively regulates lipolysis. Mechanistically, Tld1 LD targeting requires TG, and is mediated by two distinct hydrophobic regions (HRs). Molecular dynamics simulations reveal Tld1 HRs interact with TG on LDs and adopt specific conformations on TG-rich LDs versus SE-rich LDs in yeast and human cells. Tld1-deficient yeast display no defect in LD biogenesis, but exhibit elevated TG lipolysis dependent on lipase Tgl3. Remarkably, over-expression of Tld1, but not LD protein Pln1/Pet10, promotes TG accumulation without altering SE pools. Finally, we find Tld1-deficient cells display altered LD mobilization during extended yeast starvation. We propose Tld1 senses TG-rich LDs and regulates lipolysis on LD subpopulations.