Abstract
Lipid droplet (LD) biogenesis is essential for lipid homeostasis during nutrient stress, yet how lipid intermediates are spatially organized to support efficient triacylglycerol (TAG) synthesis remains unclear. Here, we identify Nvj3 as a nutrient-responsive regulator that links diacylglycerol (DAG) availability to TAG synthesis and LD formation at the endoplasmic reticulum (ER). Nvj3 is induced by glucose depletion and recruited to LD-associated ER domains. Loss of Nvj3 causes neutral lipid accumulation under steady state conditions but delays TAG synthesis under acute inducible metabolic transitions. Using controlled TAG induction systems, we show that Nvj3 is required to couple Dga1-dependent TAG synthesis to LD formation. In the absence of Nvj3, TAG accumulates but remains inefficiently packaged into LDs. Consistent with this defect, nvj3Δ cells exhibit altered phospholipid remodeling and mislocalization of DAG away from ER domains during starvation. Together, these findings establish Nvj3 as an organizer of lipid availability during metabolic stress and suggest that spatial control of DAG is a key determinant of LD biogenesis.