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Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER). Relatively little is known about how sites of LD formation are selected and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)-synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER–LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG-synthases and additional LD biogenesis factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG. We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis