Seipin luminal domain is dispensable in establishing functional ER sites of lipid droplet biogenesis

Lipid droplet (LD) biogenesis occurs in the endoplasmic reticulum (ER), the mechanisms of which is not completely known. Seipin (Fld1 in yeast) is a crucial ER membrane protein that defines LD biogenesis sites. Here, we report truncated seipin, Fld1-∆LR in yeast, and the human equivalent hSeipin-∆LR, mutants lacking the conserved luminal domain region (LR). Remarkably, Fld1-∆LR localizes to ER subdomains independently of LDs like Fld1, and complements the lack of Fld1 function. Fld1-∆LR foci represent functional LD biogenesis sites as it recruits LD biogenesis machinery, including factors Nem1, Ldb16, Pex30, and Yft2 and become enriched in diacylglycerol upon induction of LD formation. We demonstrate that unlike Fld1, Fld1-∆LR or hSeipin-∆LR rescues the LD morphology defect and terbinafine drug sensitivity of fld1∆ ldb16∆ cells like hSeipin. Mutating proline 31 to glycine (P31G) abrogates functioning of Fld1-∆LR. Interestingly, Fld1-∆LR can oligomerize and assemble into decamer like Fld1. We find that fld1∆ cells expressing Fld1-∆LR/hSeipin-∆LR resulted in increased steady state triacylglycerol (TAG) levels, suggesting crucial regulatory role of the luminal domain in TAG partitioning into LDs. Collectively, we propose a mechanistic model in which Fld1-∆LR represent the minimal region in seipin that is necessary and sufficient to organize functional LD biogenesis sites.