Seipin is an endoplasmic reticulum (ER) membrane protein implicated in lipid droplet (LD) biogenesis and mutated in severe congenital lipodystrophy (BSCL2). Here, we show that seipin is stably associated with nascent ER–LD contacts in human cells, typically via one mobile focal point per LD. Seipin appears critical for such contacts since ER–LD contacts were completely missing or morphologically aberrant in seipin knockout and BSCL2 patient cells. In parallel, LD mobility was increased and protein delivery from the ER to LDs to promote LD growth was decreased. Moreover, while growing LDs normally acquire lipid and protein constituents from the ER, this process was compromised in seipin‐deficient cells. In the absence of seipin, the initial synthesis of neutral lipids from exogenous fatty acid was normal, but fatty acid incorporation into neutral lipids in cells with pre‐existing LDs was impaired. Together, our data suggest that seipin helps to connect newly formed LDs to the ER and that by stabilizing ER–LD contacts seipin facilitates the incorporation of protein and lipid cargo into growing LDs in human cells.

Graphical AbstractHighlights d Seipin can determine the site of LD formation d Seipin-mediated ER-LD membrane contacts display a uniform neck-like architecture d Seipin at ER-LD contact facilitates continuous triglyceride transfer to LD d Acute removal of seipin reveals a principle of LD ripening via the ER SUMMARY Seipin is an oligomeric integral endoplasmic reticulum (ER) protein involved in lipid droplet (LD) biogenesis. To study the role of seipin in LD formation, we relocalized it to the nuclear envelope and found that LDs formed at these new seipin-defined sites. The sites were characterized by uniform seipinmediated ER-LD necks. At low seipin content, LDs only grew at seipin sites, and tiny, growth-incompetent LDs appeared in a Rab18-dependent manner. When seipin was removed from ER-LD contacts within 1 h, no lipid metabolic defects were observed, but LDs became heterogeneous in size. Studies in seipin-ablated cells and model membranes revealed that this heterogeneity arises via a biophysical ripening process, with triglycerides partitioning from smaller to larger LDs through droplet-bilayer contacts. These results suggest that seipin supports the formation of structurally uniform ER-LD contacts and facilitates the delivery of triglycerides from ER to LDs. This counteracts ripening-induced shrinkage of small LDs.

Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron miniIAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes. Loss-of-function methods for reducing target protein levels target DNA, RNA or protein 1. Auxin-inducible degron (AID) technology allows rapid targeted protein depletion with the smallmolecule auxin 2. To apply AID, an auxin-inducible destabilizing domain, or 'degron' , is fused to the target protein. In addition, an auxin receptor F-box protein TIR1/AFB is exogenously expressed, forming a chimeric SKP1-CUL1-F-Box (SCF) ubiquitin E3 ligase with endogenous components in eukaryotic cells. The chimeric E3 ligase recruits the degron in an auxin-dependent manner 3. Addition of indole-3-acetic acid (IAA) or other small molecules of the auxin class to the culture medium thus induces the polyubiquitination and rapid proteasomal degradation of the degron-fused protein (Fig. 1a). This approach has been used as a powerful tool to acutely deplete target proteins in studying their functions 4-7. However, current AID systems can severely degrade target proteins before IAA addition (known as 'basal degradation') and suffer from inefficient auxin-inducible depletion in a context-and target-specific manner 2,8-10. These pitfalls have substantially limited the applications of AID technology. An AID system with both minimal basal degradation and rapid auxin-inducible depletion would be desirable. In current AID systems, Oryza sativa TIR1 (OsTIR1) is the most commonly used auxin receptor F-box protein in combination with different degrons deriving from Arabidopsis thaliana IAA17 (AtIAA17) 2,4,5,7-14. We initially aimed to acutely deplete endogenous human seipin, a transmembrane protein involved in lipid droplet (LD) biogenesis. However, seipin tagged with a degron termed miniAID (composed of AtIAA17 amino acid (aa) 68-132) was severely degraded in cells expressing OsTIR1 without IAA addition. Consequently, cells exhibited defective LD biogenesis already before IAA addition, resembling a seipin knockout phenotype 15 (Supplementary Fig. 1a-c). To search for an improved AID system, we established a pipeline in human A431 cells for screening various auxin receptor F-box proteins and degrons, through co-integration into the adenoassociated virus integration site 1 (AAVS1) safe harbor locus 16 (Fig. 1b and Supplementary Note 1). We first compared OsTIR1 to five other auxin receptor F-box proteins, using miniAID as the degron. A. thaliana AFB2 (AtAFB2) was identified as the best hit: compared to OsTIR1, it displayed minimal basal depletion