ER nests are specialized ER subdomains in Arabidopsis where peroxisomes and lipid droplets form.

Organelles are defining features of eukaryotic cells, yet much remains to be learned about organelle biogenesis. Lipid droplets and peroxisomes, which play opposing roles in storing and catabolizing fats, form from a mysterious domain in the endoplasmic reticulum (ER). We used live-cell fluorescence microscopy to visualize peroxisome and lipid droplet biogenesis in young Arabidopsis seedlings, where lipid catabolism is active, and peroxisomes can be unusually large. We found that the ER domains where these organelles are born, which we term ER nests, are complex, dynamic structures that exclude general ER proteins but accumulate other proteins, including lipid biosynthetic enzymes and the COPII component SAR1. Furthermore, ER nests appear to define peroxisome-lipid droplet contact sites. Our findings provide a framework for understanding how these domains form and sort their protein components, illuminate eukaryotic lipid biosynthesis, and elucidate how distinct organelles arise from the ER.