Single-molecule analysis of protein targeting from the endoplasmic reticulum to lipid droplets

Numerous proteins migrate from the bilayer membrane of the endoplasmic reticulum (ER) to the monolayer surface of lipid droplets (LD) to perform critical metabolic functions. How cargoes move between these organelles and accumulate on LDs is unclear, particularly for mammalian cells. Here, we developed a synchronized ER-to-LD protein-trafficking assay and combined it with MINFLUX- and HILO-based single-molecule tracking and machine-learning analysis. Trafficking of a model protein LiveDrop between the ER and LDs occurs by diffusion through seipin- associated ER-LD membrane bridges that allow bidirectional protein movement. Unexpectedly, LiveDrop moves with the same apparent diffusion coefficient in the ER and LDs, but becomes molecularly confined at LD surfaces through a mechanism requiring its tryptophan residues. These data show how a specific LD-targeting membrane sequence leads to confined motion and LD protein accumulation, and directly demonstrate intra-organellar protein trafficking through membrane contact sites.