Membrane bridges and nanodomain partitioning govern membrane protein targeting to lipid droplets

Description

This data depository is created to store the raw imaging and tracking data for the manuscript titled "Membrane bridges and nanodomain partitioning govern membrane protein targeting to lipid droplets". Abstract Numerous metabolic enzymes translocate from the ER membrane bilayer to the lipid droplet (LD) monolayer, where they perform essential functions. Mislocalization of certain LD-targeted membrane proteins, including HSD17B13 and PNPLA3, is implicated in metabolic dysfunction-associated steatotic liver disease (MASLD). However, the mechanisms governing the trafficking and accumulation of ER proteins on LDs remain poorly understood. Here, using MINFLUX and HILO single-molecule tracking combined with machine learning, we show that HSD17B13, GPAT4, and the model cargo LiveDrop diffuse at comparable speeds in the ER and on LDs, but become nano-confined upon reaching the LD surface. Mechanistic dissection of LiveDrop targeting revealed that this confinement, along with protein accumulation on LDs, depends on specific residues within its targeting motif. These residues mediate preferential interactions with nanoscale membrane domains, suggesting that LD-targeted proteins selectively partition into distinct lipid-protein environments that transiently alter local motion and concentrate them at the LD surface. Single-molecule trajectories further revealed bidirectional trafficking of LiveDrop across seipin-containing ER-LD bridges, providing direct evidence for lateral protein transfer across membrane contact sites. These findings establish nanodomain-based confinement as a key mechanism driving selective protein accumulation on LDs and reveal how membrane bridges between organelles facilitate protein sorting.

Institutions

• Sloan-Kettering Institute

  NY, New York
• Harvard Medical School

  MA, Boston

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