Membrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Here, we show that LD membrane proteins can be delivered to the ER either co- or post-translationally and that their membrane-embedded region specifies pathway selection. The co-translational route for LD membrane protein biogenesis is insensitive to a small molecule inhibitor of the Sec61 translocon, Ipomoeassin F, and instead relies on the ER membrane protein complex (EMC) for membrane insertion. This route may even result in a transient exposure of the short N-termini of some LD membrane proteins to the ER lumen, followed by putative topological rearrangements that would enable their transmembrane segment to form a hairpin loop and N-termini to face the cytosol. Our study reveals an unexpected complexity to LD membrane protein biogenesis and identifies a role for the EMC during their co-translational insertion into the ER.

Membrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Here, we show that LD membrane proteins can be delivered to the ER either co- or post-translationally and that their membrane-embedded region specifies pathway selection. The co-translational route for LD membrane protein biogenesis is insensitive to a small molecule inhibitor of the Sec61 translocon, Ipomoeassin F, and instead relies on the ER membrane protein complex (EMC) for membrane insertion. Strikingly, this route can also result in a transient exposure of the short N-termini of LD membrane proteins to the ER lumen, followed by topological rearrangements that enable their transmembrane segment to form a hairpin loop and N-termini to face the cytosol. Our study reveals an unexpected complexity to LD membrane protein biogenesis and identifies a role for the EMC during their co-translational insertion into the ER.SUMMARY STATEMENTInsertion of many lipid droplet membrane proteins into the endoplasmic reticulum (ER) is co-translational, mediated by the ER membrane protein complex (EMC) and involves topology reorientation.