The surface of lipid droplets constitutes a barrier for endoplasmic reticulum residential integral membrane spanning proteins

Abstract

Lipid droplets (LDs) constitute globular subcellular structures that mainly serve to store energy in form of neutral lipids, particularly triacylglycerol and steryl esters. LDs are closely associated with the membrane of the endoplasmic reticulum (ER), and are limited by a monolayer membrane of phospholipids harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here we address the question whether integral membrane proteins could localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER resident proteins, such as Wbp1 (a N-glycosyl transferase complex subunit), Sec61 (a translocon subunit), and Pmt1 (a protein O-mannosyltransferase). The resulting fusion proteins localize to the rim of LDs in both yeast and mammalian cells. LD targeting of membrane proteins is not only observed with the PLIN3-containing fusion proteins, but also for native endogenous polytopic transmembrane proteins. These data indicate that integral membrane proteins that function in a bilayer membrane can reversibly associate with the LD surface, suggesting that the LD surface is continuous with the ER membrane. In cells lacking LDs, Wbp1 and Sec61 fusion proteins localize to the ER, forming crescent-like membrane protrusions. These ER crescents resemble sites of LD formation as they contain seipin and other LD-localized proteins, suggesting that membrane-anchored PLIN3 induces the formation of membrane subdomains.

A Unique Junctional Interface at Contact Sites Between the Endoplasmic Reticulum and Lipid Droplets

“…The ER-LD junctions therefore are strikingly different from other contact sites between organelles delineated by bilayer membranes. This architecture at the ER-LD junction constitutes a barrier for ER residential integral membrane proteins and excludes them from the LD monolayer (Khaddaj et al, 2020). Early cryo electron microscopy (EM) studies have revealed that the cytoplasmic leaflet of the ER membrane is continuous with the LD surface, and freeze fracture EM suggested that the ER membrane forms a cup-like structure that holds the LD in place (Blanchette-Mackie et al, 1995;Robenek et al, 2006).…”