The CYTOLD and ERTOLD pathways for lipid droplet-protein targeting.

Lipid droplets (LDs) are the main organelles for lipid storage, and their surfaces contain unique proteins with diverse functions, including those that facilitate the deposition and mobilization of LD lipids. Among organelles, LDs have an unusual structure with an organic, hydrophobic oil phase covered by a phospholipid monolayer. The unique properties of LD monolayer surfaces require proteins to localize to LDs by distinct mechanisms. Here we review the two pathways known to mediate direct LD protein localization: the CYTOLD pathway mediates protein targeting from the cytosol toLDs, and the ERTOLD pathway functions in protein targeting from the endoplasmic reticulum toLDs. We describe the emerging principles for each targeting pathway in animal cells and highlight open questions in the field.

Determinants of Endoplasmic Reticulum-to-Lipid Droplet Protein Targeting.

Lipid droplet (LD) formation from the endoplasmic reticulum (ER) is accompanied by the targeting and accumulation of specific hydrophobic, membrane-embedded proteins on LDs. The determinants of this process are unknown. Here, we study the hydrophobic membrane motifs of two Drosophila melanogaster proteins, GPAT4 and ALG14, that utilize this pathway, and we identify crucial sequence features that mediate LD accumulation. Molecular dynamics simulations and studies in cells reveal that LD targeting of these motifs requires deeply inserted tryptophans that have lower free energy in the LD oil phase and positively charged residues near predicted hairpin hinges that become less constrained in the LD environment. Analyzing hydrophobic motifs from similar LD-targeting proteins, it appears that the distribution of tryptophan and positively charged residues distinguishes them from non-LD-targeting membrane motifs. Our studies identify specific sequence features and principles of hydrophobic membrane motifs that mediate their accumulation on LDs.

Seipin is required for converting nascent to mature lipid droplets.

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting.

Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids, such as triacylglycerol (TG), as reservoirs of metabolic energy and membrane precursors. The Arf1/COPI protein machinery, known for its role in vesicle trafficking, regulates LD morphology, targeting of specific proteins to LDs and lipolysis through unclear mechanisms. Recent evidence shows that Arf1/COPI can bud nano-LDs (∼60 nm diameter) from phospholipid-covered oil/water interfaces in vitro. We show that Arf1/COPI proteins localize to cellular LDs, are sufficient to bud nano-LDs from cellular LDs, and are required for targeting specific TG-synthesis enzymes to LD surfaces. Cells lacking Arf1/COPI function have increased amounts of phospholipids on LDs, resulting in decreased LD surface tension and impairment to form bridges to the ER. Our findings uncover a function for Arf1/COPI proteins at LDs and suggest a model in which Arf1/COPI machinery acts to control ER-LD connections for localization of key enzymes of TG storage and catabolism. DOI: http://dx.doi.org/10.7554/eLife.01607.001.