LDAF1 Holds the Key to Seipin Function

Abstract

Seipin is an ER protein important for the assembly of cytoplasmic lipid droplets. In this issue of Developmental Cell, Chung et al. (2019) show that a stable seipin-binding protein, LDAF1/promethin, functions with seipin by attracting triacylglycerol and then allowing this lipid to accumulate and partition into nascent droplets.

Cytoplasmic lipid droplets form the energy hub of nearly all eukaryotic cells, storing energy in the form of esterified fatty acids when abundant and releasing it for oxidation either on site or in distant tissues. Thus, understanding how these organelles, which consist of a neutral lipid core surrounded by a protein-embedded phospholipid monolayer, come about is key to understanding energy homeostasis.

Lipid droplets form on the endoplasmic reticulum and most likely remain there throughout their existence. Remaining on the ER allows droplets to readily communicate with this ubiquitous membrane system, accepting nascent proteins and phospholipids from the bilayer and perhaps donating both back when droplets are spent. But how do these molecules partition into droplets in the first place? The ER transmembrane protein seipin is key to this process. Seipin was first discovered as a protein essential to adipogenesis in humans, as patients with a severe form of congenital generalized lipodystrophy had loss-of-function mutations in BSCL2, the mammalian seipin gene (Magré et al., 2001). It was quite surprising, therefore, to find the presence of seipin in fungi and plants (Szymanski et al., 2007), organisms without adipose tissue, and that seipin was essential for the biosynthesis of normal lipid droplets. The link between lipodystrophy and lipid droplet assembly is still not well understood, although regulation of a glycerol phosphate acyl transferase may be key to this association (Pagac et al., 2016).

That seipin plays an important role in droplet assembly was implied in early studies demonstrating the localization of seipin to ER/LD junctions, and phenotypes in seipin knockout cells that included droplets with aberrant morphology, neutral lipid accumulation in the ER, a sluggish rate of lipid particle production, and mistargeting of a subset of LD proteins. More directly, recent work has demonstrated the appearance of seipin to sites at an early state of droplet formation (Wang et al., 2016). Seipin consists of a large ER luminal domain with both amino- and carboxyl termini facing the cytosol. The recent publication of the atomic structure of fly and human seipin (Sui et al., 2018; Yan et al., 2018) is allowing important insights into function. Seipin forms homooligomers of 10 to 12 subunits with radial symmetry. Near the center of the oligomer is a hydrophobic α-helical domain that could integrally interact with the membrane. In this issue of Developmental Cell, Walther-Farese and colleagues show that this domain may be fundamental to seipin function (Chung et al., 2019).

Chung et al. identified a binding protein to seipin, TMEM159, also termed promethin. While several labs have identified seipin binding proteins through the years, what made this new partner particularly interesting is that its binding to seipin was dependent on the hydrophobic helical domain, not on other parts of the luminal domain or on the two transmembrane domains that tether each subunit of seipin to the membrane. The purified seipin-TMEM159 binding complex was stable, and binding was stoichiometric. The authors renamed TMEM159 Lipid Droplet Assembly Factor 1, or LDAF1. An exciting discovery was that the seipin/LDAF1 complex could bind considerable amounts of triacylglycerol (TG), while seipin alone could not. This brought up the possibility that the binding to TG could seed a new droplet from monomers diffusing in the ER.

Chung et al., using highly inclined and laminated optical sheet (HILO) microscopy, were able to track the initial steps of droplet formation over a large field of cells over several minutes. Using Perilipin 3 (Plin3) as a marker of young droplets, they demonstrated that the seipin complex, but not seipin alone, appeared at sites of future droplets, often even before Plin3 or LiveDrop, a reagent developed earlier in the lab to track small neutral lipid aggregates (Wang et al., 2016). This is consistent with the idea that the seipin complex may be seeding the aggregates itself. Once droplets appear, the authors showed that LDAF1 dissociates from seipin and climbs up upon the droplet itself.

Can LDAF1, which appears to bind TG, alone direct droplet assembly? To test this hypothesis, Chung et al. directed this molecule to ER/plasma membrane junctions with the use of a noncovalent crosslinker. Using total internal reflection (TIRF) microscopy, the authors could monitor LDAF1 close to the surface. Upon addition of crosslinker immobilizing LDAF1, seipin quickly migrated to colocalize with LDAF1, and droplet formation soon followed.

LDAF1 is a small protein of 161 amino acids, the bulk of which is four contiguous putative transmembrane sequences. These sequences may exist as two helical hairpins, and the domain proved necessary and sufficient for seipin binding and function. Binding was abolished by mutating two conserved serines in the seipin hydrophobic domain, further implicating this seipin domain in complex formation.

These observations suggest an alternative model for early droplet formation. Current thinking posits that newly synthesized monomeric neutral lipid spontaneously coalesce into small lens-shaped structures that diffuse laterally within the membrane until encountering seipin. Seipin stabilizes the growing droplet and is joined by other proteins such as perilipin or FIT2 to ensure that LDs bud toward the cytosolic side of the membrane. Furthermore, the current study suggests a broader and earlier role for seipin. LDAF1 in the complex may be the catalyst for seeding the TG aggregate from dissolved monomers. As the neutral lipid phase distends the membrane, LDAF1 and perilipin may draw the lipid toward the cytosol, while seipin and FIT2 on the luminal side may ensure outward budding. While the complex functions in droplet assembly, seipin alone continues to function at the interface between ER and mature droplet, aiding in neutral lipid and perhaps phospholipid and protein trafficking between the two compartments (Salo et al., 2016).

While this is a big step forward in our understanding, questions remain. Earlier experiments indicate that neutral lipid lenses, tagged with LiveDrop, circulate in the ER before encountering seipin. Was this an experimental artifact, or is this behavior cell specific? How does the role of LDAF1 here relate to that of yeast Ldo proteins, which may direct droplets to the ER/vacuole interface (Eisenberg-Bord et al., 2018; Teixeira et al., 2018)? Is one seipin-LDAF1 complex sufficient for formation of a droplet? What is LDAF1 doing on the droplet surface–is it working in conjunction with Plin3 to allow outward budding? And what is the extensive luminal domain of seipin doing? Is it simply blocking droplet budding into the lumen, or is its role more complicated, perhaps scaffolding other protein involved in neutral lipid synthesis? Nonetheless, with this report we begin to understand the function of seipin at atomic resolution. Answers to the questions above, and others, are now expected to come quickly.