Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1–3

Abstract

Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs in Saccharomyces cerevisiae, demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targeting in vivo and in vitro. Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.

Insertion of perilipin 3 into a glycero(phospho)lipid monolayer depends on lipid headgroup and acyl chain species

“…Indeed, we now show that the  max (a qualitative measure for the affinity of the protein for the lipid monolayer) increases upon addition of N-terminal sequences (perilipin 3C, perilipin 3B, and the full-length protein). This agrees with the cell-based studies that show that the N terminus is necessary and sufficient for LD targeting and binding (1,25,28). For example, compare the insertion isotherms ( max ) for perilipin 3D with those of perilipin 3C and 3B.…”

“…However, an overview of the literature suggests that amphipathic -helices in the N terminus of the protein are necessary and sufficient (1,25,28). This region overlaps with the so-called 11-mer repeat region.…”

“…Targeting has been investigated for perilipins 1 through 5 in cellular studies and different regions of the proteins have been identified (1,(22)(23)(24)(25)(26)(27). Based on currently available data, it appears likely that amphipathic sequences, especially the 11-mer repeat region of the proteins, target perilipins to LDs (1,23,(28)(29)(30)(31). The 11-mer repeat is a protein region with a repeating 11 amino acid pattern (32).…”

Mature lipid droplets are accessible to ER luminal proteins

“…Although the precise signal that is recognized by lipid-dropletlocalized proteins is not yet well defined, it has been speculated that some of these proteins, such as PLIN3, recognize lipid-packaging defects (Bulankina et al, 2009). This is supported by recent results indicating that amphipathic helices present in a number of lipiddroplet-localized proteins, including PLINs, are sufficient for lipid droplet targeting (Grippa et al, 2015;Rowe et al, 2016). Thus, similar to proteins containing lipid-binding amphipathic helices, as present in proteins with an ALPs motif or those of the N-BAR family, which recognize lipid packaging defects induced by membrane curvature, PLIN3 and possibly other lipid-droplettargeted proteins harboring amphipathic helices, might recognize altered spacing of phospholipid headgroups, possibly induced by the presence of cone-shaped or neutral lipids within a flat bilayer membrane (Bulankina et al, 2009;Drin and Antonny, 2010).…”

“…These observations, together with the fact that lipoproteins of animal cells, which are structurally very similar to lipid droplets, mature and assemble within the ER lumen from where they are then secreted into the extracellular space, prompted us to investigate the topology of lipid droplet formation in more detail (Fisher and Ginsberg, 2002;Hussain et al, 2003). Although it is clear that lipid droplets are accessible to soluble cytosolic factors, such as the perilipins (PLINs), which are abundant structural proteins that are targeted to the lipid droplets through conserved amphipathic helices, it is unknown whether lipid droplets are also accessible from within the lumen of the ER (Rowe et al, 2016).…”