A giant amphipathic helix from a perilipin that is adapted for coating lipid droplets

Abstract

How proteins are targeted to lipid droplets (LDs) and distinguish the LD surface from the surfaces of other organelles is poorly understood, but many contain predicted amphipathic helices (AHs) that are involved in targeting. We have focused on human perilipin 4 (Plin4), which contains an AH that is exceptional in terms of length and repetitiveness. Using model cellular systems, we show that AH length, hydrophobicity, and charge are important for AH targeting to LDs and that these properties can compensate for one another, albeit at a loss of targeting specificity. Using synthetic lipids, we show that purified Plin4 AH binds poorly to lipid bilayers but strongly interacts with pure triglycerides, acting as a coat and forming small oil droplets. Because Plin4 overexpression alleviates LD instability under conditions where their coverage by phospholipids is limiting, we propose that the Plin4 AH replaces the LD lipid monolayer, for example during LD growth.

Neutral lipids regulate amphipathic helix affinity for model lipid droplets

“…It is well known that the PL packing density correlates with the binding of AHs (Bigay et al, 2005; Čopič et al, 2018; Pranke et al, 2011; Prévost et al, 2018), but here it did not seem to explain the differential recruitment of the tested AHs. We thus sought an independent confirmation of this unanticipated finding of the unchanged PL density at the monolayer covering the different neutral lipids.…”

“…S1 D). The presence of opposite charges on the hydrophilic face of the AH might have promoted lateral AH–AH interaction and stabilized binding, as proposed for Plin4 (Čopič et al, 2018). On the contrary, when we put a charge in the hydrophobic face of the AH, by mutating the valine into arginine (V5R), we lost binding to the aLDs (Fig.…”

“…Instead, to reach binding specificity, there might be amphipathic sequences that are well-tuned in hydrophobicity to decrease the neutral lipid/water surface tension enough without binding to bilayers and designed with an optimal interaction with a given neutral lipid. This seems to be at least the case for the most abundant LD proteins, Perilipin 1–5, whose 11-mer repeat AH sequence lacks bulky hydrophobic residues but can specifically detect LD surface (Ajjaji et al, 2019; Brasaemle, 2007; Čopič et al, 2018; Rowe et al, 2016). For instance, Perilipin 3 seems to better bind to LDs in the presence of diacylglycerols (Skinner et al, 2009).…”

“…(Bigay and Antonny, 2012). On LDs, the PL packing density is so far the only lipid parameter found to regulate the binding of AHs (Bacle et al, 2017; Prévost et al, 2018; Thiam et al, 2013a), which depends also on the nature of the amphipathic sequence (Čopič et al, 2018; Prévost et al, 2018). Despite this understanding, it is still unclear how AHs specifically bind to LDs or LD subsets.…”

“…Decreasing the PL packing density at the monolayer surface of LDs accentuates the contact between hydrophobic and hydrophilic phases, which is favorable for the binding of AHs (Čopič et al, 2018; Prévost et al, 2018; Thiam et al, 2013a). Indeed, AHs are basically surfactants, and their adsorption to hydrophobic–hydrophilic interfaces is favorable for reducing interfacial energies (Thiam and Dugail, 2019).…”