Exceptional stability of a perilipin on lipid droplets depends on its polar residues, suggesting multimeric assembly

Manuel Giménez-Andrés,Juan Martin D'Ambrosio,Bruno Antonny,Sandra Antoine-Bally,Tadej Emeršič,Alenka Čopič,Jure Derganc

doi:10.7554/elife.61401

Abstract

Numerous proteins target lipid droplets (LDs) through amphipathic helices (AHs). It is generally assumed that AHs insert bulky hydrophobic residues in packing defects at the LD surface. However, this model does not explain the targeting of perilipins, the most abundant and specific amphipathic proteins of LDs, which are weakly hydrophobic. A striking example is Plin4, whose gigantic and repetitive AH lacks bulky hydrophobic residues. Using a range of complementary approaches, we show that Plin4 forms a remarkably immobile and stable protein layer at the surface of cellular or in vitro generated oil droplets, and decreases LD size. Plin4 AH stability on LDs is exquisitely sensitive to the nature and distribution of its polar residues. These results suggest that Plin4 forms stable arrangements of adjacent AHs via polar/electrostatic interactions, reminiscent of the organization of apolipoproteins in lipoprotein particles, thus pointing to a general mechanism of AH stabilization via lateral interactions.

Highlights

Lipid droplets (LDs) are cellular organelles specialized for storage of lipids and maintenance of cellular lipid homeostasis
We verified using circular dichroism (CD) that this difference was not due to poor folding of the Plin3 helix (Figure 5 – figure supplement 1B): folding of Plin3 amphipathic helices (AHs) in the presence of the helix352 inducing reagent trifluoroethanol (TFE) was similar to what we previously showed for Plin4 AH (Copic et al, 2018; see Figure 6 – figure supplement 1). 355 Centrifugation of AH-oil suspensions on sucrose gradients revealed a smaller fraction of total Plin3 AH protein associated with the oil fraction than Plin4 4mer or Plin4 12mer (Figure 5C,D)
The slow lateral diffusion and the very slow dissociation of Plin4 AH molecules at the LD surface as assessed by fluorescence recovery after photobleaching (FRAP), by microfluidics, or by exchange assays, are reminiscent of the behavior of vesicular coat components that polymerize on a membrane surface via lateral interactions (Saleem et al, 2015; Sorre et al, 2012). 517 518 We previously showed that the extreme length and the low hydrophobicity of Plin4 AH 519 contributed to the specificity of its LD targeting (Copic et al, 2018)

Summary

Introduction

Lipid droplets (LDs) are cellular organelles specialized for storage of lipids and maintenance of cellular lipid homeostasis. A prominent example is the perilipins: in mammals, this is a family of five proteins that share related structural features and are highly abundant on LDs (Sztalryd and Brasaemle, 2017). We show that one perilipin, Plin, is capable of making highly stable protein-lipid structures by forming an immobile coat on the surface of pure oil or LDs in cells using its unique AH. Extensive mutagenesis shows that the AH of Plin can form an immobile coat due to its organized structure that could enable interhelical interactions on the lipid surface. This model is supported by our measurement of Plin density, which reveals tight packing of helices on oil surface

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Conclusion