The C-Terminus of Perilipin 3 Shows Distinct Lipid Binding at Phospholipid-Oil-Aqueous Interfaces

Abstract

Lipid droplets (LDs) are ubiquitously expressed organelles; the only intracellular organelles that contain a lipid monolayer rather than a bilayer. Proteins localize and bind to this monolayer as they do to intracellular lipid bilayers. The mechanism by which cytosolic LD binding proteins recognize, and bind, to this lipid interface remains poorly understood. Amphipathic α-helix bundles form a common motif that is shared between cytosolic LD binding proteins (e.g., perilipins 2, 3, and 5) and apolipoproteins, such as apoE and apoLp-III, found on lipoprotein particles. Here, we use pendant drop tensiometry to expand our previous work on the C-terminal α-helix bundle of perilipin 3 and the full-length protein. We measure the recruitment and insertion of perilipin 3 at mixed lipid monolayers at an aqueous-phospholipid-oil interface. We find that, compared to its C-terminus alone, the full-length perilipin 3 has a higher affinity for both a neat oil/aqueous interface and a phosphatidylcholine (PC) coated oil/aqueous interface. Both the full-length protein and the C-terminus show significantly more insertion into a fully unsaturated PC monolayer, contrary to our previous results at the air-aqueous interface. Additionally, the C-terminus shows a preference for lipid monolayers containing phosphatidylethanolamine (PE), whereas the full-length protein does not. These results strongly support a model whereby both the N-terminal 11-mer repeat region and C-terminal amphipathic α-helix bundle domains of perilipin 3 have distinct lipid binding, and potentially biological roles.

Structural insights into perilipin 3 membrane association in response to diacylglycerol accumulation

“…DO phospholipids contain two unsaturated acyl chains, have increased membrane packing defects [34,[37][38][39] and are enriched in ER membranes after oleate supplementation, which stimulates LD formation [15,[40][41][42]. Initial liposome co-sedimentation experiments varied the ratio of neutral phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE), as PC and PE represent the major lipids on both the cytoplasmic face of the ER and surface of LDs, PE increases both PLIN2 binding to liposomes [43] and PLIN3 insertion into mixed lipid monolayers at phospholipid-oil interfaces [44] and the ratio of PC to PE has previously been shown to regulate protein distribution on LDs [43,45]. Under all PC-to-PE ratios tested, PLIN3 did not bind liposomes comprised solely of PC and PE (Fig.…”

Interaction of Two Amphipathic α-Helix Bundle Proteins, ApoLp-III and ApoE 3, with the Oil–Aqueous Interface

“…For example, a 5 μL drop of neat triolein in buffer yields Ne ∼ 0.5, which is insufficient for reliable interfacial tension measurements by ADSA. For a drop of the same size but coated with POPC, we estimate Ne ∼ 0.7, which is sufficiently large that the interfacial tension determined by ADSA becomes nearly independent of droplet size . In the experiments discussed here, all small droplets were both ≥6 μL and sufficiently deformed ( Ne ∼ 0.7–1).…”

“…For a drop of the same size but coated with POPC, we estimate Ne ∼ 0.7, which is sufficiently large that the interfacial tension determined by ADSA becomes nearly independent of droplet size. 38 In the experiments discussed here, all small droplets were both ≥6 μL and sufficiently deformed (Ne ∼ 0.7−1). Additional details on image analysis are given in ref 32.…”

Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knock-in zebrafish

“…Perilipins are recruited to the lipid droplet surface directly from the cytosol, mediated at least in part by the 11-mer repeat regions which fold into amphipathic helices (Rowe et al 2016, Bulankina et al 2009, Ajjaji et al 2019, McManaman et al 2003, Garcia et al 2003, Gimenez-Andres et al 2021. There is also evidence that 4-helix bundles located in the Cterminus regulate the affinity and stability of lipid droplet binding (Ajjaji et al 2019, Rowe et al 2016, Hickenbottom et al 2004, Titus et al 2021, Chong et al 2011. Perilipins act to regulate lipid storage by preventing or modulating access of lipid droplets to lipases and lipophagy machinery (Sztalryd and Brasaemle 2017, Granneman et al 2007, Wang, Bell, et al 2011, Wang et al 2009, Patel et al 2014, Granneman et al 2009, Listenberger et al 2007, Kaushik and Cuervo 2015.…”