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

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.

Triglyceride lipolysis driven by glucose restriction triggers liquid-crystalline phase transitions and proteome remodeling of lipid droplets

“…This may be attributed to protein-protein interactions or oligomeric properties of those proteins on the LD surface. In support of this, we find that artificially multimerizing Erg6 with a DsRed2 tag promotes its retention at LDs during AGR, implying that protein oligomerization enhances LD residency, as it has previously been observed for some perilipins (Kory et al, 2015), (Giménez-Andrés et al, 2021).…”

“…Previous studies dissecting LD protein targeting indicate that protein homo-oligomerization promotes a stable protein association with the LD surface, and this multimerization is thought to give perilipin proteins long-term associations with LDs (Giménez-Andrés et al, 2021). To test if protein homo-oligomerization could enhance Erg6 targeting to LDs, we tagged Erg6 with DsRed2, a well-established tetrameric fluorescent tag that we previously characterized as enabling protein oligomerization in vivo (Rogers et al, 2021).…”

Perilipin 3 promotes the formation of membrane domains enriched in diacylglycerol and lipid droplet biogenesis proteins

“…This membrane remodeling function was attributed to the C-terminal 4-helix bundle of PLIN3, which shows structural homology to the N-terminal domain of apolipoprotein E (ApoE), a protein known to promote formation of small lipidic discs (Wilson et al, 1991; Saito et al, 2001; Hickenbottom et al, 2004). In addition, PLIN4, which lacks the PAT domain, forms stable lateral arrangements of amphipathic helices on a membrane surface, suggestive of a membrane coat (Giménez-Andrés et al, 2021; Copic et al, 2018). These independent observations are consistent with a more general membrane-organizing property of the native soluble PLINs, which is likely enhanced in the membrane-anchored PLIN fusions that we employed and characterized in this study.…”

“…The amphipathic repeat segments that are present in PLIN family members are similar to those found in apolipoproteins and the Parkinson’s disease-associated alpha-synuclein. They are important for targeting the LD surface possibly by recognizing lipid packing defects in the LD monolayer (Bussell & Eliezer, 2003; Rowe et al, 2016; Copic et al, 2018; Giménez-Andrés et al, 2018; Ajjaji et al, 2019; Giménez-Andrés et al, 2021). The 4-helix bundle, on the other hand, has structural similarity to apolipoprotein E, and fragments liposomes into disc-like structures in vitro (Hickenbottom et al, 2004; Bulankina et al, 2009).…”

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

“…Lipid droplets are typically coated by one or more perilipins (PLINs), an evolutionarily related protein family defined by two conserved protein motifs, the N-terminal ~100 amino acid hydrophobic PAT domain followed by a repeating 11-mer helical motif of varying length (Kimmel and Sztalryd 2016, Lu et al 2001, Londos et al 1999, Bussell and Eliezer 2003, Miura et al 2002. 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.…”