Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial α-helical membrane anchors

Abstract

Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayer-integrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles. Systematic cysteine-scanning mutagenesis showed the predicted TMD of one candidate MIP, DHRS3, to be a partially buried amphipathic α-helix in both lipid droplet monolayers and the cytoplasmic leaflet of endoplasmic reticulum membrane bilayers. Coarse-grained molecular dynamics simulations support these observations, suggesting that this helix is most stable at the solvent-membrane interface. The simulations also predicted similar interfacial amphipathic helices when applied to seven additional MIPs from our dataset. Our findings suggest that interfacial helices may be a common motif by which MIPs are integrated into membranes, and provide high-throughput methods to identify and study MIPs.

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

“…In addition to their repetitive AH regions, other segments of Plin1, Plin2 and Plin3 have been implicated in binding to LDs, in particular the C-terminal 4-helix bundle (Subramanian et al, 2004; Mirheydari et al, 2016; Ajjaji et al, 2019), but this has not been the case for Plin4 (Copic et al, 2018). In contrast to the behavior of their AHs, the association of full-length Plin1 and Plin2 with LDs can be very stable (Targett-Adams et al, 2003; Soni et al, 2009; Pataki et al, 2018; Ajjaji et al, 2019), and the COPI machinery has been implicated in the recruitment of Plin2 to LDs by an unknown mechanism (Nakamura et al, 2004; Soni et al, 2009).…”

“…Different types of proteins have been found to associate with LDs (Brasaemle et al, 2004; Bersuker et al, 2018; Kory et al, 2015; Pataki et al, 2018; Mejhert et al, 2020). They can be either stably embedded in the LD monolayer, coming by diffusion from the endoplasmic reticulum (ER), from which LDs emerge, or they associate with LDs peripherally from the cytosol (Ohsaki et al, 2014; Bersuker and Olzmann, 2017).…”

An alternative membrane topology permits lipid droplet localization of peroxisomal fatty acyl-CoA reductase 1

“…5C,D). While the hydrophobic domain of ACSL3 allows both the N-and C-terminus to be oriented towards the cytosol at the ER and on LDs (Poppelreuther et al, 2012), there is no consensus yet whether this is due to the formation of an amphipathic helix or a hairpin motif (Kory et al, 2016;Pataki et al, 2018). Regardless, the corresponding A3Nt-HD2-GFP construct behaved virtually indistinguishably from A3Nt-GFP, overlapping extensively with LDs after incubation with OA ( Fig.…”

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

“…Different types of proteins have been found to associate with LDs ( Brasaemle et al, 2004 ; Bersuker et al, 2018 ; Kory et al, 2015 ; Pataki et al, 2018 ; Mejhert et al, 2020 ). They can be either stably embedded in the LD monolayer, coming by diffusion from the endoplasmic reticulum (ER), from which LDs emerge, or they associate with LDs peripherally from the cytosol ( Ohsaki et al, 2014 ; Bersuker and Olzmann, 2017 ).…”