Unique Motifs and Length of Hairpin in Oleosin Target the Cytosolic Side of Endoplasmic Reticulum and Budding Lipid Droplet

Abstract

Plant cytosolic lipid droplets (LDs) are covered with a layer of phospholipids and oleosin and were extensively studied before those in mammals and yeast. Oleosin has short amphipathic N-and C-terminal peptides flanking a conserved 72-residue hydrophobic hairpin, which penetrates and stabilizes the LD. Oleosin is synthesized on endoplasmic reticulum (ER) and extracts ER-budding LDs to cytosol. To delineate the mechanism of oleosin targeting ER-LD, we have expressed modifiedoleosin genes in Physcomitrella patens for transient expression and tobacco (Nicotiana tabacum) BY2 cells for stable transformation. The results have identified oleosin motifs for targeting ER-LD and oleosin as the sole molecule responsible for budding-LD entering cytosol. Both the N-terminal and C-terminal peptides are not required for the targeting. The hairpin, including its entire length, initial N-portion residues, and hairpin-loop of three Pro and one Ser residues, as well as the absence of an N-terminal ER-targeting peptide, are necessary for oleosin targeting ER and moving onto budding LDs and extracting them to cytosol. In a reverse approach, eliminations of these necessities allow the modified oleosin to enter the ER lumen and extract budding LDs to the ER lumen. Modified oleosin with an added vacuole signal peptide transports the ER-luminal LDs to vacuoles. The overall findings define the mechanism of oleosin targeting ER-LDs and extracting budding LDs to the cytosol as well as reveal potential applications.

Arabidopsis lipid droplet‐associated protein (LDAP) – interacting protein (LDIP) influences lipid droplet size and neutral lipid homeostasis in both leaves and seeds

“…LD proteins can target the LD surface either by binding the lipids of the phospholipid monolayer and/or neutral lipid core or by associating with other coat proteins (Kory et al ., ; Bersuker and Olzmann, ). In general, they are divided into two classes: class I proteins, including the oleosins, which target to the LD surface via the ER (Huang and Huang, ), and class II proteins, which target directly from the cytoplasm. When transiently over‐expressed in tobacco leaf cells, Cherry‐LDIP localized to LDs and the cytoplasm, but was never observed in association with the ER, even after extended periods of transient expression in tobacco (Figure S12) or when stably (ectopically) expressed in Arabidopsis plants (Figure S10), suggesting that LDIP is a class II‐type LD protein.…”

Plant Lipid Bodies Traffic on Actin to Plasmodesmata Motorized by Myosin XIs

“…Plant LBs can contain a variety of proteins, including OLEOSINS, CALEOSINS, STEROLEOSINS [2,5], LDAPs, and LDIP [36]. In seeds, the extraction of LBs from the ER is controlled solely by the LB-specific integral protein OLEOSIN [3,7,37]. OLEOSINs are small 15-26 kD proteins that are inserted co-translationally into the ER-membrane, with the termini located in the cytosol, and the central hydrophobic hairpin positioned under strain in the ER bilayer [37] similarly to GPAT4 [28].…”

“…In seeds, the extraction of LBs from the ER is controlled solely by the LB-specific integral protein OLEOSIN [3,7,37]. OLEOSINs are small 15-26 kD proteins that are inserted co-translationally into the ER-membrane, with the termini located in the cytosol, and the central hydrophobic hairpin positioned under strain in the ER bilayer [37] similarly to GPAT4 [28]. From this unstable position the OLEOSIN molecule will diffuse into an emerging oil lens, extracting it from the ER while relaxing its hydrophobic hairpin in the lipid core.…”

“…From this unstable position the OLEOSIN molecule will diffuse into an emerging oil lens, extracting it from the ER while relaxing its hydrophobic hairpin in the lipid core. The length of the hairpin is instrumental, as shorter hairpins reduce their LB targeting [37]. Although the Nand C-termini are not essential for targeting, they are important for providing full coverage of the LB surface, thereby preventing coalescence with adjacent LBs via steric hindrance and electronegative repulsion.…”

Adiposome Targeting and Enzymatic Activity of Lipid Droplet-Specific Proteins

“…For direct localization, specific domains have been identified that are integral monotopic structures including hydrophobic domains or/and amphipathic α-helixes (Bersuker and Olzmann, 2017;Kory et al, 2016). The targeting formats include hydrophobic hairpin, terminal hydrophobic domain, and nonterminal hydrophobic domains (Boeszoermenyi et al, 2015;Huang and Huang, 2017;Na et al, 2015). In addition, one or more amphipathic α-helixes could be also involved (Ding et al, 2012;Krahmer et al, 2011), a type of motif that is characteristic of LD resident proteins (Bulankina et al, 2009;Chong et al, 2011;Rowe et al, 2016;Subramanian et al, 2004).…”