Serine/Threonine/Tyrosine Protein Kinase Phosphorylates Oleosin, a Regulator of Lipid Metabolic Functions  

Abstract

(A.V., V.V., R.R.)Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A 2 activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A 2 activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca 2+ inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation.

Bioinformatics Reveal Five Lineages of Oleosins and the Mechanism of Lineage Evolution Related to Structure/Function from Green Algae to Seed Plants

“…d-SL2 oleosin, which is amphipathic and contains a hydrophobic hairpin, may act as a fortuitous lipid-binding mediator in transformed yeast cells in vivo and in microsomes in vitro, such that an intrinsic membraneassociated acyltransferase or phospholipase is activated and/or has its substrate specificity modified. This hypothetical mediator activity could be altered when the HX 4 D motif or the N-or C-terminal peptide was removed or after phosphorylation (Parthibane et al, 2012a(Parthibane et al, , 2012b. A demonstration that Arachis.d-SL2 oleosin alone on artificial LDs or liposomes has bifunctional activities of acyltransferase and phospholipase would eliminate the mediator possibility.…”

“…Phosphorylated perilipin interacts with lipase via a lipase moderator, a/b hydrolase domain-containing protein5, and binds to mitochondria via its C-terminal peptide; motifs in perilipin for these interactions are unknown (Wang et al, 2011). Conversely, oleosins could act only as a physical barrier preventing LDs from contacting lipase and glyoxysomes until germination and only after they have been phosphorylated or proteolyzed (Parthibane et al, 2012a;Deruyffelaere et al, 2015). Recently, a peanut oleosin (named OLE3 then and Arach.d-SL2 here) was shown to be a bifunctional enzyme exhibiting oleoyl-CoA:monoacylglycerol acyltransferase and phosphatidylcholine acylhydrolase (phospholipase) activities in transformed quadruple mutant yeast (Saccharomyces cerevisiae) in vivo or in isolated microsomes (Parthibane et al, 2012b).…”

“…Phosphorylation would modify oleosins and, thus, the LD surface (Parthibane et al, 2012a) for involvement in or avoidance of metabolic activity, gain or loss of LD stability (susceptibility to proteolysis and lipolysis via binding to the hydrolases directly or via modulators), and/or binding to subcellular structures (e.g. glyoxysomes).…”

Specialization of Oleosins in Oil Body Dynamics during Seed Development in Arabidopsis Seeds

“…It is generally agreed that oleosins cover the OB surface, with their central hydrophobic domain inserted in the TAG through the phospholipid layer . Besides their structural function in OBs, oleosins may serve as docking stations for other proteins at its surface and may participate in the biosynthesis and mobilization of plant oils (Parthibane et al, 2012a(Parthibane et al, , 2012b. Oleosins are probably involved in OB stability (Leprince et al, 1998;Shimada et al, 2008) and in the regulation of OB repulsion (Heneen et al, 2008), preventing the coalescence of OBs into a single organelle (Schmidt and Herman, 2008).…”

Genome-Wide Analysis of Oleosin Gene Family in 22 Tree Species: An Accelerator for Metabolic Engineering of BioFuel Crops and Agrigenomics Industrial Applications?

“…Rapid initiation of Arabidopsis pollination requires the oleosin-domain protein GRP17 (Mayfield and Preuss, 2000). Recent research suggests that OLEs may be bifunctional enzymes with both monoacylglycerol acyltransferase and phospholipase activities regulated by serine/threonine/tyrosine protein kinases (Parthibane et al, 2012a;2012b).…”