Role of the Proline Knot Motif in Oleosin Endoplasmic Reticulum Topology and Oil Body Targeting

Abstract

An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N-and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-P-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-P-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.

Murine Diacylglycerol Acyltransferase-2 (DGAT2) Can Catalyze Triacylglycerol Synthesis and Promote Lipid Droplet Formation Independent of Its Localization to the Endoplasmic Reticulum

“…The second region is between amino acids 327 and 350, which contains four proline residues that could be part of a proline knot motif. A proline knot is responsible for targeting oleosin to oil bodies in plants and consists of three closely spaced proline residues within a hydrophobic sequence of 75 amino acids (36).…”

Self-assembly of tunable protein suprastructures from recombinant oleosin

“…They have an N-terminal hydrophilic segment, followed by a hydrophobic core [among the longest natural hydrophobic stretches (17)] and another hydrophilic segment at the C terminus (18,19). Although the crystal structure is unknown, the molecule is believed to resemble a hairpin with the hydrophobic domain bifurcated by a proline knot, a stretch containing three prolines that induce a 180°turn in the chain (14,17,20,21). The two legs of the hairpin are helical, possibly forming a coiled-coil (17,20).…”

Leaf Oil Body Functions as a Subcellular Factory for the Production of a Phytoalexin in Arabidopsis

“…Seed oleosins have a central hydrophobic domain and a Pro knot motif responsible for targeting the protein to oil bodies (Abell et al, 1997;Chen et al, 1999;Takahashi et al, 2000;Chen and Tzen, 2001;Aubert et al, 2010), and they confer freezing tolerance to seedlings by maintaining proper oil body size within seed cells (Siloto et al, 2006;Shimada et al, 2008). Caleosins have a Ca 2+ -binding motif (an EF hand motif) in addition to the oil body-targeting domains (Abell et al, 1997;Chen et al, 1999;Takahashi et al, 2000;Chen and Tzen, 2001;Aubert et al, 2010).…”