Plant seed oil bodies comprise a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with abundant oleosins and some minor proteins. Three minor proteins, temporarily termed Sops 1-3, have been identified in sesame oil bodies. A cDNA sequence of Sop1 was obtained by PCR cloning using degenerate primers derived from two partial amino acid sequences, and subsequently confirmed via immunological recognition of its over-expressed protein in Escherichia coli. Alignment with four published homologous sequences suggests Sop1 as a putative calcium-binding protein. Immunological cross-recognition implies that this protein, tentatively named caleosin, exists in diverse seed oil bodies. Caleosin migrated faster in SDS-PAGE when incubated with Ca2+. A single copy of caleosin gene was found in sesame genome based on Southern hybridization. Northern hybridization revealed that both caleosin and oleosin genes were concurrently transcribed in maturing seeds where oil bodies are actively assembled. Hydropathy plot and secondary structure analysis suggest that caleosin comprises three structural domains, i.e., an N-terminal hydrophilic calcium-binding domain, a central hydrophobic anchoring domain, and a C-terminal hydrophilic phosphorylation domain. Compared with oleosin, a conserved proline knot-like motif is located in the central hydrophobic domain of caleosin and assumed to involve in protein assembly onto oil bodies.

Plant seeds store triacylglycerols in discrete organelles called oil bodies. An oil body stores a matrix of triacylglycerols surrounded by phospholipids and alkaline proteins termed oleosins. Oil bodies in rice seeds are present in embryos and aleurone layers. They do not coalesce in crowded environments, as observed on electron microscopy. The detected isoelectric point of purified rice oil bodies is pH 6.2. This implies that rice oil bodies possess a negatively charged surface at neutral pH. The suspension of rice oil bodies in pH 6.5 buffer induces aggregation. Presumably, the negatively charged surface causes electrostatic repulsion that maintains rice oil bodies as discrete organelles. Rice oil bodies lose their integrity on trypsin treatment. Undoubtedly, oleosins play an important role in the stability of oil bodies. There are two oleosin isoforms in rice oil bodies. Antibodies raised against these two homologous isoforms do not cross-recognize each other. Both isoforms are restricted to oil bodies, as detected on immuno-assaying. Partial amino acid sequences of these two isoforms were obtained, and compared with the deduced sequences of two maize and two rice oleosin genes. The comparison confirmed that the two major proteins in rice oil bodies are the two oleosin isoforms.

An in vitro system was established to examine the targeting of proteins to maturing seed oil bodies. Oleosin, the most abundant structural protein, and caleosin, a newly identified minor constituent in seed oil bodies, were translated in a reticulocyte lysate system and simultaneously incubated with artificial oil emulsions composed of triacylglycerol and phospholipid. The results suggest that oil body proteins could spontaneously target to artificial oil emulsions in a co-translational mode. Incorporation of oleosin to artificial oil emulsions extensively protected a fragment of approximately 8 kDa from proteinase K digestion. In a competition experiment, in vitro translated caleosin and oleosin preferentially target to artificial oil emulsions instead of microsomal membranes. In oil emulsions with neutral phospholipids, relatively low protein targeting efficiency was observed. The targeting efficiency was substantially elevated when negatively charged phospholipids were supplemented to oil emulsions to mimic the native phospholipid composition of oil bodies. Mutated caleosin lacking various structural domains or subdomains was examined for its in vitro targeting efficiency. The results indicate that the subdomain comprising the proline knot motif is crucial for caleosin targeting to oil bodies. A model of direct targeting of oil-body proteins to maturing oil bodies is proposed.

The oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosin isoforms with molecular masses of 18 and 16 kDa are present in rice oil bodies. Chicken antibodies raised against oleosin 18 kDa and rabbit antibodies raised against oleosin 16 kDa did not cross-recognize these two homologous isoforms. This peculiar non-cross recognition was used to locate the two oleosin isoforms on the surface of oil bodies via immunofluorescence detection using anti-chicken IgG conjugated with FITC (fluorescein isothiocyanate) and anti-rabbit IgG conjugated with Texas-Red. The results revealed that both oleosin isoforms resided on each oil body in vivo and in vitro. Artificial oil bodies were reconstituted via sonication using triacylglycerol, phospholipid, and oleosins. The results indicated that the two rice oleosin isoforms could stabilize artificial oil bodies individually whereas oleosin 16 kDa provided better stability to the organelles than oleosin 18 kDa.

The oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosins are present in the oil bodies of diverse angiosperms, and classified as high and low Mr isoforms according to their relative molecular masses in each species. In sesame oil bodies, besides the two ubiquitous oleosin isoforms (17 and 15 kDa), an additional minor oleosin (15.5 kDa) was revealed on Tricine SDS-PAGE. A full-length cDNA fragment was cloned, sequenced and deduced to be a putative oleosin of 15,446 Da. The gene was constructed in a fusion or non-fusion vector and then over-expressed with different efficiency in Escherichia coli. All three oleosins purified from sesame oil bodies were subjected to immunoassaying using antibodies raised against the over-expressed oleosin. The results confirmed that this gene encodes the sesame 15.5 kDa oleosin. Sequence comparisons with other known oleosins revealed that sesame 15.5 kDa oleosin does not represent a new oleosin isoform class but may have been derived through gene duplication and truncation of sesame 17 kDa oleosin, and possesses the minimal structure of the high Mr oleosin isoform. A conserved amphipathic alpha-helix is predicted in sesame 15.5 kDa oleosin, which may imply a potential biological function associated with this isoform.