Cloning and secondary structure analysis of caleosin, a unique calcium-binding protein in oil bodies of plant seeds

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 iden tified in sesame oil bodies. A cDNA sequence of Sop1 was obtained by PCR cl oning using degenerate primers derived from two partial amino acid sequence s, and subsequently confirmed via immunological recognition of its over-exp ressed protein in Escherichia coli, Alignment with four published homologou s sequences suggests Sop1 as a putative calcium-binding protein, Immunologi cal cross-recognition implies that this protein, tentatively named caleosin , exists in diverse seed oil bodies, Caleosin migrated faster in SDS-PAGE w hen incubated with Ca2+. A single copy of caleosin gene was found in sesame genome based on Southern hybridization. Northern hybridization revealed th at both caleosin and oleosin genes were concurrently transcribed in maturin g seeds where oil bodies are actively assembled, Hydropathy plot and second ary structure analysis suggest that caleosin comprises three structural dom ains, i.e,, an N-terminal hydrophilic calcium-binding domain, a central hyd rophobic anchoring domain, and a C-terminal hydrophilic phosphorylation dom ain. Compared with oleosin, a conserved proline knot-like motif is located in the central hydrophobic domain of caleosin and assumed to involve in pro tein assembly onto oil bodies.