Ubiquitination of oleosin-H and caleosin in sesame oil bodies after seed germination

Sesame (Sesamum indicum L.) seed oil bodies are composed of triacylglycerols encapsulated by a monolayer of phospholipids embedded with three classes of proteins, oleosin, caleosin and steroleosin. Among proteins extracted from sesame oil bodies after germination, laddering bands higher than the original antigens were recognized by antibodies against oleosin-H (17 kDa) and caleosin (27 kDa), but not those against oleosin-L (15 kDa), steroleosin-A (39 kDa) and steroleosin-B (41 kDa). Regardless the original antigens, the lowest but relatively abundant laddering band (32 kDa) detected by antibodies against oleosin-H and that (42 kDa) detected by antibodies against caleosin were eluted from SDS-PAGE gels, and then subjected to mass spectrometric analyses. The results showed that the 32 kDa and 42 kDa bands were ubiquitinated oleosin-H and caleosin, respectively. The ubiquitination was further confirmed by immunological detection using antibodies against ubiquitin. Ubiquitination sites were found at three lysine residues (130, 143 and 145) of oleosin-H and two lysine residues (165 and 235) of caleosin. Two ubiquitination sites of oleosin-H, Lys143 and Lys145, were located in the extra 18-residue segment found only in oleosin-H, but not oleosin-L isoforms.