Localization of seed oil body proteins in tobacco protoplasts reveals specific mechanisms of protein targeting to leaf lipid droplets.

Oleosin, caleosin and steroleosin, are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene, tagged with fluorescent proteins, was transiently expressed into tobacco protoplasts, and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localisation of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to LDs, whereas steroleosin, characterised by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum (ER). Protoplasts fractionation on sucrose gradients indicated that both oleosin and caleosin-GFP peaked at different fractions than where steroleosin-GFP or the ER marker BiP, were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together our results pointed for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.