The biogenesis of the plant seed oil body: Oleosin protein is synthesised by ER-bound ribosomes

An in vitro system was developed to demonstrate that the oleosin protein is synthesised on bound ribosomes and co-translationally inserted into the endoplasmic reticulum (ER) membrane. Microsomes were isolated with their associated bound ribosomes and transcripts from the developing embryos of sunflower (Helianthus annuus L.), and used to programme a cell-free translation system. The presence of the oleosin within the population of polypeptides synthesised by these rough membranes was demonstrated by immunoprecipitation with a specific oleosin antibody. Displacement of the bound ribosomes by the chelation of Mg2+ was also shown to displace the oleosin transcript. In vitro synthesised oleosin was shown to insert into sunflower microsomal membranes, with a portion of the protein being resistant to protease digestion. Radio-sequencing was used to determine the N-terminus of this fragment, providing new insights into the topology of the oleosin protein.