No abstract

Two distinct cDNAs encoding oleosins (oil body proteins) have been identified by degenerate PCR as transcripts present in the developing seeds of sunflower (Helianthus annuus L. cv. Dwarf Sunbred). One (pSOM) of these is closely related to a reported sunflower oleosin, whilst the other (pSO5) has not been previously described. Different expression patterns were observed for the two cDNAs, pSO5 being expressed earlier than pSOM in seed maturation and oil deposition. The results support the contention that oleosin proteins are synthesised either during or closely after the formation of the oil body. Translation in vitro of synthetic oleosin transcripts was enhanced by the addition of microsomes but suppressed by the addition of purified signal recognition particle (SRP) complex. Deletion of 62 amino acid residues at the C-terminus of the oleosin did not alter the in vitro targeting of the protein to the microsomal membrane. Taken together these data support the idea that oleosins are targeted to the ER membrane as part of oil body biogenesis.

Oil-bodies, from the immature cotyledons of sunflower (Helianthus annuus L.), were difficult to purify to homogeneity using conventional techniques. The major protein contaminants were albumin and globulin storage proteins. A protocol has been developed, therefore, based upon the stringent washing of the oil-body fraction in 9 M urea, which effectively removed almost all the contaminating protein as judged by SDS/PAGE. The urea-washed oil-bodies were enriched in two major proteins of M(r) 19000 and 20000. These proteins were oleosins as demonstrated by their amino acid compositions and the sequence analysis of peptides produced by CNBr cleavage. Far-UV CD spectra of the oleosins in trifluoroethanol, trifluoroethanol/water mixtures and as mixed micelles in SDS, were typical of alpha-helical proteins with alpha-helical contents of some 55%. The phospholipid content of the urea-washed preparations was less than 0.1% of that required to form a half-unit membrane surrounding the oil-body. The oil-body surface therefore appears to be an unusual and novel structure, covered largely by an oleosin protein coat or pellicle rather than a conventional fluid membrane, half-unit or otherwise.

No abstract

No abstract