ROLE OF THE PROLINE KNOT MOTIF IN OLEOSIN ENDOPLASMIC-RETICULUM TOPOLOGY AND OIL BODY TARGETING

An Arabidopsis oleosin was used as a model to study oleosin topology a nd targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteina se K mapping of the membrane topology. Oleosin maintains a conformatio n with a membrane-integrated hydrophobic domain flanked by N- and C-te rminal domains located on the outer microsome surface. This is a uniqu e membrane topology on the endoplasmic reticulum (ER). Three universal ly conserved proline residues within the ''proline knot'' motif of the oleosin hydrophobic domain were substituted by leucine residues. Afte r in vitro translation, only minor differences in proteinase K protect ion could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on t he ER was observed between the two oleosin forms. However, as an oleos in-beta-glucuronidase translational fusion, the proline knot variant f ailed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expre ssing oleosin-beta-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the nati ve form. Therefore, the proline knot motif is not important for ER int egration and the determination of topology but is required for oil bod y targeting. The loss of the proline knot results in an intrinsic inst ability in the oleosin polypeptide during trafficking.