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Role of the Proline Knot Motif in Oleosin Endoplasmic Reticulum Topology and Oil Body Targeting

Ben M. Abell, Larry A. Holbrook, Malleva Abenes, Denis J. Murphy, Matthew J. Hills and Maurice M. Moloney
The Plant Cell
Vol. 9, No. 8 (Aug., 1997), pp. 1481-1493
DOI: 10.2307/3870397
Stable URL: http://www.jstor.org/stable/3870397
Page Count: 13
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Role of the Proline Knot Motif in Oleosin Endoplasmic Reticulum Topology and Oil Body Targeting
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Abstract

An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N- and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-β-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-β-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native from. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.

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