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 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-beta-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-beta-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. 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.
Abell
B M
BM
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Holbrook
L A
LA
Abenes
M
M
Murphy
D J
DJ
Hills
M J
MJ
Moloney
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029681:Arabidopsis Proteins; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D014644:Genetic Variation; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010938:Plant Oils / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011392:Proline / Q000737:chemistry; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013025:Glycine max
D029681:Arabidopsis Proteins; D017931:DNA Primers; D018744:DNA, Plant; D008565:Membrane Proteins; D010938:Plant Oils; D010940:Plant Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C079353:oleosin protein, Arabidopsis; D011392:Proline