title: Membrane protein topology of oleosin is constrained by its long hydrophobic domain.
abstract: Oleosin proteins from Arabidopsis assume a unique endoplasmic reticulum (ER) topology with a membrane-integrated hydrophobic (H) domain of 72 residues, flanked by two cytosolic hydrophilic domains. We have investigated the targeting and topological determinants present within the oleosin polypeptide sequence using ER-derived canine pancreatic microsomes. Our data indicate that oleosins are integrated into membranes by a cotranslational, translocon-mediated pathway. This is supported by the identification of two independent functional signal sequences in the H domain, and by demonstrating the involvement of the SRP receptor in membrane targeting. Oleosin topology was manipulated by the addition of an N-terminal cleavable signal sequence, resulting in translocation of the N terminus to the microsomal lumen. Surprisingly, the C terminus failed to translocate. Inhibition of C-terminal translocation was not dependent on either the sequence of hydrophobic segments in the H domain, the central proline knot motif or charges flanking the H domain. Therefore, the topological constraint results from the length and/or the hydrophobicity of the H domain, implying a general case that long hydrophobic spans are unable to translocate their C terminus to the ER lumen.
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary AB T2N 1N4, Canada.
authors.forename: Ben M
authors.initials: BM
authors.lastname: Abell
authors.forename: Stephen
authors.initials: S
authors.lastname: High
authors.forename: Maurice M
authors.initials: MM
authors.lastname: Moloney
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis; D029681:Arabidopsis Proteins; D002254:Carbonates / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010179:Pancreas / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
chemical_list: D029681:Arabidopsis Proteins; D002254:Carbonates; D010940:Plant Proteins; C079353:oleosin protein, Arabidopsis; C005686:sodium carbonate; D004247:DNA