Expression and in vitro targeting of a sunflower oleosin

Thoyts, P. J. E.; Millichip, Mark; Stobart, A. K.; Griffiths, W. T.; Shewry, P. R.; Napier, Johnathan A.

doi:10.1007/bf00043664

Cited by 51 publications

(67 citation statements)

References 20 publications

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“…Pathway of Oleosin-Evidence to date suggests that oleosins are integrated into ER membranes co-translationally, following a SRP-and Sec61-dependent pathway (27)(28)(29)34). To place oleosin topology in context, we needed to define the targeting pathway of oleosin into our experimental system of canine pancreatic membrane.…”

Section: Er Targetingmentioning

confidence: 99%

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Membrane Protein Topology of Oleosin Is Constrained by Its Long Hydrophobic Domain

Abell

High

Moloney

2002

Journal of Biological Chemistry

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Oleosin proteins from Arabidopsis assume a unique endoplasmic reticulum (ER) topology with a membraneintegrated 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.Membrane proteins targeted to the ER 1 generally display a uniform and consistent topology, in which the orientation of membrane spans is determined by the interaction between nascent polypeptides and the translocon (1, 2). A set of general rules has been established that are useful for prediction of unknown topologies. These rules also further our understanding of the underlying mechanisms of membrane insertion. In the simplest case, individual membrane spans can be orientated by sequential insertion, resulting in adoption of the opposite orientation to the previous span (3). The presence of charged residues in flanking regions is a critical influence on the orientation of individual membrane spans; positively charged residues show a strong preference for retention in the cytosol, while negatively charged residues have a weak affinity for translocation to the ER lumen (4 -7). Variants of a yeast protein, Ste2p, a G protein-coupled pheromone receptor, strictly followed the "positive inside" rule for the first transmembrane span (8). The N terminus is translocated only if the charge difference is reversed by removal of all the N-terminal positive charges or addition of C-terminal negative charges. ER membrane does not support a transmembrane potential, suggesting that this preference may be due to charged residues interacting with phospholipids (9). In the case of signal-anchors, translocation of the N terminus can be blocked by the adoption of a fully folded conformation (10). Prediction of topology can also be aided by analyzing the membrane span itself. Transmembrane spans up to 25 leucine residues increasingly favored a topology with the N terminus in the lumen as the polyleucine chain was lengthened (11). The interaction between multiple topology determinants is ...

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Section: Er Targetingmentioning

confidence: 99%

“…Current evidence suggests that oleosin targets to the ER by an SRP-mediated pathway; oleosin mRNA is found in ERbound polyribosomes (27), and exogenous SRP inhibits oleosin translation in vitro (28). Oleosin expression in yeast mutants also indicates a requirement for SRP and Sec61 (29).…”

mentioning

confidence: 99%

Membrane Protein Topology of Oleosin Is Constrained by Its Long Hydrophobic Domain

Abell

High

Moloney

2002

Journal of Biological Chemistry

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“…Subsequent studies showed that in vitrc-translated oleosin could be targeted to canine pancreatic microsomes but not to erythrocytes, plastids, heat-inactivated canine pancreatic microsomes, or oil bodies (Hills et al, 1993;Loer and Herman, 1993). Although these experiments did not demonstrate the pathway of ER targeting, it has been found that the signal recognition particle is capable of causing translational arrest in vitro (Thoyts et al, 1995). This provides preliminary evidence for a cotranslational, signal recognition particle-mediated targeting pathway but does not rule out post-translational ER targeting of oleosins (Ng et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

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

Abell¹,

Holbrook²,

Abenes³

et al. 1997

The Plant Cell

105

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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-P-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-P-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.

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“…In developing oilseeds, oleosin is localized on the surface of the oil body, which is the main site of triacylglycerol storage [7]. There is evidence to suggest that oleosin is co-translationally integrated into rough endoplasmic reticulum (rER) membranes [8,9]. It is also known from biochemical experiments that biogenesis of oil bodies parallels the localisation of oleosins in rER membranes [10,11].…”

Section: Introductionmentioning

confidence: 99%

“…It is also known from biochemical experiments that biogenesis of oil bodies parallels the localisation of oleosins in rER membranes [10,11]. Even though signal sequence for targeting of oleosin to the sites where oil bodies are formed is not known [8,10,12,13] These data indicate that there is dynamic interaction between sites of oil body formation and oleosin synthesis [14,15]. Oleosins are unusual proteins because they possess three different functional domains: a 70-80-residue uninterrupted hydrophobic domain is flanked by relatively polar amino-and carboxy-terminal domains.…”

Section: Introductionmentioning

confidence: 99%

Amino-Terminus of Oleosin Protein Defines the Size of Oil Bodies - Topological Model of Oleosin-Oil Body Complex

J¹,

P²

2015

Saha, J Plant Biochem Physiol

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Lipids formed in seeds of oilseed plant are stored in specific cytosolic structures called oil bodies, which are covered with oleosin proteins. Disruption of oleosin layer by heat treatment makes lipids susceptible to oxidation. By analyzing the amino acid sequence of Arabidopsis oleosin, unique domains were identified within the aminoterminus of the protein. Removal of these domains affected both the size of oil bodies and their susceptibility to lipid-specific stain, Nile Blue A. Amino-terminally truncated oleosin mutant, which was in fusion to green fluorescent protein (GFP), confined to rough endoplasmic reticulum (rER) suggesting that the amino-terminus of oleosin is required for the efficient lateral transportation of oleosin proteins from rER to oil bodies. The size of oil bodies covered by amino-terminal mutants was at least 5 times larger than native oil bodies. Based on the observations, a topological model was generated, which could explain how oleosin maintains and protects the integrity of oil bodies during seed storage. Citation: Wahlroos T, Soukka J, Denesyuk A, Susi P (2015) Amino-Terminus of Oleosin Protein Defines the Size of Oil Bodies -Topological Model of Oleosin-Oil Body Complex. J Plant Biochem Physiol 3: 155. Amino-Terminus of Oleosin

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Expression and in vitro targeting of a sunflower oleosin

Cited by 51 publications

References 20 publications

Membrane Protein Topology of Oleosin Is Constrained by Its Long Hydrophobic Domain

Membrane Protein Topology of Oleosin Is Constrained by Its Long Hydrophobic Domain

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

Amino-Terminus of Oleosin Protein Defines the Size of Oil Bodies - Topological Model of Oleosin-Oil Body Complex

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