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 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 ...

Endoplasmic Reticulum, Oleosins, and Oils in Seeds and Tapetum Cells

“…The mRNA for the synthesis of oleosin is associated with the RER. Translation of oleosin mRNA in an in vitro synthesis system is retarded or enhanced when SRP or microsomes are added, respectively (Loer and Herman, 1993;Thoyts et al, 1995;Abell et al, 2002;Beaudoin and Napier, 2002). The findings suggest that translation of the oleosin mRNA pauses after binding of the SRP to the nascent peptide and accelerates when the newly synthesized oleosins are incorporated into the ER.…”

The adsorption of biological peptides and proteins at the oil/water interface. A potentially important but largely unexplored field

“…Oleosin consists of N-and C-terminal charged amphipathic a helices (AaH) domains that lie on the surface of the droplet and a central ?60aa hydrophobic b sheet that penetrates into the core lipid. This b sheet interacts with adjacent b sheet central domains to produce an almost rigid surface (18,19). Interdigitated between and probably under the terminal N-and C-umbrella of the oleosin molecule are phospholipids rendered resistant to hydrolysis (7).…”

Biogenesis and functions of lipid droplets in plants

“…Like most membrane-bound proteins, oleosins are initially inserted into the ER in a cotranslational manner (42)(43)(44), after which they adopt an orientation that includes their N and C termini facing toward the cytosol and a large hydrophobic domain intercalated within the ER bilayer ( 45 ). A conserved, so-called "proline knot" at the middle of the hydrophobic domain is essential for targeting (partitioning) the oleosin protein to LDs ( 45 ) ( Fig.…”