Membrane topology and sequence requirements for oil body targeting of oleosin

Oleosin protein is targeted to oil bodies via the endoplasmic reticulum (ER) and consists of a lipid-submerged hydrophobic (H) domain that is flanked by cytosolic hydrophilic domains. We investigated the relationship between oleosin ER topology and its subsequent ability to target to oil bodies. Oleosin variants were created to yield differing ER membrane topologies and tagged with a reporter enzyme. Localisation was assessed by fractionation after transient expression in embryonic cells. Membrane-straddled topologies with N-terminal sequence in the ER lumen and C-terminal sequence in the cytosol were unable to target to oil bodies efficiently. Similarly, a translocated topology with only ER membrane and lumenal sequence was unable to target to oil bodies efficiently. Both topology variants accumulated proportionately higher in ER microsomal fractions, demonstrating a block in transferring from ER to oil bodies. The residual oil body accumulation for the inverted topology was shown to be because of partial adoption of native ER membrane topology, using a reporter variant, which becomes inactivated by ER-mediated glycosylation. In addition, the importance of H domain sequence for oil body targeting was assessed using variants that maintain native ER topology. The central proline knot motif (PKM) has previously been shown to be critical for oil body targeting, but here the arms of the H domain flanking this motif were shown to be interchangeable with only a moderate reduction in oil body targeting. We conclude that oil body targeting of oleosin depends on a specific ER membrane topology but does not require a specific sequence in the H domain flanking arms.     

Purification and structural characterization of the central hydrophobic domain of oleosin

The oil bodies of rapeseeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with abundant structural alkaline proteins termed oleosins and some other minor proteins. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain flanked by relatively polar C- and N-terminal domains. Although the hydrophilic N-terminal domain had been studied, the structural feature of the central hydrophobic domain remains unclear due to its high hydrophobicity. In the present study, we reported the generation, purification, and characterization of a 9-kDa central hydrophobic domain from rapeseed oleosin (19 kDa). The 9-kDa central hydrophobic domain was produced by selectively degrading the N and C termini with enzymes and then purifying the digest by SDS-PAGE and electroelution. We have also reconstituted the central domain into liposomes and synthetic oil bodies to determine the secondary structure of the domain using CD and Fourier transform infrared (FTIR) spectroscopy. The spectra obtained from CD and FTIR were analyzed with reference to structural information of the N-terminal domain and the full-length rapeseed oleosin. Both CD and FTIR analysis revealed that 50-63% of the domain was composed of beta-sheet structure. Detailed analysis of the FTIR spectra indicated that 80% of the beta-sheet structure, present in the central domain, was arranged in parallel to the intermolecular beta-sheet structure. Therefore, interactions between adjacent oleosin proteins would give rise to a stable beta-sheet structure that would extend around the surface of the seed oil bodies stabilizing them in emulsion systems. The strategies used in our present study are significant in that it could be generally used to study difficult proteins with different independent structural domains, especially with long hydrophobic domains.     

Characterization and modelling of the hydrophobic domain of a sunflower oleosin

The oleosins are a group of hydrophobic proteins present on the surface of oil bodies in seeds, where they are thought to prevent coalescence. They contain a central hydrophobic domain of 68-74 residues that is thought to form a loop into the triacylglycerol matrix of the oil body, but the conformation adopted by this sequence is uncertain. We have therefore expressed an oleosin cDNA from sunflower (Helianthus annuus L.) in Escherichia coli as a fusion with maltose-binding protein (MBP) and isolated a peptide corresponding to the hydrophobic domain by sequential digestion with factor Xa (to remove the MBP) followed by trypsin and Staphylococcus V8 protease to remove the N- and C-terminal domains of the oleosin. Circular dichroism spectroscopy of the peptide in two solvent systems chosen to mimic the environment within the oil body (trifluoroethanol and SDS) demonstrated high proportions of alpha-helical structure, with no beta-sheet. A model was therefore developed in which the domain forms an alpha-helical hairpin structure, the two helices being separated by a turn region. We consider that this model is consistent with our current knowledge of oleosin structure and properties.     

Solubilization conditions for hydrophobic membrane protein,oleosin, in soybeans

Oleosins are amphipathic proteins found in soybeans that give stability to oil-bodies by surrounding them. Furthermore, oleosins have broad biotechnological applications in protein purification and in immobilization as a tagging parther. In this study we provide the proper combinations of detergents and chaotropes for solubilization of oleosins. The solubilization effects of each detergent were compared by the results of SDS-PAGE analysis, from the density measurement of each band using an image analysis program. Among the twelve chemicals tested, 3-(decyldimethylammonio) propanesulfonale inner salt (SB 3–10), nonaethylene glycol octylphenyl ether (NP-40), and n-dodecyl-β-d-maltoside (DM) were determined to be effective for oleosin solubilization. In addition, urea, thiourea, and reducing agents played roles in enhancing the effects of the detergents. The best results were obtained with 2.5% of each detergent in combination with 4 M urea and 2 M thiourea at room temperature. The zwitterionic detergent, SB 3–10, is recommended as the best non-denaturant for the solubilization of soybean oleosins with the advantage of its easy removal by dialysis due to its low molecular weight and a high critical micelle concentration.     

The S-layer protein of Corynebacterium glutamicum is anchored to the cell wall by its C-terminal hydrophobic domain

PS2 is the S-layer protein of Corynebacterium glutamicum . The S-layer may be detached from the cell as organized sheets by detergents at room temperature. The solubilization of PS2 in the form of monomers requires detergent treatment at high temperature (70°C), conditions under which the protein is denatured. Treatment of the cells with proteinase K or trypsin results in the detachment of the organized S-layer, which remains organized. Because we show that trypsin cleaves the C-terminal part of the protein, we conclude that this domain is involved in the association of the S-layer to the cell but is not essential in the interaction between individual PS2 proteins within the S-layer. A modified form of PS2, deleted of its C-terminal hydrophobic sequence, was constructed. The protein is almost unable to form an organized S-layer and is mainly released into the medium. We suggest that PS2 is anchored via its C-terminal hydrophobic sequence to a hydrophobic layer of the wall of the bacterium located some distance above the cytoplasmic membrane.     

Improved membrane protein topology prediction by domain assignments

Topology predictions for integral membrane proteins can be substantially improved if parts of the protein can be constrained to a given in/out location relative to the membrane using experimental data or other information. Here, we have identified a set of 367 domains in the SMART database that, when found in soluble proteins, have compartment-specific localization of a kind relevant for membrane protein topology prediction. Using these domains as prediction constraints, we are able to provide high-quality topology models for 11% of the membrane proteins extracted from 38 eukaryotic genomes. Two-thirds of these proteins are single spanning, a group of proteins for which current topology prediction methods perform particularly poorly.     

Membrane topology of the Escherichia coli ExbD protein.

The ExbD protein is involved in the energy-coupled transport of ferric siderophores, vitamin B12, and B-group colicins across the outer membrane of Escherichia coli. In order to study ExbD membrane topology, ExbD-beta-lactamase fusion proteins were constructed. Cells expressing beta-lactamase fusions to residues 53, 57, 70, 76, 78, 80, 92, 121, and 134 of ExbD displayed high levels of ampicillin resistance, whereas fusions to residues 9 and 19 conferred no ampicillin resistance. It is concluded that the only hydrophobic segment of ExbD, encompassing residues 23 to 43, forms a transmembrane domain and that residues 1 to 22 are located in the cytoplasm and residues 44 to 141 are located in the periplasm.     

Microtubule-associated protein tau is phosphorylated by protein kinase C on its tubulin binding domain.

We have analyzed the in vitro phosphorylation of tau protein by Ca2+/calmodulin-dependent protein kinase, casein kinase II, and proline-directed serine/threonine protein kinase. These kinases phosphorylate tau protein in sites localized in different regions of the molecule, as determined by peptide mapping analyses. Focusing on the phosphorylation of tau by protein kinase C, it was calculated as an incorporation of 4 mol of phosphate/mol of tau. Limited proteolysis assays suggest that the phosphorylation sites could be located within the tubulin-binding domain. Direct phosphorylation of synthetic peptides corresponding to the cysteine-containing tubulin-binding region present in both fetal and adult tau isoforms demonstrates that serine 313 is modified by protein kinase C. Phosphorylation of the synthetic peptide by protein kinase C diminishes its binding to tubulin, as compared with the unphosphorylated peptide.     

Membrane localization and topology of a viral assembly protein.

The gene I protein (pI) of the filamentous bacteriophage f1 is required for the assembly of this virus. Antibodies specific to either the amino or carboxyl terminus of this protein were used to determine the location and topology of the gene I protein in f1-infected bacteria. pI is anchored in the inner membrane of Escherichia coli cells via a 20-amino-acid hydrophobic stretch, with its carboxyl-terminal 75 residues located in the periplasm and its amino-terminal 253 amino acids residing in the cytoplasm. By using the carboxyl-terminal pI antibody, a smaller protein, pI*, is also detected in f1-infected cells at a ratio of one to two molecules per molecule of pI. Analysis of proteins produced from a gene I amber mutant plasmid or bacteriophage suggests that pI* is most likely the result of an in-frame internal translational initiation event at methionine 241 of the 348-amino-acid pI. pI* is shown to be an integral inner membrane protein inserted in the same orientation as pI. The relation of the cellular locations of pI and pI* to some of the proposed functions of pI is discussed.     

The folding of an immunoglobulin-like Greek key protein is defined by a common-core nucleus and regions constrained by topology

TNfn3, the third fibronectin type III domain of human tenascin, is an immunoglobulin-like protein that is a good model for experimental and theoretical analyses of Greek key folding. The third fibronectin type III domain of human tenascin folds and unfolds in a two-state fashion over a range of temperature and pH values, and in the presence of stabilising salts. Here, we present a high resolution protein engineering analysis of the single rate determining transition state. The 48 mutations report on the contribution of side-chains at 32 sites in the core and loop regions. Three areas in the protein exhibit high Phi-values, indicating that they are partially structured in the transition state. First, a common-core ring of four positions in the central strands B, C, E and F, that are in close contact, form a nucleus of tertiary interactions. The two other regions that appear well-formed are the C' region and the E-F loop. The Phi-values gradually decrease away from these regions such that the very ends of the two terminal strands A and G, have Phi-values of zero. We propose a model for the folding of immunoglobulin-like proteins in which the common-core "ring" forms the nucleus for folding, whilst the C' and E-F regions are constrained by topology to pack early. Folding characteristics of a group of structurally related proteins appear to support this model.     

Membrane topology of the human seipin protein

The Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene encodes an integral membrane protein, called seipin, of unknown function localized to the endoplasmic reticulum of eukaryotic cells. Seipin is associated with the heterogeneous genetic disease BSCL2, and mutations in an N-glycosylation motif links the protein to two other disorders, autosomal-dominant distal hereditary motor neuropathy type V and Silver syndrome. Here, we report a topological study of seipin using an in vitro topology mapping assay. Our results suggest that the predominant form of seipin is 462 residues long and has an N(cyt)-C(cyt) orientation with a long luminal loop between the two transmembrane helices.     

Microdeletions within the hydrophobic core region of cellular prion protein alter its topology and metabolism

The cellular prion protein (PrP^C) is a GPI-anchored cell-surface protein. A small subset of PrP^C molecules, however, can be integrated into the ER-membrane via a transmembrane domain (TM), which also harbors the most highly conserved regions of PrP^C, termed the hydrophobic core (HC). A mutation in HC is associated with prion disease resulting in an enhanced formation of a transmembrane form (^CtmPrP), which has thus been postulated to be a neurotoxic molecule besides PrP^Sc. To elucidate a possible physiological function of the transmembrane domain, we created a set of mutants carrying microdeletions of 2-8 aminoacids within HC between position 114 and 121. Here, we show that these mutations display reduced propensity for transmembrane topology. In addition, the mutants exhibited alterations in the formation of the C1 proteolytic fragment, which is generated by α-cleavage during normal PrP^C metabolism, indicating that HC might function as recognition site for the protease(s) responsible for PrP^C α-cleavage. Interestingly, the mutant G113V, corresponding to a hereditary form of prion disease in humans, displayed increased ^CtmPrP topology and decreased α-cleavage in our in vitro assay. In conclusion, HC represents an essential determinant for transmembrane PrP topology, whereas the high evolutionary conservation of this region is rather based upon preservation of PrP^C α-cleavage, thus highlighting the biological importance of this cleavage.     

Nucleolin is an Ag-NOR protein; this property is determined by its amino-terminal domain independently of its phosphorylation state.

The Ag-NOR proteins are defined as markers of "active" ribosomal genes. They correspond to a set of proteins specifically located in the nucleolar organizer regions (NORs), but have not yet been clearly identified. We adapted the specific detection method of the Ag-NOR proteins to Western blots in order to identify these proteins. Using a purified protein, Western blots, and immunological characterization, the present study brings the first direct evidence leading to the identity of one Ag-NOR protein. We found that nucleolin is specifically revealed by Ag-NOR staining. Using different nucleolin fragments generated by CNBr cleavage and by overexpression in Escherichia coli, we demonstrate that the amino-terminal domain of nucleolin and not the carboxy-part of the protein is involved in silver staining. Moreover, as the pattern of staining does not vary using casein kinase II- and cdc2-phosphorylated nucleolin or dephosphorylated nucleolin, we conclude that the reduction of the silver ions is not linked to the phosphorylation state of the molecule. We propose that the concentration of acidic amino acids in the amino-terminal domain of nucleolin is responsible for Ag-NOR staining. This hypothesis is also supported by the finding that poly L-glutamic acid peptides are silver stained. These results provide data that can be used to explain the specificity of Ag-NOR staining. Furthermore, we clearly establish that proteolysis of the amino-terminal Ag-NOR-sensitive part of nucleolin occurs in vitro, leading to the accumulation of the carboxy-terminal Ag-NOR-negative part of the protein. We argue that this cleavage occurs in vivo as already proposed, bearing in mind that nucleolin is present in the fibrillar and in the granular component of the nucleolus, whereas no Ag-NOR staining is observed in the latter nucleolar component.     

Membrane protein insertion regulated by bringing electrostatic and hydrophobic interactions into play. A case study with the translocation domain of diphtheria toxin

The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.     

Membrane topology influences N-glycosylation of the prion protein

The glycosylation state of the glycosyl-phosphatidylinositol (GPI) anchored cellular prion protein (PrPC) can influence the formation of the disease form of the protein responsible for the neurodegenerative spongiform encephalopathies. We have investigated the role of membrane topology in the N-glycosylation of PrP by expressing a C-terminal transmembrane anchored form, PrP-CTM, an N-terminal transmembrane anchored form, PrP-NTM, a double-anchored form, PrP-DA, and a truncated form, PrPDeltaGPI, in human neuroblastoma SH-SY5Y cells. Wild-type PrP, PrP- CTM and PrP-DA were membrane anchored and present on the cell surface as glycosylated forms. In contrast, PrP-NTM, although membrane anchored and localized at the cell surface, was not N-glycosylated. PrPDeltaGPI was secreted from the cells into the medium in a hydrophilic form that was unglycosylated. The 4-fold slower rate at which PrPDeltaGPI was trafficked through the cell compared with wild-type PrP was due to the absence of the GPI anchor not the lack of N-glycans. Retention of PrPDeltaGPI in the endoplasmic reticulum did not lead to its glycosylation. These results indicate that C-terminal membrane anchorage is required for N-glycosylation of PrP.     

The cellular protein level of parkin is regulated by its ubiquitin-like domain

Parkin is a ubiquitin-protein isopeptide ligase (E3) involved in ubiquitin/proteasome-mediated protein degradation. Mutations in the parkin gene cause a loss-of-function and/or alter protein levels of parkin. As a result, the toxic build-up of parkin substrates is thought to lead to autosomal recessive juvenile Parkinsonism. To identify a role for the ubiquitin-like domain (ULD) of parkin, we created a number of hemagglutinin (HA)-tagged parkin constructs using mutational and structural information. Western blotting and immunocytochemistry showed a much stronger expression level for HA-parkin residues 77-465 (without ULD) than HA-parkin full-length (with ULD). The deletion of ULD in Drosophila parkin also caused a sharp increase in expression of the truncated form, suggesting that the function of the ULD of parkin is conserved across species. By progressive deletion analysis of parkin ULD, we found that residues 1-6 of human parkin play a crucial role in controlling the expression levels of this gene. HA-parkin residues 77-465 showed ubiquitination in vivo, demonstrating that the ULD is not critical for parkin auto-ubiquitination; ubiquitination seemed to cluster on the central domain of parkin (residues 77-313). These effects were specific for the ULD of parkin and not transfection-, toxic-, epitope tag-, and/or vector-dependent. Taken together, these data suggest that the 76 most NH(2)-terminal residues (ULD) dramatically regulate the protein levels of parkin.     

Membrane topology of helix 0 of the Epsin N-terminal homology domain

Specific interaction of the epsin N-terminal homology (ENTH) domain with the plasma membrane appears to bridge other related proteins to the specific regions of the membrane that are invaginated to form endocytic vesicles. An additional a-helix, referred to as helix 0 (H0), is formed in the presence of the soluble ligand inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] at the N terminus of the ENTH domain (amino acid residues 3-15). The ENTH domain alone and full-length epsin cause tubulation of liposomes made of brain lipids. Thus, it is believed that H0 is membrane-inserted when it is coordinated with the phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], resulting in membrane deformation as well as recruitment of accessory factors to the membrane. However, formation of H0 in a real biological membrane has not been demonstrated. In the present study, the membrane structure of H0 was determined by measurement of electron paramagnetic resonance (EPR) nitroxide accessibility. H0 was located at the phosphate head-group region of the membrane. Moreover, EPR line-shape analysis indicated that no pre-formed H0-like structure were present on normal acidic membranes. PtdIns(4,5)P2 was necessary and sufficient for interaction of the H0 region with the membrane. H0 was stable only in the membrane. In conclusion, the H0 region of the ENTH domain has an intrinsic ability to form H0 in a PtdIns(4,5)P2-containing membrane, perhaps functioning as a sensor of membrane patches enriched with PtdIns(4,5)P2 that will initiate curvature to form endocytic vesicles.     

Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain

Ca(2+)-independent or novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown function. Removal of the C2 domain of the Aplysia nPKC Apl II allows activation of the enzyme at lower concentrations of phosphatidylserine, suggesting an inhibitory role for the C2 domain in enzyme activation. However, the mechanism for C2 domain-mediated inhibition is not known. Mapping of the autophosphorylation sites for protein kinase C (PKC) Apl II reveals four phosphopeptides in the regulatory domain of PKC Apl II, two of which are in the C2 domain at serine 2 and serine 36. Unlike most PKC autophosphorylation sites, these serines could be phosphorylated in trans. Interestingly, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes in vitro. In cells, PKC Apl II phosphorylation at serine 36 was increased by PKC activators, and PKC phosphorylated at this position translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. We suggest that translocation of nPKCs is regulated by phosphorylation of the C2 domain.     

Selective membrane permeabilization by the rotavirus VP5* protein is abrogated by mutations in an internal hydrophobic domain

Rotavirus infectivity is dependent on the proteolytic cleavage of the VP4 spike protein into VP8* and VP5* proteins. Proteolytically activated virus, as well as expressed VP5*, permeabilizes membranes, suggesting that cleavage exposes a membrane-interactive domain of VP5* which effects rapid viral entry. The VP5* protein contains a single long hydrophobic domain (VP5*-HD, residues 385 to 404) at an internal site. In order to address the role of the VP5*-HD in permeabilizing cellular membranes, we analyzed the entry of o-nitrophenyl-beta-D-galactopyranoside (ONPG) into cells induced to express VP5* or mutated VP5* polypeptides. Following IPTG (isopropyl-beta-D-thiogalactopyranoside) induction, VP5* and VP5* truncations containing the VP5*-HD permeabilized cells to the entry and cleavage of ONPG, while VP8* and control proteins had no effect on cellular permeability. Expression of VP5* deletions containing residues 265 to 474 or 265 to 404 permeabilized cells; however, C-terminal truncations which remove the conserved GGA (residues 399 to 401) within the HD abolished membrane permeability. Site-directed mutagenesis of the VP5-HD further demonstrated a requirement for residues within the HD for VP5*-induced membrane permeability. Functional analysis of mutant VP5*s indicate that conserved glycines within the HD are required and suggest that a random coiled structure rather than the strictly hydrophobic character of the domain is required for permeability. Expressed VP5* did not alter bacterial growth kinetics or lyse bacteria following induction. Instead, VP5*-mediated size-selective membrane permeability, releasing 376-Da carboxyfluorescein but not 4-kDa fluorescein isothiocyanate-dextran from preloaded liposomes. These findings suggest that the fundamental role for VP5* in the rotavirus entry process may be to expose triple-layered particles to low [Ca](i), which uncoats the virus, rather than to effect the detergent-like lysis of early endosomal membranes.