Monotopic Membrane Protein Research Articles

Conformation, pore-forming activity, and antigenicity of synthetic peptide analogues of a spiralin putative amphipathic α helix

Current models depict spiralin as a bitopic transmembrane protein with the transbilayer domain being an amphipathic α helix. However, though secondary structure prediction methods suggest a helical conformation for the hypothetical transmembrane segment of spiralin, no potential transmembrane helices could be detected in this protein using the method of Von Heijne ( Von Heijne, G. (1992) J. Mol. Biol. 225, 487–494). Therefore, we have reconsidered the spiralin topological model by investigating the properties of the chemically synthesized peptides SM-BC3 (LNAVNTYATLAKAVLDAIQN-NH 2) and SC-R8A2 (LNAVNTYATLASAVLEAIKN-NH 2), corresponding to the hypothetical transmembrane segments of spiralins of two distinct spiroplasma species. The hydrophobic moment plot method suggests that these spiralin amino acid stretches are class G amphipathic α helices (i.e., helices localized on the surface of a globular protein domain). Circular dichroism spectra showed that both peptides have little ordered structure in aqueous solutions but adopt a mainly helical conformation in the presence of 25% trifluoroethanol or in detergent micelles (up to 74% α helix). Both peptides formed concentration- and voltage-dependent pores in planar lipid bilayers with a unitary conductance of 130 pS in 1 M KCl and with mean numbers of monomers per conducting aggregates of 6 for SC-R8A2 and 9 for SM-BC3. However, the two peptides displayed a haemolytic activity only at high concentrations (≫ 250 μM) and reacted with antibodies raised against membrane-bound spiralin. Together with previously published results, these data suggest that spiralin is a monotopic membrane protein anchored at the surface of the spiroplasma cell and that the 20-residue amphipathic segment is most probably a class G helix containing a B-cell epitope.

Overexpression of TGN38/41 leads to mislocalisation of γ-adaptin

TGN38 and TGN41 are isoforms of a monotopic integral membrane protein which recycles between the trans Golgi network (TGN) and the cell surface, but which, at steady state, is predominantly located in the TGN. Full-length and truncated versions of rat TGN38/41 have been expressed in monkey (COS) and human (Heb7a) cells under the control of the heavy metal inducible Metallothionein IIA promoter. This has allowed the regulated expression of TGN38/41 protein constructs to different levels in the transfected cells. These studies show that (i) controlled overexpression of TGN38/41 results in mislocalisation to parts of the endocytic pathway, (ii) a truncated version of TGN38/41, lacking the cytoplasmic domain, remains in the TGN, and (iii) there is a direct or indirect interaction between the cytoplasmic domain of TGN38/41 and γ-adaptin.

Prostaglandin H synthase: Implications for membrane structure

The crystal structure of the membrane protein prostaglandin H synthase (PGHS) provides strong evidence for the existence of monotopic membrane proteins: PGHS seems to interact with the membrane via a motif of amphipathic helices positioned parallel to the plane of the membrane. The orientation of this unique membrane binding motif is fixed in space by an epidermial growth factor(EGF)-like module on its amino-terminal end and by the catalytic domain at its carboxy-terminal end. The catalytic domain of PGHS has a high structural homology to other mammalian heme peroxidases.

The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1.

The three-dimensional structure of prostaglandin H2 synthase-1, an integral membrane protein, has been determined at 3.5 A resolution by X-ray crystallography. This bifunctional enzyme comprises three independent folding units: an epidermal growth factor domain, a membrane-binding motif and an enzymatic domain. Two adjacent but spatially distinct active sites were found for its haem-dependent peroxidase and cyclooxygenase activities. The cyclooxygenase active site is created by a long, hydrophobic channel that is the site of non-steroidal anti-inflammatory drug binding. The conformation of the membrane-binding motif strongly suggests that the enzyme integrates into only one leaflet of the lipid bilayer and is thus a monotopic membrane protein.

Evaluation of transmembrane helix prediction methods using the recently defined NMR structures of the coat proteins from bacteriophages M13 and Pf1

Currently, there are a large number of hydropathy scales available to predict the presence of transmembrane segments within integral membrane proteins. These scales and their subsequent numerical manipulations provide an aid in the determination of topology in transmembrane proteins. In order to analyse the accuracy of these procedures to correctly identify the boundaries of a transmembrane segment, 13 methods were applied to the amino-acid sequence of the coat proteins from the bacteriophages Pf1 and M13. These monotopic integral membrane proteins have been incorporated into detergent micelles and their structures have recently been solved using NMR. The predicted regions were then compared to their NMR-determined structures. All methods were able to detect a transmembrane region within the protein sequence. However, there was considerable differences in their accuracy in determining the boundaries of the main transmembrane α-helix. Surprisingly, the methods which worked the best for Pf1 coat protein had poor accuracy in identifying the transmembrane region correctly in the M13 protein. It was concluded that a number of methods should be utilized in order to obtain a clear model of transmembrane protein topology, and that regardless of how closely related two proteins are, a different conclusion may be obtained from different prediction procedures.

The hydrophobic membrane-spanning sequences of the gp52 glycoprotein are required for the pathogenicity of Friend spleen focus-forming virus

Friend spleen focus-forming virus (SFFV) codes for a transport-defective envelope glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 is a monotopic integral membrane protein anchored in the membrane by a stretch of hydrophobic amino acid residues located near the carboxy terminus of the molecule. We have constructed a mutant SFFV envelope gene in which the sequences that code for the hydrophobic membrane-spanning domain have been deleted, and we expressed this gene by using recombinant vaccinia virus vectors or retroviral vectors. The mutant SFFV envelope gene was found to encode a truncated glycoprotein (gp52t) which was also transport defective; a majority of gp52t remained cell associated, while a small proportion of the molecules underwent oligosaccharide processing. The processed form of gp52t was secreted from the cells. Retroviral vectors carrying the mutant SFFV envelope gene were found to be nonpathogenic in adult mice. These results indicate that the hydrophobic membrane-spanning region of gp52 is required for pathogenicity of SFFV and suggest that these sequences may play a role in signal transduction. The results also indicate that the transport defect of SFFV gp52 is due to structural features of the ectodomain of the molecule.

Membrane association and defective transport of spleen focus-forming virus glycoproteins.

The gp52 glycoprotein of the spleen focus-forming virus found in the Friend and Rauscher complexes of murine leukemia viruses (MuLV) has been previously identified as a recombinant molecule involving substitutions and deletions of the MuLV env gene. Unlike the MuLV structural glycoproteins, gp52 is defective in its transport to the cell surface. We have studied aspects of the intracellular transport and membrane association of gp52 to investigate the possible mechanisms underlying the defective transport process. It was found that a panel of monoclonal antibodies to different epitopes of p 15E, as well as an antiserum to a synthetic peptide corresponding to the carboxy terminus of MuLV envelope precursors, failed to react with gp52. Despite the possible absence of membrane-anchoring regions of MuLV envelope proteins known to reside on p 15E, gp52 was not found to be secreted into the culture fluids. Detergent extraction studies indicated that gp52 is associated with the membranes and not the contents of microsomal vesicles in speen focus-forming virus-infected cells. gp65, the processed form of gp52, could be labeled with [3H]palmitic acid, suggesting a membrane association. To determine whether a spontaneous denaturation occurs leading to aggregation and defective transport of gp52, we studied the surface expression of gp52 in cells grown at different temperatures, as well as the solubility of gp52 in low concentrations of Triton X-100. No evidence of aggregation or of a temperature-dependent difference in transport was obtained. gp52 appears to be a monotopic integral membrane protein, unlike MuLV envelope proteins which are bitopic integral membrane proteins; proteolytic digestion of intact microsomal vesicles did not reveal a detectable cytoplasmic tail under conditions where this could be demonstrated on MuLV envelope precursors. We suggest that a loss of putative signals involved in mediating intracellular transport is a likely cause for the defective transport of the spleen focus-forming virus glycoproteins.