Membrane topology mapping of vitamin K epoxide reductase by in vitro translation/cotranslocation

Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10-29, 101-123, and 127-149, and membrane-integration of residues 75-97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM.     

Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation

The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.     

Molecular Analysis of Ems-Induced Frizzled Mutations in Drosophila Melanogaster

The frizzled (fz) gene of Drosophila is essential for the development of normal tissue polarity in the adult cuticle of Drosophila. In fz mutants the parallel array of hairs and bristles that decorate the cuticle is disrupted. Previous studies have shown that fz encodes a membrane protein with seven putative transmembrane domains, and that it has a complex role in the development of tissue polarity, as there exist both cell-autonomous and cell nonautonomous alleles. We have now examined a larger number of alleles and found that 15 of 19 alleles display cell nonautonomy. We have examined these and other alleles by Western blot analysis and found that most fz mutations result in altered amounts of Fz protein, and many also result in a Fz protein that migrates aberrantly in SDS-PAGE. We have sequenced a subset of these alleles. Cell nonautonomous fz alleles were found to be associated with mutations that altered amino acids in all regions of the Fz protein. Notably, the four cell-autonomous mutations were all in a proline residue located in the presumptive first cytoplasmic loop of the protein. We have also cloned and sequenced the fz gene from D. virilis. Conceptual translation of the D. virilis open reading frame indicates that the Fz protein is unusually well conserved. Indeed, in the putative cytoplasmic domains the Fz proteins of the two species are identical.     

Characterization of membrane association domains within the Tomato ringspot nepovirus X2 protein, an endoplasmic reticulum-targeted polytopic membrane protein

Replication of nepoviruses (family Comoviridae) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleoside triphosphate-binding protein (NTB) of Tomato ringspot nepovirus is an integral membrane protein with two ER-targeting sequences and have suggested that it anchors the viral replication complex (VRC) to the membranes. A second highly hydrophobic protein domain (X2) is located immediately upstream of the NTB domain in the RNA1-encoded polyprotein. X2 shares conserved sequence motifs with the comovirus 32-kDa protein, an ER-targeted protein implicated in VRC assembly. In this study, we examined the ability of X2 to associate with intracellular membranes. The X2 protein was fused to the green fluorescent protein and expressed in Nicotiana benthamiana by agroinfiltration. Confocal microscopy and membrane flotation experiments suggested that X2 is targeted to ER membranes. Mutagenesis studies revealed that X2 contains multiple ER-targeting domains, including two C-terminal transmembrane helices and a less-well-defined domain further upstream. To investigate the topology of the protein in the membrane, in vitro glycosylation assays were conducted using X2 derivatives that contained N-glycosylation sites introduced at the N or C termini of the protein. The results led us to propose a topological model for X2 in which the protein traverses the membrane three times, with the N terminus oriented in the lumen and the C terminus exposed to the cytoplasmic face. Taken together, our results indicate that X2 is an ER-targeted polytopic membrane protein and raises the possibility that it acts as a second membrane anchor for the VRC.     

Topology models for 37 Saccharomyces cerevisiae membrane proteins based on C-terminal reporter fusions and predictions

We provide experimentally based topology models for 37 integral membrane proteins from Saccharomyces cerevisiae. A C-terminal fusion to a dual Suc2/His4C topology reporter has been used to determine the location of the C terminus of each protein relative to the endoplasmic reticulum membrane, and this information is used in conjunction with theoretical topology prediction methods to arrive at a final topology model. We propose that this approach may be used to produce reliable topology models on a proteome-wide scale.     

Membrane topology of the murine fatty acid transport protein 1

The murine fatty acid transport protein (FATP1) was identified in an expression cloning screen for proteins that facilitate transport of fatty acids across the plasma membranes of mammalian cells. Hydropathy analysis of this protein suggests a model in which FATP1 has multiple membrane-spanning domains. To test this model, we inserted a hemagglutinin epitope tag at the amino terminus or a FLAG tag at the carboxyl terminus of the FATP1 cDNA and expressed these constructs in NIH 3T3 cells. Both tagged constructs produce proteins of the expected molecular masses and are functional in fatty acid import assays. Indirect immunofluorescence studies with selective permeabilization conditions and protease protection studies of sealed membrane vesicles from cells expressing epitope-tagged FATP1 were performed. These experiments show that the extreme amino terminus of tagged FATP1 is oriented toward the extracellular space, whereas the carboxyl terminus faces the cytosol. Additionally, enhanced green fluorescent protein fusion constructs containing predicted membrane-associated or soluble portions of FATP1 were expressed in Cos7 cells and analyzed by immunofluorescence and subcellular fractionation. These experiments demonstrate that amino acids 1-51, 52-100, and 101-190 contain signals for integral association with the membrane, whereas residues 258-313 and 314-475 are only peripherally membrane-associated. Amino acid residues 191-257 and 476-646 do not direct membrane association and likely face the cytosol. Taken together, these data support a model of FATP1 as a polytopic membrane protein with at least one transmembrane and multiple membrane-associated domains. This study provides the first experimental evidence for topology of a member of the family of plasma membrane fatty acid transport proteins.     

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE*

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N_cyt/C_exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.     

Topology of the transposon Tn10-encoded tetracycline resistance protein within the inner membrane of Escherichia coli

The transposon Tn10-encoded tetracycline resistance protein TetA is an integral membrane protein responsible for the export of tetracycline from the cytoplasmic to the periplasmic side of the inner membrane of Gram-negative bacteria. From a plot of the average hydrophobicity along the sequence of this protein, a two-dimensional membrane topology with 12 transmembrane domains may be predicted. Using plasmid-bearing Escherichia coli maxicells we specifically radiolabeled the TetA protein. The amino terminus of this membrane protein was shown not to be processed, and its location on the inner side of the cytoplasmic membrane was demonstrated by a newly developed use of a chemical method. Spheroplasts and inside-out vesicles of the TetA protein synthesizing maxicells were subjected to limited digestion by proteases of different specificities. The TetA protein was not accessible to proteases from the periplasmic side. On the inner side of the cytoplasmic membrane, the carboxyl terminus and four sites accessible to endoproteases could be identified. The cleavage sites are proposed to be localized between amino acid residues 60-70, 110-130, 180-200, and at amino acid 327. These results allow the definition of a model for the two-dimensional topology of the TetA protein.     

Topology of Streptococcus pneumoniae CpsC,a Polysaccharide Copolymerase and Bacterial Protein Tyrosine Kinase Adaptor Protein

In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. In Streptococcus pneumoniae, this transmembrane adaptor is CpsC, with the C terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest that CpsC has two transmembrane domains, with the N and C termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal hemagglutinin (HA)-tagged Cps2C protein in S. pneumoniae strain D39. Incubation of both protoplasts and membranes with carboxypeptidase B (CP-B) resulted in complete degradation of HA-Cps2C in all cases, indicating that the C terminus of Cps2C was likely extracytoplasmic and hence that the protein''s topology was not as predicted. Similar results were seen with membranes from S. pneumoniae strain TIGR4, indicating that Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C terminus by flow cytometry indicated that it was extracytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this effect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C terminus flipping across the cytoplasmic membrane, where it interacts with CpsD in order to regulate tyrosine kinase activity.     

Subcellular localization and membrane topology of the melon ethylene receptor CmERS1

Ethylene receptors are multispanning membrane proteins that negatively regulate ethylene responses via the formation of a signaling complex with downstream elements. To better understand their biochemical functions, we investigated the membrane topology and subcellular localization of CmERS1, a melon (Cucumis melo) ethylene receptor that has three putative transmembrane domains at the N terminus. Analyses using membrane fractionation and green fluorescent protein imaging approaches indicate that CmERS1 is predominantly associated with the endoplasmic reticulum (ER) membrane. Detergent treatments of melon microsomes showed that the receptor protein is integrally bound to the ER membrane. A protease protection assay and N-glycosylation analysis were used to determine membrane topology. The results indicate that CmERS1 spans the membrane three times, with its N terminus facing the luminal space and the large C-terminal portion lying on the cytosolic side of the ER membrane. This orientation provides a platform for interaction with the cytosolic signaling elements. The three N-terminal transmembrane segments were found to function as topogenic sequences to determine the final topology. High conservation of these topogenic sequences in all ethylene receptor homologs identified thus far suggests that these proteins may share the same membrane topology.     

Molecular determinants of the sensory and motor neuron-derived factor insertion into plasma membrane

The sensory and motor neuron-derived factor (SMDF) is a type III neuregulin that regulates development and proliferation of Schwann cells. Although SMDF has been shown to be a type II protein, the molecular determinants of membrane biogenesis, insertion, and topology remain elusive. Here we used heterologous expression of a yellow fluorescent protein-SMDF fusion protein along with a stepwise deletion strategy to show that the apolar/uncharged segment (Ile(76)-Val(100)) acts as an internal, uncleaved membrane insertion signal that defines the topology of the protein. Unexpectedly, removal of the transmembrane segment (TM) did not eliminate completely membrane association of C-terminal fragments. TM-deleted fusion proteins, bearing the amino acid segment (Ser(283)-Glu(296)) located downstream to the epidermal growth factor-like motif, strongly interacted with plasma membrane fractions. However, synthetic peptides patterned after this segment did not insert into artificial lipid vesicles, suggesting that membrane interaction of the SMDF C terminus may be the result of a post-translational modification. Subcellular localization studies demonstrated that the 40-kDa form, but not the 83-kDa form, of SMDF was segregated into lipid rafts. Deletion of the N-terminal TM did not affect the interaction of the protein with these lipid microdomains. In contrast, association with membrane rafts was abolished completely by truncation of the protein C terminus. Collectively, these findings are consistent with a topological model for SMDF in which the protein associates with the plasma membrane through both the TM and the C-terminal end domains resembling the topology of other type III neuregulins. The TM defines its characteristic type II membrane topology, whereas the C terminus is a newly recognized anchoring motif that determines its compartmentalization into lipid rafts. The differential localization of the 40- and 83-kDa forms of the neuregulin into rafts and non-raft domains implies a central role in the protein biological activity.     

During apoptosis bcl-2 changes membrane topology at both the endoplasmic reticulum and mitochondria

In healthy cells the antiapoptotic protein Bcl-2 adopts a topology typical of tail-anchored proteins with only the hydrophobic carboxyl terminus inserted into the membrane, as shown by labeling cell lysates with a membrane-impermeant sulfhydryl-specific reagent. Induction of apoptosis in cells triggered a change in the conformation of Bcl-2 such that cysteine 158 near the base of helix 5 inserted into the lipid bilayer of both endoplasmic reticulum and mitochondria where it was protected from labeling. Addition of a peptide corresponding to the BH3 domain of the proapoptotic protein Bim to cell lysates triggered a similar conformational change in Bcl-2, demonstrating that preexisting, membrane-bound Bcl-2 proteins change topology.     

Location and unusual membrane topology of the immunity protein of the Escherichia coli phage T4.

The immunity protein (Imm) encoded by the Escherichia coli phage T4 effects exclusion of phage superinfecting cells already infected with T4. The 83-residue polypeptide possesses two long lipophilic areas (from residues 3 to 32 and 37 to 65) interrupted by a hydrophilic stretch including two positively charged residues. The charge distribution of the protein very strongly suggested that it is a plasma membrane protein with the C terminus facing the periplasm. While it could be shown that the expected location was correct, fusions of Imm to alkaline phosphatase or beta-galactosidase showed that the C terminus was at the cytosolic side of the membrane. Also, concerning function, there was almost no structural specificity to this part of the protein. Even removal of the two positively charged residues did not completely abolish function. Evidence suggesting that Imm is associated with the membrane at specific sites is presented. It is proposed that Imm is localized to the membrane with the help of a receptor and that, therefore, it does not follow the established rules for the topology of other membrane proteins. The results also suggest that Imm acts indirectly, possibly by altering the conformation of a component of a phage DNA injection site.     

Membrane assembly of the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred orientation.

The M protein of mouse hepatitis virus strain A59 is a triple-spanning membrane protein which assembles with an uncleaved internal signal sequence, adopting an NexoCcyt orientation. To study the insertion mechanism of this protein, domains potentially involved in topogenesis were deleted and the effects analyzed in topogenesis were deleted and the effects analyzed in several ways. Mutant proteins were synthesized in a cell-free translation system in the presence of microsomal membranes, and their integration and topology were determined by alkaline extraction and by protease-protection experiments. By expression in COS-1 and Madin-Darby canine kidney-II cells, the topology of the mutant proteins was also analyzed in vivo. Glycosylation was used as a biochemical marker to assess the disposition of the NH2 terminus. An indirect immunofluorescence assay on semi-intact Madin-Darby canine kidney-II cells using domain-specific antibodies served to identify the cytoplasmically exposed domains. The results show that each membrane-spanning domain acts independently as an insertion and anchor signal and adopts an intrinsic preferred orientation in the lipid bilayer which corresponds to the disposition of the transmembrane domain in the wild-type assembled protein. These observations provide further insight into the mechanism of membrane integration of multispanning proteins. A model for the insertion of the coronavirus M protein is proposed.     

Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion

Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected Drosophila melanogaster cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses. To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast Saccharomyces cerevisiae, a host able to support full FHV RNA replication and virion formation. Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in Drosophila cells, and that this mitochondrial localization was independent of viral RNA synthesis. Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain. Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization. Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion. We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain.     

Novel topological features of FhaC, the outer membrane transporter involved in the secretion of the Bordetella pertussis filamentous hemagglutinin

Many pathogenic Gram-negative bacteria secrete virulence factors across the cell envelope into the extracellular milieu. The secretion of filamentous hemagglutinin (FHA) by Bordetella pertussis depends on the pore-forming outer membrane protein FhaC, which belongs to a growing family of protein transporters. Protein alignment and secondary structure predictions indicated that FhaC is likely to be a beta-barrel protein with an odd number of transmembrane beta-strands connected by large surface loops and short periplasmic turns. The membrane topology of FhaC was investigated by random insertion of the c-Myc epitope and the tobacco etch virus protease-specific cleavage sequence. FhaC was fairly permissive to short linker insertions. Furthermore, FhaC appeared to undergo conformational changes upon FHA secretion. Surface detection of the inserted sequences indicated that several predicted loops in the C-terminal moiety as well as the N terminus of the protein are exposed. However, a large surface-predicted region in the N-terminal moiety of FhaC was inaccessible from the surface. In addition, the activity and the stability of the protein were affected by insertions in that region, indicating that it may have important structural and/or functional roles. The surface exposure of the N terminus and the presence of an odd number of beta-strands are novel features for beta-barrel outer membrane proteins.     

The Role of Proline in the Membrane Re-entrant Helix of Caveolin-1

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103–122. We have performed an in silico analysis of the conformational preference of this segment of caveolin-1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro¹¹⁰ residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the P110A mutant of caveolin-1, with a FLAG tag at the N terminus, in HEK 293 cells. We evaluate the topology of the proteins with confocal immunofluorescence microscopy in these cells. We find that FLAG tag at the N terminus of the wild type caveolin-1 is not reactive with antibodies unless the cell membrane is permeabilized with detergent. This indicates that in these cells, the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG tag at the N terminus of the P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. We also find that the P110A mutation causes a large reduction of endocytosis of caveolae, cellular lipid accumulation, and lipid droplet formulation. In addition, we find that this mutation markedly reduces the ability of caveolin-1 to form structures with the characteristic morphology of caveolae or to partition into the detergent-resistant membranes of these cells. Thus, the single Pro residue in the membrane-inserting segment of caveolin-1 plays an important role in both the membrane topology and localization of the protein as well as its functions.     

Study of membrane orientation and glycosylated extracellular loops of mouse P-glycoprotein by in vitro translation.

Increased expression of P-glycoprotein (Pgp) has been demonstrated to cause multidrug resistance (MDR) in vitro, and it may be responsible for chemotherapy failure in a number of human cancers. Pgp is a plasma membrane protein thought to function as an energy-dependent drug transporter. From its deduced protein sequence the topology of Pgp was proposed to contain 12 transmembrane domains with six extracellular loops and two cytoplasmic ATP-binding sites. To investigate further the membrane orientation of Pgp, we have expressed a full length cDNA of mouse mdr1, as well as its truncated forms, in a cell-free system supplemented with dog pancreatic microsomal membranes (RM). We determined which domains of the in vitro-synthesized Pgp had transversed the RM membranes by analyzing their resistance to protease digestion and their glycosylation state. To our surprise, this system revealed that a significant portion of in vitro-synthesized Pgp molecules has an additional glycosylated domain in the C-terminal half. Previously, only the first predicted extracellular loop near the N terminus had been thought to be glycosylated. Furthermore, we discovered that Pgp has at least two functional signal recognition particle/docking protein dependent signal sequences, one at the N-terminal half and the other at the C-terminal half. These findings suggest a new topological model for in vitro synthesized P-glycoprotein which may be relevant to its in vivo topology.     

Experimentally based topology models for E. coli inner membrane proteins

Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a protein's C terminus and topology prediction. Here, we show that determination of the location of a protein's C terminus, rather than some internal loop, is the best strategy for large-scale topology mapping studies. We further report experimentally based topology models for 31 Escherichia coli inner membrane proteins, using methodology suitable for genome-scale studies.