Membrane topology of Escherichia coli diacylglycerol kinase.

The topology of Escherichia coli diacylglycerol kinase (DAGK) within the cytoplasmic membrane was elucidated by a combined approach involving both multiple aligned sequence analysis and fusion protein experiments. Hydropathy plots of the five prokaryotic DAGK sequences available were uniform in their prediction of three transmembrane segments. The hydropathy predictions were experimentally tested genetically by fusing C-terminal deletion derivatives of DAGK to beta-lactamase and beta-galactosidase. Following expression, the enzymatic activities of the chimeric proteins were measured and used to determine the cellular location of the fusion junction. These studies confirmed the hydropathy predictions for DAGK with respect to the number and approximate sequence locations of the transmembrane segments. Further analysis of the aligned DAGK sequences detected probable alpha-helical N-terminal capping motifs and two amphipathic alpha-helices within the enzyme. The combined fusion and sequence data indicate that DAGK is a polytopic integral membrane protein with three transmembrane segments with the N terminus of the protein in the cytoplasm, the C terminus in the periplasmic space, and two amphipathic helices near the cytoplasmic surface.     

Role of a small cytoplasmic domain in the establishment of serine chemoreceptor membrane topology.

The Escherichia coli serine chemoreceptor takes on a simple membrane topology with two transmembrane segments separating cytoplasmically disposed N and C termini from a central periplasmic domain. We investigated the role of the small N-terminal cytoplasmic domain in membrane insertion using alkaline phosphatase gene fusions. Mutations eliminating the positive charge of the domain altered insertion dramatically, with reciprocal effects on hybrids with periplasmic and C-terminal cytoplasmic fusion junctions. Efficient export of the normally cytoplasmic C-terminal domain required that, in addition to the N-terminal changes, a short amphiphatic sequence at the beginning of the C-terminal domain be also absent. These findings document the importance of the positive character of the N-terminal domain in chemoreceptor membrane insertion and imply that partially redundant sequence information controls the orientation of the second transmembrane segment.     

Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa

The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.     

Membrane topology of the Streptococcus pneumoniae FtsW division protein

The topology of FtsW from Streptococcus pneumoniae, an essential membrane protein involved in bacterial cell division, was predicted by computational methods and probed by the alkaline phosphatase fusion and cysteine accessibility techniques. Consistent results were obtained for the seven N-terminal membrane-spanning segments. However, the results from alkaline phosphatase fusions did not confirm the hydropathy analysis of the C-terminal part of FtsW, whereas the accessibility of introduced cysteine residues was in agreement with the theoretical prediction. Based on the combined results, we propose the first topological model of FtsW, featuring 10 membrane-spanning segments, a large extracytoplasmic loop, and both N and C termini located in the cytoplasm.     

A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template. This GlpT structure represents the inward (or cytoplasm)-facing conformation predicted by the alternating access model for transport. LacZ-PhoA fusion analysis and site-directed fluorescence labeling substantiated the membrane topology and orientation predicted by this model and most hydropathy analyses. The model predicts the presence of a proton pathway within the N-terminal six-helix bundle of ProP (as opposed to the corresponding pathway found within the C-terminal helix bundle of its paralogue, LacY). Replacement of residues within the N-terminal helix bundle impaired the osmotic activation of ProP, providing the first indication that residues outside the C-terminal domain are involved in osmosensing. Some residues that were accessible from the periplasmic side, as predicted by the structural model, were more susceptible to covalent labeling in permeabilized membrane fractions than in intact bacteria. These residues may be accessible from the cytoplasmic side in structures not represented by our current model, or their limited exposure in vivo may reflect constraints on transporter structure that are related to its osmosensory mechanism.     

Transmembrane segment (TMS) VIII of the Na(+)/Citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia coli membrane.

The amino acid sequence of the sodium ion-dependent citrate transporter CitS of K. pneumoniae contains 12 hydrophobic stretches that could form membrane-spanning segments. A previous analysis of the membrane topology in Escherichia coli using the PhoA gene fusion technique indicated that only nine of these hydrophobic segments span the membrane, while three segments, Vb, VIII and IX, were predicted to have a periplasmic location (Van Geest, M., and Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582-25589). A topology study of C-terminally truncated CitS molecules in dog pancreas microsomes revealed that the protein traverses the endoplasmic reticulum membrane 11 times. In agreement with the PhoA fusion data, segment Vb was predicted to have a periplasmic location, but, in contrast, segments VIII and IX were found to be membrane-spanning (Van Geest, M., Nilsson, I., von Heijne, G., and Lolkema, J. S. (1999) J. Biol. Chem. 274, 2816-2823). In the present study, using site-directed Cys labeling, the topology of segments VIII and IX in the full-length CitS protein was determined in the E. coli membrane. Engineered cysteine residues in the loop between the two segments were accessible to a membrane-impermeable thiol reagent exclusively from the cytoplasmic side of the membrane, demonstrating that transmembrane segments (TMSs) VIII and IX are both membrane-spanning. It follows that the folding of CitS in the E. coli and endoplasmic reticulum membrane is the same. Cysteine accessibility studies of CitS-PhoA fusion molecules demonstrated that in the E. coli membrane segment VIII is exported to the periplasm in the absence of the C-terminal CitS sequences, thus explaining why the PhoA fusions do not correctly predict the topology. An engineered cysteine residue downstream of TMS VIII moved from a periplasmic to a cytoplasmic location when the fusion protein containing TMSs I-VIII was extended with segment IX. Thus, downstream segment IX is both essential and sufficient for the insertion of segment VIII of CitS in the E. coli membrane.     

Membrane topology of the pBR322 tetracycline resistance protein. TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion.

The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter. Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA. We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA. In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, several TetA-PhoA fusions have unexpected properties. One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity. However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity. In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287). We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5. (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions. We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance. The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e. long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.     

Topological analysis of GtrA and GtrB proteins encoded by the serotype-converting cassette of Shigella flexneri

Serotype conversion (O-antigen glucosylation) in Shigella flexneri is mediated by temperate bacteriophages, which encode a three-gene cluster that contains gtrA, gtrB, and gtr([type]). Sequence analysis has revealed that gtrA and gtrB are conserved and readily interchangeable between serotypes. The gtr([type]) is unique in each serotype and responsible for specifically mediating conversion by the addition of a glucosyl group to the O-antigen units. Analysis of the GtrA and GtrB amino acid sequence using computer prediction programs indicated that GtrA and GtrB have four and two transmembrane segments, respectively. The topology model of GtrA was analyzed by constructing consecutive sandwich fusions using a dual reporter PhoA/LacZ at predetermined positions targeting each of the 3 cytoplasmic and 2 periplasmic hypothetical loops. The topology of GtrB was determined by constructing C-terminal truncated fusions of GtrB to full-length PhoA and LacZ by a PCR-mediated method. These approaches revealed that GtrA consists of four transmembrane segments with both the N-terminal and C-terminal ends in the cytoplasm. Accordingly, GtrB consists of two transmembrane segments with both ends also in the cytoplasm. Furthermore, membrane anchorage of the extended N-terminal end of GtrB was found to be important in catalysis. This study completes the topology of all three proteins (GtrA, GtrB, and the gtr([type]): GtrV) involved in the glucosyltransferase activity that results in serotype conversion of S. flexneri. A model is proposed showing how both O-antigen synthesis and modification take place in S. flexneri.     

Cleavage of urokinase receptor regulates its interaction with integrins in thyroid cells

The membrane topology of a resistance-nodulation-division (RND) family transporter, MexD of Pseudomonas aeruginosa, was determined. Although it had been predicted previously that most RND proteins contain 12 transmembrane helices, three independent computer programs used in the present study predicted that MexD possessed 11, 14 or 17 transmembrane segments. To investigate the topology of MexD more thoroughly, 25 MexD-PhoA (alkaline phosphatase) and 18 MexD-Bla (beta-lactamase) fusion plasmids were constructed and analyzed. The resulting topological model had just 12 transmembrane helices and two periplasmic loops of about 300 residues between helices 1 and 2 and helices 7 and 8. It is therefore proposed that the N- and C-termini are located in the cytoplasm and the predicted orientation is consistent with the 'positive-inside rule'. This topological model can be applied to other RND proteins.     

Membrane topology of PssT,the transmembrane protein component of the type I exopolysaccharide transport system in Rhizobium leguminosarum bv. trifolii strain TA1

The pssT gene was identified as the fourth gene located upstream of the pssNOP gene cluster possibly involved in the biosynthesis, polymerization, and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum bv. trifolii strain TA1. The hydropathy profile and homology searches indicated that PssT belongs to the polysaccharide-specific transport family of proteins, a component of the type I system of the polysaccharide transport. The predicted membrane topology of the PssT protein was examined with a series of PssT-PhoA fusion proteins and a complementary set of PssT-LacZ fusions. The results generally support a predicted topological model for PssT consisting of 12 transmembrane segments, with amino and carboxyl termini located in the cytoplasm. A mutant lacking the C-terminal part of PssT produced increased amounts of total EPS with an altered distribution of high- and low-molecular-weight forms in comparison to the wild-type RtTA1 strain. The PssT mutant produced an increased number of nitrogen fixing nodules on clover.     

Defining the regions of Escherichia coli YidC that contribute to activity

The YidC/Oxa1/Alb3 family of proteins catalyzes membrane protein insertion in bacteria, mitochondria, and chloroplasts. In this study, we investigated which regions of the bacterial YidC protein are important for its function in membrane protein biogenesis. In Escherichia coli, YidC spans the membrane six times, with a large 319-residue periplasmic domain following the first transmembrane domain. We found that this large periplasmic domain is not required for YidC function and that the residues in the exposed hydrophilic loops or C-terminal tail are not critical for YidC activity. Rather, the five C-terminal transmembrane segments that contain the three consensus sequences in the YidC/Oxa1/Alb3 family are important for its function. However, by systematically replacing all the residues in transmembrane segment (TM) 2, TM3, and TM6 with serine and by swapping TM4 and TM5 with unrelated transmembrane segments, we show that the precise sequence of these transmembrane regions is not essential for in vivo YidC activity. Single serine mutations in TM2, TM3, and TM6 impaired the membrane insertion of the Sec-independent procoat-leader peptidase protein. We propose that the five C-terminal transmembrane segments of YidC function as a platform for the translocating substrate protein to support its insertion into the membrane.     

Topological characterization of the essential Escherichia coli cell division protein FtsW

The membrane topology of Escherichia coli FtsW, a 46-kDa essential protein, was analyzed using a set of 28 ftsW-alkaline phosphatase (ftsW-phoA) and nine ftsW-beta-lactamase (ftsW-bla) gene fusions obtained by in vivo and in vitro methods. The alkaline phosphatase activities or resistance pattern of cells expressing the FtsW-PhoA or FtsW-Bla fusions confirmed only eight out of 10 transmembrane segments predicted by computational methods. After comparison with the recent topology of Streptococcus pneumoniae FtsW, we could identify all the fusions in absolute agreement with the predicted model: N-terminal and C-terminal ends in the cytoplasm, 10 transmembrane segments and one large loop of 67 amino acids (E240-E306) located in the periplasm.     

Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II.

A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC'::pho'A gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yield approximately 1/20th as much activity. Replacement of pho'A with lac'Z in nine of the fusions confirmed the topology, as beta-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membrane-spanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus delta pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.     

Transfer of electrons across the cytoplasmic membrane by DsbD, a membrane protein involved in thiol-disulphide exchange and protein folding in the bacterial periplasm

Reduction of non-native protein disulphides in the periplasm of Escherichia coli is catalysed by three enzymes, DsbC, DsbG and DsbE, each of which harbours a catalytic Cys-X-X-Cys dithiol motif. This dithiol motif requires continuous reduction for activity. Genetic evidence suggests that the source of periplasmic reducing power resides within the cytoplasm, provided by thioredoxin (trxA) and thioredoxin reductase (trxB). Cytoplasmic electrons donated by thioredoxin are thought to be transferred into the periplasm via the DsbD membrane protein. To understand the molecular nature of electron transfer, we have analysed the membrane topology of DsbD. DsbD is exported by an N-terminal signal peptide. The N- and C-terminal domains are positioned in the periplasmic space, connected by eight transmembrane segments. Electron transfer was shown to require five cysteine sulphydryl of DsbD. Trans complementation of mutant DsbD molecules revealed intermolecular electron transfer. We discuss a model whereby the membrane-embedded disulphides of DsbD accept electrons from cytoplasmic thioredoxin and transfer them to the C-terminal periplasmic dithiol motif of DsbD.     

Tertiary contacts of helix V in the lactose permease determined by site-directed chemical cross-linking in situ

The six N-terminal transmembrane helices (N6) and the six C-terminal transmembrane helices (C6) in lactose permease, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices V and VII, VIII, or X was studied by thiol-specific chemical cross-linking. The results demonstrate that Cys residues in the periplasmic half of helix V cross-link with Cys residues in the periplasmic half of helix VII. In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices V and VII. Moreover, Cys residues on one entire face of helix V cross-link with Cys residues on one face of helix VIII. Finally, paired Cys residues at the cytoplasmic ends of helices V and X cross-link, but no cross-linking is observed when paired Cys residues are placed at the periplasmic ends of the two helices. Taken together, the results indicate that the periplasmic halves of helices V and VII are in close proximity and that the two helices tilt away from one another toward the cytoplasmic side of the membrane. Furthermore, helices V and VIII are in close proximity throughout their lengths and do not tilt appreciably with respect to one another, and helices V and X are in close proximity at the cytoplasmic but not at the periplasmic face of the membrane.     

The PecM protein of the phytopathogenic bacterium Erwinia chrysanthemi, membrane topology and possible involvement in the efflux of the blue pigment indigoidine

The pecS regulatory locus negatively modulates the expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Previous studies have shown that PecS down-regulates the expression of both pecSand pecMgenes and that PecM is required for full PecS activity. Computer-aided hydropathy analysis of PecM predicted the presence of between 8 to 10 potential transmembrane segments. We analyzed the membrane topology of PecM using the beta-lactamase gene fusion system and obtained the following unique characteristics. PecM contains 10 membrane spanning segments, with both the amino and carboxyl termini located in the cytoplasmic side of the inner membrane. The fourth periplasmic loop, which has a relatively long hydrophilic domain containing 17 amino acid residues, may play an important role in PecM function. The topological model obtained for PecM can be applied to PecM homologues in other bacteria. Measurement of the extrusion of the blue pigment indigoidine by the E. chrysanthemi derivative isogenic mutants pecS, pecM and pecS-pecM revealed that PecM is required for complete efflux of the pigment. Its relation to other efflux systems and its potential physiological role are discussed.     

Investigation of Myelin/Oligodendrocyte Glycoprotein Membrane Topology

Abstract: Myelin/oligodendrocyte glycoprotein (MOG) is a CNS-specific integral membrane protein that is an atypical member of the immunoglobulin (Ig) superfamily with two potential transmembrane domains based upon hydropathy analysis. With only one other exception, all Ig family members possess a single or no membrane spanning region. In order to analyze MOG membrane topology, we prepared stably transfected cells that express mouse MOG and used three domain-specific antisera to ascertain the localization of these hydrophilic domains. As expected, MOG's glycosylated N-terminal Ig-like domain was identified as extracellular, because membrane permeabilization was not required for immunoreactivity with the MOG_1–125 antiserum. In contrast, both MOG_154–169 and MOG_198–218 antisera stained cells only upon permeabilization. These data indicate that only MOG's N-terminal hydrophobic domain spans the lipid bilayer, and we propose that MOG's C-terminal hydrophobic domain associates with the cytoplasmic face of the plasma membrane. As for MOG's second hydrophobic domain, it is clear that either orientation (transmembrane versus membrane-associated) would be unique among Ig-like proteins, and the implications of our proposed topology for MOG in oligodendroglial plasma membrane are discussed.     

Membrane topology of ABC-type macrolide antibiotic exporter MacB in Escherichia coli

MacB is an ABC-type membrane protein that exports only macrolide compounds containing 14- and 15-membered lactones, cooperating with a membrane fusion protein, MacA, and a multifunctional outer membrane channel, TolC. We determined the membrane topology of MacB by means of site-specific competitive chemical modification of single cysteine mutants. As a result, it was revealed that MacB is composed of four transmembrane (TM) segments with a cytoplasmic N-terminal nucleotide binding domain of about 270 amino acid residues and a periplasmic large hydrophilic polypeptide between TM segments 1 and 2 of about 200 amino acid residues.     

Integration of deletion mutants of bovine rhodopsin into the membrane of the endoplasmic reticulum

Newly synthesized eukaryotic membrane proteins must be integrated into the membrane of the endoplasmic reticulum with the correct topology to enable the subsequent acquisition of the correctly folded, functional conformation. Here, an analysis is presented of N-terminal glycosylation and steady-state membrane orientation of a series of truncation mutants of the seven-helix protein rhodopsin expressed in COS-1 cells. Mutants containing one, three, or five N-terminal transmembrane segments of rhodopsin, as well as mutants containing only the first transmembrane segment, but with hydrophilic extensions at the C-terminus were studied. The findings demonstrate that the C-terminal transmembrane segments play a crucial role in determining the final orientation of rhodopsin, and that the commitment to the correct orientation occurs only after the synthesis of at least three transmembrane segments. The experiments also suggest that the molecular machinery involved in the integration of a newly synthesized seven-helix membrane protein into the endoplasmic reticulum membrane is sensitive to the overall hydrophobicity of the sequence that follows the first transmembrane segment.