Incorporation of transmembrane peptides from the vacuolar H(+)-ATPase in phospholipid membranes: spin-label electron paramagnetic resonance and polarized infrared spectroscopy

Peptides were designed that are based on candidate transmembrane sequences of the V o-sector from the vacuolar H (+)-ATPase of Saccharomyces cerevisiae. Spin-label EPR studies of lipid-protein interactions were used to characterize the state of oligomerization, and polarized IR spectroscopy was used to determine the secondary structure and orientation, of these peptides in lipid bilayer membranes. Peptides corresponding to the second and fourth transmembrane domains (TM2 and TM4) of proteolipid subunit c (Vma3p) and of the putative seventh transmembrane domain (TM7) of subunit a (Vph1p) are wholly, or predominantly, alpha-helical in membranes of dioleoyl phosphatidylcholine. All three peptides self-assemble into oligomers of different sizes, in which the helices are differently inclined with respect to the membrane normal. The coassembly of rotor (Vma3p TM4) and stator (Vph1p TM7) peptides, which respectively contain the glutamate and arginine residues essential to proton transport by the rotary ATPase mechanism, is demonstrated from changes in the lipid interaction stoichiometry and helix orientation. Concanamycin, a potent V-ATPase inhibitor, and a 5-(2-indolyl)-2,4-pentadienoyl inhibitor that exhibits selectivity for the osteoclast subtype, interact with the membrane-incorporated Vma3p TM4 peptide, as evidenced by changes in helix orientation; concanamycin additionally interacts with Vph1p TM7, suggesting that both stator and rotor elements contribute to the inhibitor site within the membrane. Comparison of the peptide behavior in lipid bilayers is made with membranous subunit c assemblies of the 16-kDa proteolipid from Nephrops norvegicus, which can substitute functionally for Vma3p in S. cerevisiae.     

Analysis of the membrane topology of transmembrane segments in the C-terminal hydrophobic domain of the yeast vacuolar ATPase subunit a (Vph1p) by chemical modification

The integral V(0) domain of the vacuolar (H(+))-ATPases (V-ATPases) provides the pathway by which protons are transported across the membrane. Subunit a is a 100-kDa integral subunit of V(0) that plays an essential role in proton translocation. To better define the membrane topology of subunit a, unique cysteine residues were introduced into a Cys-less form of the yeast subunit a (Vph1p) and the accessibility of these cysteine residues to modification by the membrane permeant reagent N-ethylmaleimide (NEM) and the membrane impermeant reagent polyethyleneglycol maleimide (PEG-mal) in the presence and absence of the protein denaturant SDS was assessed. Thirty Vph1p mutants containing unique cysteine residues were constructed and analyzed. Cysteines introduced between residues 670 and 710 and between 807 and 840 were modified by PEG-mal in the absence of SDS, indicating a cytoplasmic orientation. Cysteines introduced between residues 602 and 620 and between residues 744 and 761 were modified by NEM but not PEG-mal in the absence of SDS, suggesting a lumenal orientation. Finally, cysteines introduced at residues 638, 645, 648, 723, 726, 734, and at nine positions between residue 766 and 804 were modified by NEM and PEG-mal only in the presence of SDS, consistent with their presence within the membrane or at a protein-protein interface. The results support an eight transmembrane helix (TM) model of subunit a in which the C terminus is located on the cytoplasmic side of the membrane and provide information on the location of hydrophilic loops separating TM6, 7, and 8.     

Modeling the transmembrane domain of bacterial chemoreceptors

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.     

Definition of membrane topology and identification of residues important for transport in subunit a of the vacuolar ATPase

Subunit a of the vacuolar H(+)-ATPases plays an important role in proton transport. This membrane-integral 100-kDa subunit is thought to form or contribute to proton-conducting hemichannels that allow protons to gain access to and leave buried carboxyl groups on the proteolipid subunits (c, c', and c″) during proton translocation. We previously demonstrated that subunit a contains a large N-terminal cytoplasmic domain followed by a C-terminal domain containing eight transmembrane (TM) helices. TM7 contains a buried arginine residue (Arg-735) that is essential for proton transport and is located on a helical face that interacts with the proteolipid ring. To further define the topology of the C-terminal domain, the accessibility of 30 unique cysteine residues to the membrane-permeant reagent N-ethylmaleimide and the membrane-impermeant reagent polyethyleneglycol maleimide was determined. The results further define the borders of transmembrane segments in subunit a. To identify additional buried polar and charged residues important in proton transport, 25 sites were individually mutated to hydrophobic amino acids, and the effect on proton transport was determined. These and previous results identify a set of residues important for proton transport located on the cytoplasmic half of TM7 and TM8 and the lumenal half of TM3, TM4, and TM7. Based upon these data, we propose a tentative model in which the cytoplasmic hemichannel is located at the interface of TM7 and TM8 of subunit a and the proteolipid ring, whereas the lumenal hemichannel is located within subunit a at the interface of TM3, TM4, and TM7.     

Interferon-induced Transmembrane Protein 3 Is a Type II Transmembrane Protein

The interferon-induced transmembrane (IFITM) proteins are a family of small membrane proteins that inhibit the cellular entry of several genera of viruses. These proteins had been predicted to adopt a two-pass, type III transmembrane topology with an intracellular loop, two transmembrane helices (TM1 and TM2), and extracellular N and C termini. Recent work, however, supports an intramembrane topology for the helices with cytosolic orientation of both termini. Here we determined the topology of murine Ifitm3. We found that the N terminus of Ifitm3 could be stained by antibodies at the cell surface but that this conformation was cell type-dependent and represented a minority of the total plasma membrane pool. In contrast, the C terminus was readily accessible to antibodies at the cell surface and extracellular C termini comprised most or all of those present at the plasma membrane. The addition of a C-terminal KDEL endoplasmic reticulum retention motif to Ifitm3 resulted in sequestration of Ifitm3 in the ER, demonstrating an ER-luminal orientation of the C terminus. C-terminal, but not N-terminal, epitope tags were also degraded within lysosomes, consistent with their luminal orientation. Furthermore, epitope-tagged Ifitm3 TM2 functioned as a signal anchor sequence when expressed in isolation. Collectively, our results demonstrate a type II transmembrane topology for Ifitm3 and will provide insight into its interaction with potential targets and cofactors.     

Production and initial structural characterization of the TM4TM5 helix‐loop‐helix domain of the translocator protein

Mainly present in the mitochondria, the translocator protein, TSPO, previously known as the peripheral benzodiazepine receptor, is a small essential membrane protein, involved in the translocation of cholesterol across mitochondrial membranes, a rate determining step in steroids biosynthesis. We previously reported the structure of five fragments encompassing the five putative transmembrane helices and showed that each of these fragments constitutes an autonomous folding unit. To further characterize the structural determinants responsible for helix–helix association of this membrane protein, we now investigate the folding of double transmembrane domains in various detergent micelles. Herein, we present the successful biosynthesis of a double transmembrane domain encompassing the last two C‐terminal helices (TM4TM5). For optimal production of this domain in Escherichia coli, the evaluation of various peptide constructs, including TM4TM5 fused to different purification tags or to solubilizing proteins, was necessary. The protocol of production of TM4TM5 with more than 95% purity is reported. This domain was further characterized using circular dichroism and solution state NMR. Far‐UV circular dichroism studies indicate that the secondary structure of TM4TM5 is highly helical when solubilized in various detergent micelles including n‐dodecyl‐β‐d ‐maltoside, n‐octyl‐β‐d ‐glucoside, n‐dodecylphosphocholine, 1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine (DHPC), and 1‐palmitoyl‐2‐hydroxy‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol). In addition, the solubilization conditions of the domain were optimized for NMR experiments, and preliminary analysis indicates that TM4TM5 adopts a stable tertiary fold within the TM4TM5‐DHPC complex. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

N‐linked glycosylation of a subunit isoforms is critical for vertebrate vacuolar H+‐ATPase (V‐ATPase) biosynthesis

The a subunit of the V₀ membrane‐integrated sector of human V‐ATPase has four isoforms, a1‐a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N‐glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N‐glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope‐tagged human a1‐a3 subunits, with or without mutations that convert Asn to Gln at putative N‐glycosylation sites. Expression and N‐glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N‐glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild‐type a subunits after enzymatic deglycosylation. Cycloheximide‐chase experiments showed that unglycosylated subunits were turned over at a higher rate than N‐glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co‐immunoprecipitation studies showed that they were unable to associate with the V‐ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N‐glycosylation is crucial in all four human V‐ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V‐ATPase complexes.     

A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations.

A structural model of the transmembrane portion of the acetylcholine receptor was developed from sequences of all its subunits by using transfer energy calculations to locate transmembrane alpha-helices and to calculate which helical side chains should be in contact with water inside the channel, with portions of other transmembrane helices, or with lipid hydrocarbon chains. "Knobs-into-holes" side chain packing calculations were used with other factors to stack the transmembrane alpha-helices together. In the model each subunit has the following structures in order along the sequence from the NH2 terminus: a large extracellular domain of undetermined structure, a short apolar alpha-helix that lies on the extracellular lipid surface of the membrane; three apolar transmembrane alpha-helices (I, II, and III), a cytoplasmic domain of undetermined structure, an amphipathic transmembrane alpha-helix (L) that forms the channel lining, a short extracellular alpha-helix, another apolar transmembrane alpha-helix (IV), and a small cytoplasmic domain formed by the COOH-terminal end of the chain. Three concentric layers form the pore. A bundle of five amphipathic L helices forms the channel lining. This bundle is surrounded by a bundle of 10 alternating II and III helices. Helices I and IV cover portions of the outer surface of the bundle formed by helices II and III. Positions of disulfide bridges are predicted and a mechanism for opening and closing conformational changes is proposed that requires tilting transmembrane helices and possibly a thiol-disulfide interchange reaction.     

Transmembrane topography of the 100-kDa a subunit (Vph1p) of the yeast vacuolar proton-translocating ATPase.

The membrane topography of the yeast vacuolar proton-translocating ATPase a subunit (Vph1p) has been investigated using cysteine-scanning mutagenesis. A Cys-less form of Vph1p lacking the seven endogenous cysteines was constructed and shown to have 80% of wild type activity. Single cysteine residues were introduced at 13 sites within the Cys-less mutant, with 12 mutants showing greater than 70% of wild type activity. To evaluate their disposition with respect to the membrane, vacuoles were treated in the presence or absence of the impermeant sulfhydryl reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) followed by the membrane permeable sulfhydryl reagent 3-(N-maleimidylpropionyl) biocytin (MPB). Three of the 12 active cysteine mutants were not labeled by MPB. The mutants E3C, D89C, T161C, S266C, N447C, K450C, and S703C were labeled by MPB in an AMS-protectable manner, suggesting a cytoplasmic orientation, whereas G602C and S840C showed minimal protection by AMS, suggesting a lumenal orientation. Factor Xa cleavage sites were introduced at His-499, Leu-560, and Pro-606. Cleavage at 560 was observed in the absence of detergent, suggesting a cytoplasmic orientation for this site. Based on these results, we propose a model of the a subunit containing nine transmembrane segments, with the amino terminus facing the cytoplasm and the carboxyl terminus facing the lumen.     

The crystal structure of the CmABCB1 G132V mutant,which favors the outward‐facing state,reveals the mechanism of the pivotal joint between TM1 and TM3

CmABCB1 is a homologue of human P‐glycoprotein, which extrudes various substrates by iterative cycles of conformational changes between the inward‐ and outward‐facing states. Comparison of the inward‐ and outward‐facing structures of CmABCB1 suggested that pivotal joints in the transmembrane domain regulate the tilt of transmembrane helices. Transmembrane helix 1 (TM1) forms a tight helix–helix contact with TM3 at the TM1–3 joint. Mutation of Gly132 to valine at the TM1–3 joint, G132V, caused a 10‐fold increase in ATPase activity, but the mechanism underlying this change remains unclear. Here, we report a crystal structure of the outward‐facing state of the CmABCB1 G132V mutant at a 2.15 Å resolution. We observed structural displacements between the outward‐facing states of G132V and the previous one at the region around the TM1–3 joint, and a significant expansion at the extracellular gate. We hypothesize that steric hindrance caused by the Val substitution shifted the conformational equilibrium toward the outward‐facing state, favoring the dimeric state of the nucleotide‐binding domains and thereby increasing the ATPase activity of the G132V mutant.     

Coupled proteolytic and mass spectrometry studies indicate a novel topology for the glycine receptor

Members of the heteropentameric ligand-gated ion channel superfamily rapidly mediate signaling across the synaptic cleft. Sequence analysis and limited experimental studies have yielded a topological model containing four transmembrane alpha-helices, labeled M1 to M4, and a large soluble, extracellular N-terminal domain. This model persists to date despite some recent structural studies that suggest it may be inappropriate. In this study, the topology of the glycine receptor was probed by limited proteolysis coupled to mass spectrometry. Of particular note, accessible cleavage sites within the putative M1 and M3 transmembrane helices were identified. Membrane-associated fragments within the postulated globular extracellular N-terminal domain were also observed. This report presents several key details incorporated in a new topological model and is the first direct experimental evidence that a subset of the transmembrane regions are too short to be membrane-spanning alpha-helices; rather, these regions are proposed to be a mix of alpha-helices and beta-sheets. This report is also the first to exploit the capability of mass spectrometry to probe critically the topology of a class of membrane proteins of unknown structure.     

Subcellular distribution and membrane topology of the mammalian concentrative Na+-nucleoside cotransporter rCNT1

The rat transporter rCNT1 is the archetype of a family of concentrative nucleoside transporters (CNTs) found both in eukaryotes and in prokaryotes. In the present study we have used antibodies to investigate the subcellular distribution and membrane topology of this protein. rCNT1 was found to be expressed predominantly in the brush-border membranes of the polarized epithelial cells of rat jejunum and renal cortical tubules and in the bile canalicular membranes of liver parenchymal cells, consistent with roles in the absorption of dietary nucleosides, of nucleosides in the glomerular filtrate, or of nucleosides arising from the action of extracellular nucleotidases, respectively. The effect of endoglycosidase F treatment on wild-type and mutant rCNT1 expressed in Xenopus oocytes revealed that the recombinant transporter could be glycosylated at either or both of Asn605 and Asn643, indicating that its C terminus is extracellular. In contrast, potential N-glycosylation sites introduced near the N terminus, or between putative transmembrane (TM) helices 4 and 5, were not glycosylated. The deduced orientation of the N terminus in the cytoplasm was confirmed by immunocytochemistry on intact and saponin-permeabilized Chinese hamster ovary cells expressing recombinant rCNT1. These results, in conjunction with extensive analyses of CNT family protein sequences using predictive algorithms, lead us to propose a revised topological model, in which rCNT1 possesses 13 TM helices with the hydrophilic N-terminal and C-terminal domains on the cytoplasmic and extracellular sides of the membrane, respectively. Furthermore, we show that the first three TM helices, which are absent from prokaryote CNTs, are not essential for transporter function; truncated proteins lacking these helices, derived either from rCNT1 or from its human homolog hCNT1, were found to retain significant sodium-dependent uridine transport activity when expressed in oocytes.     

Topology, subcellular localization, and sequence diversity of the Mlo family in plants

Barley Mlo defines the founder of a novel class of plant integral membrane proteins. Lack of the wild type protein leads to broad spectrum disease resistance against the pathogenic powdery mildew fungus and deregulated leaf cell death. Scanning N-glycosylation mutagenesis and Mlo-Lep fusion proteins demonstrated that Mlo is membrane-anchored by 7 transmembrane (TM) helices such that the N terminus is located extracellularly and the C terminus intracellularly. Fractionation of leaf cells and immunoblotting localized the protein to the plant plasma membrane. A genome-wide search for Mlo sequence-related genes in Arabidopsis thaliana revealed approximately 35 family members, the only abundant gene family encoding 7 TM proteins in higher plants. The sequence variability of Mlo family members within a single species, their topology and subcellular localization are reminiscent of the most abundant class of metazoan 7 TM receptors, the G-protein-coupled receptors.     

Crystal structure of yeast V1‐ATPase in the autoinhibited state

Vacuolar ATPases (V‐ATPases) are essential proton pumps that acidify the lumen of subcellular organelles in all eukaryotic cells and the extracellular space in some tissues. V‐ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, wherein the soluble catalytic sector, V₁, is released from the membrane and its MgATPase activity silenced. The crystal structure of yeast V₁ presented here shows that activity silencing involves a large conformational change of subunit H, with its C‐terminal domain rotating ~150° from a position near the membrane in holo V‐ATPase to a position at the bottom of V₁ near an open catalytic site. Together with biochemical data, the structure supports a mechanistic model wherein subunit H inhibits ATPase activity by stabilizing an open catalytic site that results in tight binding of inhibitory ADP at another site.     

Detection of drug‐induced conformational change of a transmembrane protein in lipid bilayers using site‐directed spin labeling

As a target of antiviral drugs, the influenza A M2 protein has been the focus of numerous structural studies and has been extensively explored as a model ion channel. In this study, we capitalize on the expanding body of high‐resolution structural data available for the M2 protein to design and interpret site‐directed spin‐labeling electron paramagnetic resonance spectroscopy experiments on drug‐induced conformational changes of the M2 protein embedded in lipid bilayers. We obtained data in the presence of adamantane drugs for two different M2 constructs (M2TM 22–46 and M2TMC 23–60). M2TM peptides were spin labeled at the N‐terminal end of the transmembrane domain. M2TMC peptides were spin labeled site specifically at cysteine residues substituted for amino acids within the transmembrane domain (L36, I39, I42, and L43) and the C‐terminal amphipathic helix (L46, F47, F48, C50, I51, Y52, R53, F54, F55, and E56). Addition of adamantane drugs brought about significant changes in measured electron paramagnetic resonance spectroscopy environmental parameters consistent with narrowing of the transmembrane channel pore and closer packing of the C‐terminal amphipathic helices.     

Membrane dynamics of γ‐secretase with the anterior pharynx‐defective 1B subunit

The four‐subunit protease complex γ‐secretase cleaves many single‐pass transmembrane (TM) substrates, including Notch and β‐amyloid precursor protein to generate amyloid‐β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx‐defective 1 (APH‐1) and presenilin (PS) exist in two homologous forms APH1‐A and APH1‐B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ‐secretase medicine. Here, we developed the first complete structural model of the APH‐1B subunit using the published cryo‐electron microscopy (cryo‐EM) structures of APH1‐A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all‐atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH‐1B alone and in γ‐secretase without and with substrate C83‐bound. We show that APH‐1B adopts a 7TM topology with a water channel topology similar to APH‐1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo‐EM structures with APH‐1A, however with subtle differences: The substrate‐bound APH‐1B γ‐secretase was quite stable, but some TM helices of PS1 and APH‐1B rearranged in the membrane consistent with the disorder seen in the cryo‐EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH‐1B, that is, it represents a more closed state, due to interactions with the C‐terminal fragment of PS1. Our structural‐dynamic model of APH‐1B alone and in γ‐secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.     

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V₁ domain that hydrolyzes ATP and an integral V₀ domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V₀) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V₁V₀ complex and the free V₀ domain to identify residues protected from modification by the presence of V₁. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V₁ domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H-V-ATPase

Vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an α-helical conformation for peptide MTM7 and in DMSO three α-helical regions are identified by 2D ¹H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an α-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Topological characterization of the essential Escherichia coli cell division protein FtsN.

Genetic and biochemical approaches were used to analyze a topological model for FtsN, a 36-kDa protein with a putative transmembrane segment near the N terminus, and to ascertain the requirements of the putative cytoplasmic and membrane-spanning domains for the function of this protein. Analysis of FtsN-PhoA fusions revealed that the putative transmembrane segment of FtsN could act as a translocation signal. Protease accessibility studies of FtsN in spheroblasts and inverted membrane vesicles confirmed that FtsN had a simple bitopic topology with a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large periplasmic carboxy terminus. To ascertain the functional requirements of the N-terminal segments of FtsN, various constructs were made. Deletion of the N-terminal cytoplasmic and membrane-spanning domains led to intracellular localization of the carboxy domain, instability,and loss of function. Replacement of the N-terminal cytoplasmic and membrane-spanning domains with a membrane-spanning domain from MalG restored subcellular localization and function. These N-terminal domains of FtsN could also be replaced by the cleavable MalE signal sequence with restoration of subcellular localization and function. It is concluded that the N-terminal, cytoplasmic, and transmembrane domains of FtsN are not required for function of the carboxy domain other than to transport it to the periplasm. FtsQ and FtsI were also analyzed.