The membrane-bound proteins of periplasmic permeases form a complex. Identification of the histidine permease HisQMP complex

The membrane-bound proteins of periplasmic transport systems have been hypothesized to form a complex with relatively little experimental support. Here we present experimental evidence that HisQ, HisM, and HisP, the membrane-bound proteins of the periplasmic histidine transport system of Salmonella typhimurium, form such a complex. We have developed antibodies specific to each of these proteins to aid in their characterization. Extractions with urea, alkaline pH, or Triton X-114 show that HisQ and HisM are integral membrane proteins. By these tests HisP displays an unusual behavior, being associated with the membrane whether or not HisQ and HisM are present and despite its hydrophilic sequence. However, the nature of HisPs interaction with the membrane is shown to vary depending on the presence of HisQ and HisM. In their absence, HisP is somewhat peripherally associated with the membrane, while in their presence it binds much more tightly, indicating that it forms a complex in association with HisQ and HisM. This is demonstrated by the coimmunoprecipitation of all three proteins by antibodies directed against any one of them. Chemical cross-linking allowed the characterization of the subunit stoichiometry of the complex as two HisPs to one HisQ and one HisM. Within this complex all three proteins probably contact each other and the two HisPs form a dimer. We hypothesize that HisQ and HisM with their multiple membrane-spanning segments form a "channel" within which the HisP subunits are located.     

Modulation of ATPase activity by physical disengagement of the ATP-binding domains of an ABC transporter, the histidine permease.

The membrane-bound complex of the prokaryotic histidine permease, a periplasmic protein-dependent ABC transporter, is composed of two hydrophobic subunits, HisQ and HisM, and two identical ATP-binding subunits, HisP, and is energized by ATP hydrolysis. The soluble periplasmic binding protein, HisJ, creates a signal that induces ATP hydrolysis by HisP. The crystal structure of HisP has been resolved and shown to have an "L" shape, with one of its arms (arm I) being involved in ATP binding and the other one (arm II) being proposed to interact with the hydrophobic subunits (Hung, L.-W., Wang, I. X., Nikaido, K., Liu, P.-Q., Ames, G. F.-L., and Kim, S.-H. (1998) Nature 396, 703-707). Here we study the basis for the defect of several HisP mutants that have an altered signaling pathway and hydrolyze ATP constitutively. We use biochemical approaches to show that they produce a loosely assembled membrane complex, in which the mutant HisP subunits are disengaged from HisQ and HisM, suggesting that the residues involved are important in the interaction between HisP and the hydrophobic subunits. In addition, the mutant HisPs are shown to have lower affinity for ADP and to display no cooperativity for ATP. All of the residues affected in these HisP mutants are located in arm II of the crystal structure of HisP, thus supporting the proposed function of arm II of HisP as interacting with HisQ and HisM. A revised model involving a cycle of disengagement and reengagement of HisP is proposed as a general mechanism of action for ABC transporters.     

Reconstitution of periplasmic transport in inside-out membrane vesicles. Energization by ATP

The periplasmic histidine permease of Salmonella typhimurium has been reconstituted in inside-out vesicles (IOV) of Escherichia coli by disrupting the cells with a French press in the presence of a high concentration of the periplasmic histidine-binding protein, HisJ. Efflux from IOV, which is equivalent to uptake in whole cells, is induced by ATP. The reconstituted system depends on the presence of the membrane-bound permease proteins, HisQ, HisM, and HisP, and does not function if reconstitution is performed in the presence of a mutant HisJ protein, HisJ5625, that can bind histidine normally but can't interact properly with the membrane complex. Efflux is not induced by the nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate, supporting the contention that ATP hydrolysis is necessary. 8-Azido ATP inactivates IOV, indicating that the ATP effect occurs through the HisP protein, which has previously been shown to be modified by 8-azido ATP (Hobson, A., Weatherwax, R., and Ames, G.F.-L. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 733-7337). The estimated Km of the vesicles for ATP is about 200 microM. Vanadate, an inhibitor of phosphohydrolase enzymes, inhibits ATP-induced efflux. We conclude that ATP is likely to be the proximal energy source for periplasmic permeases.     

Reconstitution of the histidine periplasmic transport system in membrane vesicles. Energy coupling and interaction between the binding protein and the membrane complex

The periplasmic histidine transport system of Salmonella typhimurium has been reconstituted in isolated right-side-out membrane vesicles. The reconstituted system is entirely dependent on both the periplasmic protein, HisJ, and the membrane-bound complex, composed of proteins HisQ, HisM, and HisP. Transport is also dependent on the presence of ascorbate and phenazine methosulfate, which provide the energy for transport. Ascorbate oxidation generates a proton-motive-force, which allows ATP synthesis. ATP (or a cogenerated molecule) appears to be the immediate energy donor. Dissipation of the proton-motive-force or reduction of the level of ATP by a variety of treatments results in inhibition of transport. Vanadate inhibits transport, indicating that ATP utilization is necessary to energize transport. The interaction between liganded HisJ and the membrane complex has been measured directly: it displays Michaelis-Menten type kinetics, with a K1/2 of approximately 65 microM. The significance of this finding in terms of transport properties of whole cells is discussed.     

Structure and mechanism of bacterial periplasmic transport systems

Bacterial periplasmic transport systems are complex, multicomponent permeases, present in Gram-negative bacteria. Many such permeases have been analyzed to various levels of detail. A generalized picture has emerged indicating that their overall structure consists of four proteins, one of which is a soluble periplasmic protein that binds the substrate and the other three are membrane bound. The liganded periplasmic protein interacts with the membrane components, which presumably form a complex, and which by a series of conformational changes allow the formation of an entry pathway for the substrate. The two extreme alternatives for such pathway involve either the formation of a nonspecific hydrophilic pore or the development of a ligand-binding site(s) on the membrane-bound complex. One of the membrane-bound components from each system constitutes a family of highly homologous proteins containing sequence domains characteristic of nucleotide-binding sites. Indeed, in several cases, they have been shown to bind ATP, which is thus postulated to be involved in the energy-coupling mechanism. Interestingly, eukaryotic proteins homologous to this family of proteins have been identified (mammalianmdr genes and Drosophilawhite locus), thus indicating that they perform a universal function, presumably related to energy coupling in membrane-related processes. The mechanism of energy coupling in periplasmic permeases is discussed.     

Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli.

We have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.     

Sequence relationships between integral inner membrane proteins of binding protein-dependent transport systems: evolution by recurrent gene duplications.

Periplasmic binding protein-dependent transport systems are composed of a periplasmic substrate-binding protein, a set of 2 (sometimes 1) very hydrophobic integral membrane proteins, and 1 (sometimes 2) hydrophilic peripheral membrane protein that binds and hydrolyzes ATP. These systems are members of the superfamily of ABC transporters. We performed a molecular phylogenetic analysis of the sequences of 70 hydrophobic membrane proteins of these transport systems in order to investigate their evolutionary history. Proteins were grouped into 8 clusters. Within each cluster, protein sequences displayed significant similarities, suggesting that they derive from a common ancestor. Most clusters contained proteins from systems transporting analogous substrates such as monosaccharides, oligopeptides, or hydrophobic amino acids, but this was not a general rule. Proteins from diverse bacteria are found within each cluster, suggesting that the ancestors of current clusters were present before the divergence of bacterial groups. The phylogenetic trees computed for hydrophobic membrane proteins of these permeases are similar to those described for the periplasmic substrate-binding proteins. This result suggests that the genetic regions encoding binding protein-dependent permeases evolved as whole units. Based on the results of the classification of the proteins and on the reconstructed phylogenetic trees, we propose an evolutionary scheme for periplasmic permeases. According to this model, it is probable that these transport systems derive from an ancestral system having only 1 hydrophobic membrane protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion.

Leader peptidase of Escherichia coli spans the plasma membrane twice with its amino terminus on the periplasmic surface of the membrane and its large carboxyl-terminal domain protruding into the periplasm. To monitor the transfer of the amino terminus of leader peptidase to the periplasm, we have constructed a fusion protein between the 18-residue amino-terminal periplasmic domain of Pf3 bacteriophage coat protein and the beginning of leader peptidase. We find that neither the SecA or SecY proteins nor a transmembrane electrochemical potential is required for insertion of the amino terminus, while the transfer of the carboxyl-terminal domain of leader peptidase has these requirements. The first 35 residues of leader peptidase, which include the first hydrophobic domain and the carboxyl-terminal positively charged cluster, are sufficient to insert the amino terminus. When positively charged residues are introduced before the first transmembrane segment, translocation of the amino terminus is abolished. These studies in protein membrane topogenesis, showing that there are different requirements for amino and carboxyl termini insertion, indicate that multiple mechanisms exist even within the same protein.     

Identification of the Periplasmic Cobalamin-Binding Protein BtuF of Escherichia coli

Cells of Escherichia coli take up vitamin B(12) (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in SALMONELLA: The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His(6) tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.     

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.     

Membrane topology of the Drosophila vesicular glutamate transporter

The vesicular glutamate transporters (VGLUTs) are responsible for packaging glutamate into synaptic vesicles, and are part of a family of structurally related proteins that mediate organic anion transport. Standard computer-based predictions of transmembrane domains have led to divergent topological models, indicating the need for experimentally derived predictions. Here we present data on the topology of the VGLUT ortholog from Drosophila melanogaster (DVGLUT). Using immunofluorescence assays of DVGLUT transiently localized to the plasma membrane of heterologously transfected cells, we have determined the accessibility of epitope tags inserted into the lumenal/extracellular face of the protein. Using immunoisolation, we have identified complementary tagged sites that face the cytoplasm. Our data show that DVGLUT contains 10 hydrophobic regions that completely span the membrane (TMs 1-10) and that the amino and carboxyl termini are cytosolic. Importantly, between TMs 4 and 5 is an unforeseen cytosolic loop of some 50 residues. Other domains exposed to the cytosol include loops between TMs 6-7 and 8-9, and regions C-terminal to TM2 and N-terminal to TM3. Between TM2 and 3 is a potentially hydrophobic, but topologically ambiguous region. Lumenal domains include sequences between TMs 1-2, 3-4, 5-6, 7-8 and 9-10. These data provide a basis for determining structure-function relationships for DVGLUT and other related proteins.     

Bacterial periplasmic permeases belong to a family of transport proteins operating from Escherichia coli to human: Traffic ATPases

Bacterial periplasmic transport systems are complex permeases composed of a soluble substrate-binding receptor and a membrane-bound complex containing 2-4 proteins. Recent developments have clearly demonstrated that these permeases are energized by the hydrolysis of ATP. Several in vitro systems have allowed a detailed study of the essential parameters functioning in these permeases. Several of the component proteins have been shown to interact with each other and the actual substrate for the transport process has been shown to be the liganded soluble receptor. The affinity of this substrate for the membrane complex is approximately 10 microM. The involvement of ATP in energy coupling is mediated by one of the proteins in the membrane complex. For each specific permease, this protein is a member of a family of conserved proteins which bind ATP. The similarity between the members of this family is high and extends itself beyond the consensus motifs for ATP binding. Interestingly, over the last few years, several eukaryotic membrane-bound proteins have been discovered which bear a high level of homology to the family of the conserved components of bacterial periplasmic permeases. Most of these proteins are known to, or can be inferred to participate in a transport process, such as in the case of the multidrug resistance protein (MDR), the STE6 gene product of yeast, and possibly the cystic fibrosis protein. This homology suggests a similarity in the mechanism of action and possibly a common evolutionary origin. This exciting development will stimulate progress in both the prokaryotic and eukaryotic areas of research by the use of overlapping procedures and model building. We propose that this universal class of permeases be called 'Traffic ATPases' to distinguish them from other types of transport systems, and to highlight their involvement in the transport of a vast variety of substrates in either direction relative to the cell interior and their use of ATP as energy source.     

The nucleotide-binding site of HisP, a membrane protein of the histidine permease. Identification of amino acid residues photoaffinity labeled by 8-azido-ATP

The periplasmic histidine transport system (permease) of Escherichia coli and Salmonella typhimurium is composed of a soluble, histidine-binding receptor located in the periplasm and a complex of three membrane-bound proteins of which one, HisP, was shown previously to bind ATP. These permeases are energized by ATP. HisP is a member of a family of membrane transport proteins which is conserved in all periplasmic permeases and is presumed to be involved in coupling the energy of ATP to periplasmic transport. In this paper the nature of the ATP-binding site of HisP has been explored by identification of some of the residues that come into contact with ATP. HisP was derivatized with 8-azido-ATP (N3ATP). Both the underivatized and the derivatized forms of HisP were solubilized, purified, and digested with trypsin. The resulting tryptic peptides were resolved by high pressure liquid chromatography, and peptides modified by N3ATP were isolated and sequenced. Two peptides, X and Z, spanning amino acid residues 16-23 and 31-45, were found to contain sites of N3ATP attachment at His19 and Ser41, respectively. Both peptides are close to the amino-terminal end of HisP; peptide Z is located in one of the well conserved regions comprising the nucleotide-binding consensus motifs of the energy-coupling components of these permeases. These consensus motifs are found in many purine nucleotide-binding proteins. The relationship between the location of these residues and the overall structure of the ATP-binding site is discussed.     

Topological Analysis of DcuA, an Anaerobic C4-Dicarboxylate Transporter of Escherichia coli

Escherichia coli possesses three independent anaerobic C₄-dicarboxylate transport systems encoded by the dcuA, dcuB, and dcuC genes. The dcuA and dcuB genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity. A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S. Six, S. C. Andrews, G. Unden, and J. R. Guest, J. Bacteriol. 176:6470–6478, 1994). These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a β-lactamase protein lacking the N-terminal signal peptide. The resulting topological model differs from those previously predicted. It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6. The N and C termini are located in the periplasm and the predicted orientation is consistent with the “positive-inside rule.” Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region. The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB. Overproduction of DcuA to 15% of inner-membrane protein was obtained with the lacUV5-promoter-based plasmid, pYZ4.     

Nucleotide binding by membrane components of bacterial periplasmic binding protein-dependent transport systems.

Bacterial periplasmic binding protein-dependent transport systems require the function of a specific substrate-binding protein, located in the periplasm, and several membrane-bound components. We present evidence for a nucleotide-binding site on one of the membrane components from each of three independent transport systems, the hisP, malK and oppD proteins of the histidine, maltose and oligopeptide permeases, respectively. The amino acid sequence of the oppD protein has been determined and this protein is shown to share extensive homology with the hisP and malK proteins. Three lines of evidence lead us to propose the existence of a nucleotide-binding site on each of these proteins. A consensus nucleotide-binding sequence can be identified in the same relative position in each of the three proteins. The oppD protein binds to a Cibacron Blue affinity column and can be eluted by ATP but not by CTP or NADH. The oppD protein is labelled specifically by the nucleotide affinity analogue 5'-p-fluorosulphonylbenzoyladenosine. The identification of a nucleotide-binding site provides strong evidence that transport by periplasmic binding protein-dependent systems is energized directly by the hydrolysis of ATP or a closely related nucleotide. The hisP, malK and oppD proteins are thus responsible for energy-coupling to their respective transport systems.     

Membrane topology analysis of Escherichia coli K-12 Mtr permease by alkaline phosphatase and beta-galactosidase fusions.

The mtr gene of Escherichia coli K-12 encodes an inner membrane protein which is responsible for the active transport of trypotophan into the cell. It has been proposed that the Mtr permease has a novel structure consisting of 11 hydrophobic transmembrane spans, with a cytoplasmically disposed amino terminus and a carboxyl terminus located in the periplasmic space (J.P. Sarsero, P. J. Wookey, P. Gollnick, C. Yanofsky, and A.J. Pittard, J. Bacteriol. 173:3231-3234, 1991). The validity of this model was examined by the construction of fusion proteins between the Mtr permease and alkaline phosphatase or beta-galactosidase. In addition to the conventional methods, in which the reporter enzyme replaces a carboxyl-terminal portion of the membrane protein, the recently developed alkaline phosphatase sandwich fusion technique was utilized, in which alkaline phosphatase is inserted into an otherwise intact membrane protein. A cluster of alkaline phosphatase fusions to the carboxyl-terminal end of the Mtr permease exhibited high levels of alkaline phosphatase activity, giving support to the proposition of a periplasmically located carboxyl terminus. The majority of fusion proteins produced enzymatic activities which were in agreement with the positions of the fusion sites on the proposed topological model of the permease. The synthesis of a small cluster of hybrid proteins, whose enzymatic activity did not agree with the location of their fusion sites within putative transmembrane span VIII or the preceding periplasmic loop, was not detected by immunological techniques and did not necessitate modification of the proposed model in this region. Slight alterations may need to be made in the positioning of the carboxyl-terminal end of transmembrane span X.     

A novel ubiquitous family of putative efflux transporters

We describe a novel family of putative efflux transporters (PET) found in bacteria, yeast and plants. None of the members of the PET family has been functionally characterized. The bacterial and yeast proteins display a duplicated internal repeat element consisting of an N-terminal hydrophobic sequence of about 170 residues, exhibiting six putative transmembrane alpha-helical spanners (TMSs), followed by a large (230 residue), C-terminal, hydrophilic, cytoplasmic domain. The plant proteins exhibit only one such unit, but they have a larger C-terminal cytoplasmic domain. Arabidopsis thaliana encodes at least seven paralogues of the PET family. The gram-negative bacterial proteins are sometimes encoded by genes that are found in operons that also contain genes that encode membrane fusion proteins. This fact strongly suggests that PET family proteins are efflux pumps. The sequence, topological and phylogenetic characteristics of these proteins as well as the operonic structures of their encoded genes when relevant are described.     

Nucleotide sequences of the tolA and tolB genes and localization of their products, components of a multistep translocation system in Escherichia coli.

Various mutations in the tolQRAB gene cluster of Escherichia coli render the bacteria tolerant to high concentrations of the E, A, or K colicins as well as tolerant to infection by the single-stranded filamentous bacteriophage. The nucleotide sequence of a 2.8-kilobase fragment containing the tolA and tolB genes was determined. This sequence predicts TolA to be a 421-amino-acid protein of molecular mass 44,190 daltons. Studies using minicells show it to be associated with the inner membrane, presumably via a 21-amino-acid hydrophobic sequence between residues 13 and 35. The remaining 387 residues on the carboxyl side of this region are located in the periplasm. Within this region of TolA is a 230-residue portion that is predicted to form a very long helical segment. This region is rich in alanine, lysine, and glutamic and aspartic acids. The TolB protein is predicted to contain 431 amino acids. Localization studies using minicells show two proteins encoded by this open reading frame. The larger protein of 47.5 kilodaltons appears to be associated with the membrane fractions. The smaller protein is 43 kilodaltons in size and is found with the periplasmic components of the cell.     

A topological analysis of subunit alpha from Escherichia coli F1F0-ATP synthase predicts eight transmembrane segments

The membrane topology of subunit alpha from the Escherichia coli F1F0-ATP synthase was studied using a gene fusion technique. Fusion proteins linking different amino-terminal fragments of the alpha subunit with an enzymatically active fragment of alkaline phosphatase were constructed by both random transposition of TnphoA and site-directed mutagenesis. Those proteins with high levels of alkaline phosphatase activity are predicted to define periplasmic domains of alpha, and this was confirmed by testing for cell growth in minimal medium supplemented with polyphosphate (P greater than 75) as the sole source of phosphate. The enzymatic activity of some fusion proteins was shown to be sensitive to glucose present in the growth medium. Results from subcellular fractionation experiments suggest that these fusion proteins may be inactive even though they have a periplasmic alkaline phosphatase. The enzymatic activity appears dependent upon proteolytic release of the alkaline phosphatase moiety from its alpha subunit membrane anchor and suggests the target of glucose repression may be a protease present in the periplasm. For the topological analysis of the alpha subunit, a total of 28 unique fusion proteins were studied and the results were consistent with a model of alpha containing eight transmembrane segments, including periplasmic amino and carboxyl termini. Surprisingly, separate periplasmic domains were identified near amino acids 200, 233, and 270. These results suggest the flanking membrane spans are only 10-15 amino acids in length and not able to span a standard 30 A bilayer in an alpha-helical conformation. These short spans may have interesting mechanistic implications for the function of F0, because they contain several amino acids which appear critical for proton translocation. Finally, a fusion of alkaline phosphatase at amino acid 271, the carboxyl-terminal residue, but not at amino acid 260, was able to complement the strain RH305 (uncB-) for growth on succinate and suggests the last 11 amino acids of the alpha subunit are critical to the function of F1F0-ATP synthase.