Hydrophilic Subunits Research Articles

Structural organization of the major subunits in cyanobacterial photosystem 1. Localization of subunits PsaC, -D, -E, -F, and -J.

Based on an improved isolation procedure using perfusion chromatography, trimeric Photosystem 1 (PS1) complexes have been isolated from various deletion mutants of the mesophilic cyanobacterium Synechocystis PCC 6803. These mutants are only deficient in the deleted subunits, which was carefully checked by high resolution gel electrophoresis in combination with immunoblotting. These highly purified and well characterized PS1 particles were then examined by electron microscopy, followed by computer-aided image processing with single particle averaging techniques as described earlier (Kruip, J., Boekema, E. J., Bald, D., Boonstra, A. F., and Rögner, M. (1993) J. Biol. Chem. 268, 23353-23360). This precise methodological approach allowed a confident localization of the PS1 subunits PsaC, -D, -E, -F, and -J; it also shows shape and size of these subunits once integrated in the PS1 complex. Subunits PsaC, -D, and -E form a ridge on the stromal site, with PsaE toward the edge of each monomer within the trimer and PsaD extending toward the trimeric center, leaving PsaC in between. PsaF (near PsaE) and PsaJ are close together on the outer edge of each monomer; their proximity is also supported by chemical cross-linking, using the zero-length cross-linker EDC. This localization of PsaF contradicts the position suggested by the published low resolution x-ray analysis and shows for the first time the existence of at least one transmembrane alpha-helix for PsaF. A topographic three-dimensional map has been drawn from this set of results showing the location of the major PS1 subunits (besides PsaA and PsaB). These data also led to the assignment of electron density in the recent medium resolution x-ray structure for PS1 (Krauss, N., Schubert, W.-D., Klukas, O., Fromme, P., Witt, H. T., Saenger, W. (1996) Nat. Struct. Biol. 3, 965-973).

Characterization and Subunit Structure of the ATP Synthase of the Halophilic Archaeon Haloferax volcanii and Organization of the ATP Synthase Genes

The archaeal ATPase of the halophile Haloferax volcanii synthesizes ATP at the expense of a proton gradient, as shown by sensitivity to the uncoupler carboxyl cyanide p-trifluoromethoxyphenylhydrazone, to the ionophore nigericin, and to the proton channel-modifying reagent N,N'-dicyclohexylcarbodiimide. The conditions for an optimally active ATP synthase have been determined. We were able to purify the enzyme complex and to identify the larger subunits with antisera raised against synthetic peptides. To identify additional subunits of this enzyme complex, we cloned and sequenced a gene cluster encoding five hydrophilic subunits of the A1 part of the proton-translocating archaeal ATP synthase. Initiation, termination, and ribosome-binding sequences as well as the result of a single transcript suggest that the ATPase genes are organized in an operon. The calculated molecular masses of the deduced gene products are 22. 0 kDa (subunit D), 38.7 kDa (subunit C), 11.6 kDa (subunit E), 52.0 kDa (subunit B), and 64.5 kDa (subunit A). The described operon contains genes in the order D, C, E, B, and A; it contains no gene for the hydrophobic, so-called proteolipid (subunit c, the proton-conducting subunit of the A0 part). This subunit has been isolated and purified; its molecular mass as deduced by SDS-polyacrylamide gel electrophoresis is 9.7 kDa.

Subunit Structure and Organization of the Genes of the A1A0 ATPase from the Archaeon Methanosarcina mazei Gö1

The proton-translocating A1A0 ATP synthase/hydrolase of Methanosarcina mazei Gö1 was purified and shown to consist of six subunits of molecular masses of 65, 49, 40, 36, 25, and 7 kDa. Electron microscopy revealed that this enzyme is organized in two domains, the hydrophilic A1 and the hydrophobic A0 domain, which are connected by a stalk. Genes coding for seven hydrophilic subunits were cloned and sequenced. From these data it is evident that the 65-, 49-, 40- and 25-kDa subunits are encoded by ahaA, ahaB, ahaC, and ahaD, respectively; they are part of the A1 domain or the stalk. In addition there are three more genes, ahaE, ahaF, and ahaG, encoding hydrophilic subunits, which were apparently lost during the purification of the protein. The A0 domain consists of at least the 7-kDa proteolipid and the 36-kDa subunit for which the genes have not yet been found. In summary, it is proposed that the A1A0 ATPase of Methanosarcina mazei Gö1 contains at least nine subunits, of which seven are located in A1 and/or the stalk and two in A0.

Membrane Topology of the Mannose Transporter of Escherichia coli K12

The mannose transporter of the bacterial phosphotransferase system mediates carbohydrate transport across the cytoplasmic membrane concomitant with carbohydrate phosphorylation. It also functions as a receptor for bacterial chemotaxis [Adler.J. & Epstein, W. (1974) Proc. Natl Acad. Sci. USA 71. 2895-2899] and is required for infection of the cell by bacteriophage lambda where it most likely functions as a pore for penetration of phage DNA [Elliott, J. & Arber, W. (1978) Mol. & Gen. Genet. 161, 1-8]. The transporter consists of two transmembrane subunits (27-kDa IICMan and 31-kDa IIDMan) and a hydrophilic subunit (35-kDa IIABMan). Protein fusions of IICMan and IIDMan with beta-galactosidase (LacZ) and with alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the two proteins. Protein fusions were obtained by progressively deleting the manY and manZ genes from their 3' ends and ligating them to lacZ and 'phoA that lack promotor and leader sequences. Based on the analysis of 30 IICMan-PhoA. 10 IICMan-LacZ, 12 IIDMan-PhoA, and 30 IIDMan-LacZ fusions, it is predicted that IICMan has six membrane-spanning segments with the N- and C-termini on the cytoplasmic face of the membrane. IIDMan is anchored in the membrane by a single membrane-spanning segment at the end of the C-terminus, while most of the protein (250 residues) protrudes into the cytoplasm.

The Maltose Transport System of Escherichia coli Displays Positive Cooperativity in ATP Hydrolysis

Maltose transport across the cytoplasmic membrane of Escherichia coli is catalyzed by a periplasmic binding protein-dependent transport system and energized by ATP. The maltose system, a member of the ATP-binding cassette or ABC transport family, contains two copies of an ATP-binding protein in a complex with two integral membrane proteins. ATP hydrolysis by the transport complex can be assayed following reconstitution into proteoliposomes in the presence of maltose binding protein and maltose. Mutations in the transport complex that permit binding protein-independent transport render ATP hydrolysis constitutive so that hydrolysis can also be assayed with the transport complex in detergent solution. We have used both of these systems to study the role of two ATP binding sites in ATP hydrolysis. We found that both the wild-type and the binding protein-independent systems hydrolyzed ATP with positive cooperativity, suggesting that the two ATP binding sites interact. Vanadate inhibited the ATPase activity of the transport complex with 50% inhibition occurring at 10 mum vanadate. In detergent solution, the degree of cooperativity in the binding protein-independent complex decreased with increasing pH. The loss of cooperativity was accompanied by a decrease in ATPase activity and a decrease in sensitivity to vanadate. Because reconstitution of the complex into a lipid bilayer prevented the loss of cooperativity, we expect that ATP hydrolysis is cooperative in vivo. The mutations leading to binding protein-independent transport do not significantly alter the affinity, cooperativity, vanadate sensitivity, or substrate specificity of the ATP binding sites during hydrolysis. These results justify the use of the binding protein-independent system to investigate the mechanism of transport and hydrolysis.

Sialylated Oligosaccharide-specific Plant Lectin from Japanese Elderberry (Sambucus sieboldiana) Bark Tissue Has a Homologous Structure to Type II Ribosome-inactivating Proteins, Ricin and Abrin: cDNA CLONING AND MOLECULAR MODELING STUDY

Bark lectins from the elderberry species belonging to the genus Sambucus have a unique carbohydrate binding specificity for sialylated glycoconjugates containing NeuAc(alpha 2-6)Gal/GalNAc sequence. To elucidate the structure of the elderberry lectin, a cDNA library was constructed from the mRNA isolated from the bark tissue of Japanese elderberry (Sambucus sieboldiana) with lambda gt11 phage and screened with anti-S. sieboldiana agglutinin (SSA) antibody. The nucleotide sequence of a cDNA clone encoding full-length SSA (LecSSA1) showed the presence of an open reading frame with 1902 base pairs, which corresponded to 570 amino acid residues. This open reading frame encoded a signal peptide and a linker region (19 amino acid residues) between the two subunits of SSA, the hydrophobic (A-chain) and hydrophilic (B-chain) subunits. This indicates that SSA is synthesized as a preproprotein and post-translationally cleaved into two mature subunits. Homology searching as well as molecular modeling studies unexpectedly revealed that each subunit of SSA has a highly homologous structure to the galactose-specific lectin subunit and ribosome-inactivating subunit of plant toxic proteins such as ricin and abrin, indicating a close evolutionary relationship between these carbohydrate-binding proteins.

A two state lattice model of membrane proteins: Configuration as a function of sequence

A two state lattice model of soluble proteins is extended to model membrane proteins. The relationship between the structure of model proteins and their sequences is investigated as a function of the relative energy of hydrophobic type interactions. Relative energies of the interactions of hydrophobic and hydrophilic subunits with the solvent, the membrane, and with one another were chosen to mimic, within the simple model, their experimental counterparts. It is found that this reasonable energy parameterization produces model membrane proteins which share many characteristics with real membrane proteins, while other parameter sets fail in this regard. Consideration of the results obtained with the reasonable parameter sets leads to predictions about membrane proteins. Among these are that a single sequence may give a proteinlike native state in both aqueous and membrane environments.

Acetylcholinesterase in tentacles of octopus vulgaris (cephalopoda. histochemical localization and characterization of a specific high salt-soluble and heparin-soluble fraction of globular forms

Transverse sections of Octopus tentacles were stained for acetylcholinesterase (AChE) activity. An intense staining, that was suppressed by preincubation in 10 −5 M eserine, was detected in a number of neuronal cells, nerve fibres and neuromuscular junctions of intrinsic muscles of the arm. Octopus acetylcholinesterase was found as two molecular forms: an amphiphilic dimeric form (G2) sensitive to phosphatidylinositol phospholipase C and a hydrophilic tetrameric (G4) form. Sequential solubilization revealed that a significant portion of both G2 and G4 forms was recovered only in a high salt-soluble fraction (1 M NaCl, no detergent). Heparin (2 mg/ml) was able to solubilize G2 and G4 forms with the same efficiency than 1 M NaCl. The solubilizing effect of heparin was concentration-dependent and was reduced by protamine (2 mg/ml). This suggests that heparin operates through the dissociation of ionic interactions existing in situ between globular forms of AChE and cellular or extracellular polyanionic components. Interaction of AChE molecular forms with heparin has been reported so far in only a few instances and its physiological meaning is uncertain. G2 and G4 forms, interacting or not with heparin, all belong to a single pharmacological class of AChE. This suggests the existence of a single AChE gene. Amphiphilic and hydrophilic subunits thus likely result either from the processing of a single AChE transcript by alternative splicing (as in vertebrate AChE) or from a post-translation modification of a single catalytic peptide.

The Function of Wolinella succinogenes psr Genes in Electron Transport with Polysulphide as the Terminal Electron Acceptor

The membrane-integrated polysulphide reductase (Psr) of Wolinella succinogenes is part of the electron transport chain catalyzing polysulphide reduction by formate or hydrogen. The isolated enzyme catalyzes sulphide oxidation by dimethylnaphthoquinone. The two hydrophilic subunits, PsrA and PsrB of the enzyme, are encoded by genes that form an apparent operon psrABC together with a third gene. Using homologous recombination, three deletion mutants of W. succinogenes were constructed that lack psrC, psrBC or the whole psr operon. The mutants grown with formate and fumarate were fractionated, and the cell fractions were analyzed for the presence of PsrA and enzyme activity. It was concluded that: (a) polysulphide reductase is a constituent of the wild-type chain catalyzing electron transport from formate to polysulphide; (b) the gene psrC encodes a subunit that anchors the enzyme in the membrane and is required for electron transport; (c) PsrA which probably carries the substrate site, is exposed to the bacterial periplasm; (d) PsrA and PsrB are required for the activity of sulphide oxidation with 2,3-dimethyl-1,4-naphthoquinone. Surprisingly, the psrABC mutant could grow with formate and polysulphide. The membrane fraction of the mutant grown under these conditions contained an enzyme that replaced polysulphide reductase in electron transport, and catalyzed sulphide oxidation with 2,3-dimethyl-1,4-naphthoquinone.

The function of Wolinella succinogenes psr genes in electron transport with polysulphide as the terminal electron acceptor.

The membrane-integrated polysulphide reductase (Psr) of Wolinella succinogenes is part of the electron transport chain catalyzing polysulphide reduction by formate or hydrogen. The isolated enzyme catalyzes sulphide oxidation by dimethylnaphthoquinone. The two hydrophilic subunits, PsrA and PsrB of the enzyme, are encoded by genes that form an apparent operon psrABC together with a third gene. Using homologous recombination, three deletion mutants of W. succinogenes were constructed that lack psrC, psrBC or the whole psr operon. The mutants grown with formate and fumarate were fractionated, and the cell fractions were analyzed for the presence of PsrA and enzyme activity. It was concluded that: (a) polysulphide reductase is a constituent of the wild-type chain catalyzing electron transport from formate to polysulphide; (b) the gene psrC encodes a subunit that anchors the enzyme in the membrane and is required for electron transport; (c) PsrA which probably carries the substrate site, is exposed to the bacterial periplasm; (d) PsrA and PsrB are required for the activity of sulphide oxidation with 2,3-dimethyl-1,4-naphthoquinone. Surprisingly, the delta psrABC mutant could grow with formate and polysulphide. The membrane fraction of the mutant grown under these conditions contained an enzyme that replaced polysulphide reductase in electron transport, and catalyzed sulphide oxidation with 2,3-dimethyl-1,4-naphthoquinone.

Functional Reconstitution of the Purified Mannose Phosphotransferase System of Escherichia coli into Phospholipid Vesicles

The mannose transporter complex acts by a mechanism which couples translocation with phosphorylation of the substrate. It consists of a hydrophilic subunit (IIABMan) and two transmembrane subunits (IICMan, IIDMan). The purified complex was reconstituted into phospholipid vesicles by octyl glucoside dilution. Glucose export was measured with proteoliposomes which were loaded with radiolabeled glucose and to which purified IIABMan, cytoplasmic phosphorylcarrier proteins, and P-enolpyruvate were added from the outside. Vectorial transport was accompanied by stoichiometric phosphorylation of the transported sugar. Glucose added to the outside of the proteoliposomes was also phosphorylated rapidly but did not compete with vectorial export and phosphorylation of internal glucose. Glucose uptake was measured with proteoliposomes which were loaded with the cytoplasmic phosphoryl carrier proteins and P-enolpyruvate and to which glucose was added from the outside. Vectorial import and phosphorylation occurred with a higher specificity (Km 30 +/- 6 microM, kcat 401 +/- 32 pmol of Glc/micrograms of IICDMan/min) than nonvectorial phosphorylation (Km 201 +/- 43 microM, kcat 975 +/- 88 pmol of Glc/micrograms of IICDMan/min). A new plasmid pTSHIC9 for the controlled overexpression of the cytoplasmic phosphoryl carrier proteins, enzyme I, HPr, and IIAGlc, and a simplified procedure for the purification of these proteins are also described.

Purification and characterization of the catalytic moiety of vacuolar-type Na(+)-ATPase from Enterococcus hirae.

We have previously reported the molecular cloning and sequences of the ntp genes for Enterococcus hirae Na(+)-translocating ATPase [Takase, K., Kakinuma, S., Yamato, I., Konishi, K., Igarashi, K., and Kakinuma, Y. (1994) J. Biol. Chem. 269, 11037-11044]; the expected structure of this enzyme complex resembles those of the vacuolar H(+)-ATPase complexes in eukaryotes. In this paper we report purification and characterization of the catalytic moiety of Na(+)-ATPase, whose molecular size was about 400 kDa, consisting of polypeptides of 69 kDa (NtpA), 52 kDa (NtpB), and 29 kDa (NtpD) with a probable stoichiometry of 3:3:1. Purified enzyme hydrolyzed GTP as the best substrate (GTP > CTP > UTP > ATP), and the activity was maximal at around pH 6.0. The activity was not stimulated by sodium ions, and was selectively inhibited by nitrate. These properties were different from those of membrane-bound Na(+)-ATPase, suggesting that a significant conformational change of the catalytic moiety may take place upon dissociation from the membrane-embedded moiety and probably also loss of other hydrophilic subunits. Antiserum against purified enzyme inhibited the Na(+)-stimulated ATPase activity of the membranes. Immunoblotting analysis revealed that the change in the amounts of A and B subunits of the membranes paralleled that of the Na(+)-ATPase activity. Furthermore, the A subunit was missing in the membranes of a Na(+)-ATPase mutant, and recovered in those of its revertant. These immunochemical data are consistent with the notion that this enzyme is the hydrophilic catalytic moiety of the V-type Na(+)-ATPase in E. hirae.

Protein structural similarities predicted by a sequence‐structure compatibility method

A method for protein structure prediction has been developed, which evaluates the compatibility of an amino acid sequence with known 3-dimensional structures and identifies the most likely structure. The method was applied to a large number of sequences in a database, and the structures of the following proteins were predicted: (1) shikimate kinase (SKase), (2) the hydrophilic subunit of mannose permease (IIABMan), (3) rat tyrosine aminotransferase (Tyr AT), and (4) threonine dehydratase (TDH). The functional and evolutionary implications of the predictions are discussed. (1) The structural similarity between SKase and adenylate kinase was predicted. Alignment of their sequences reveals that the ATP-binding type A sequence motif and 2 ATP-binding arginine residues are conserved. The prediction suggests a similarity in their functional mechanisms as well as an evolutionary relationship. (2) The structural similarity between IIABMan and galactose/glucose-binding protein (GGBP) was predicted. The IIA and IIB domains are aligned with the N- and C-terminal domains of GGBP, respectively. The 2 phosphorylated residues, His 10 and His 175, of IIABMan are threaded onto loops located in the substrate-binding cleft of GGBP. The prediction accounts for the phosphoryl transfer from His 10 to His 175, and to the sugar substrate. (3) The structural similarity between rat Tyr AT and Escherichia coli aspartate AT was predicted, as well as (4) the structural similarity between TDH and the tryptophan synthase beta subunit. Predictions (3) and (4) support the previous predictions based on observations of the functional similarities between the proteins.

Independent folding of the domains in the hydrophilic subunit IIABman of the mannose transporter of Escherichia coli.

The active form of the hydrophilic subunit (IIABman) of the mannose transporter of Escherichia coli is a homodimer of two 35-kDa subunits. Each subunit consists of two distinct domains, IIA and IIB, which can be separated by limited trypsin digestion. Separation of tryptic fragments yields monomers of IIB and dimers of IIA, which are active and stable. To test whether the domains fold as independent units, the effects of guanidine hydrochloride (GuHCl) and temperature on the structural stability of the intact IIABman were compared with those of the isolated fragments. Equilibrium GuHCl-induced reversible unfolding, measured by circular dichroism and tryptophan fluorescence, showed a biphasic transition for intact IIABman and monophasic transitions for each isolated fragment. The midpoint transitions of the isolated IIB and IIA fragments (at 1.0 and 2.3 M GuHCl) coincide with the first and second transitions of intact IIABman. Analytical ultracentrifugation studies suggested that dissociation precedes the unfolding of IIA. Thermal unfolding of IIABman, monitored by differential scanning calorimetry, showed two well-separated transitions near 52 and 95 degrees C which corresponded to the midpoint transitions of the isolated IIB and IIA fragments. The combined results demonstrate an independent stepwise unfolding of the domains in IIABman as well as the absence of stabilizing interdomain interactions. The lack of interdomain interactions suggests an unrestricted domain motion. This may play an important role in the phosphoryl transfer reaction which is catalyzed by the binding of IIABman to a phosphoryl carrier protein HPr (via the IIA domain) and to the transmembrane subunits of the mannose transporter (via the IIB domain).

The glucose transporter of Escherichia coli. Overexpression, purification, and characterization of functional domains.

The glucose transporter of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar. It consists of a transmembrane subunit (IICBGlc) and a hydrophilic subunit (IIAGlc). The IICBGlc subunit consists of two domains. The NH2-terminal IIC domain (residues 1-386) spans the membrane eight times and contains the substrate binding site. The COOH-terminal hydrophilic IIB domain (residues 391-476) is accessible from the cytoplasmic side of the membrane. It contains the phosphorylation site (Cys421) and together with the IIC domain catalyzes the transfer of phosphoryl groups from the IIAGlc subunit to the transported solute. Starting from a plasmid vector containing ptsG under an inducible promoter, the IIB and the IIC domains have been subcloned separately, overexpressed in Escherichia coli, and purified by Ni2+ chelate affinity chromatography. Approximately 40 mg of IIBGlc-6H and 4 mg of IICGlc-6H could be purified from 1 liter of culture. Cells expressing IIBGlc-6H and IICGlc-6H separately have a three times longer generation time on glucose minimal medium than cells expressing wild-type IICBGlc. The rate of IIBGlc-6H phosphorylation determined in a nitrocellulose filter binding assay is indistinguishable from wild-type IICBGlc. The in vitro specific activity of IICGlc-6H in the presence of excess IIBGlc-6H is 2% of the control. IIBGlc-6H also complements the activity of a IICBGlc mutant with an inactive IIB domain (C421S) indicating that IIC and IIB are flexibly linked such that a free IIB domain can displace an inactive IIB domain from its contact site on the IIC domain. Based on this work, the secondary structure of the IIBGlc domain has been determined by isotope-edited NMR spectroscopy (Golic Grdadolnik, S., Eberstadt, M., Gemmecker, G., Kessler, H., Buhr, A., and Erni, B. (1994) Eur. J. Biochem. 219, 945-952).

Predicted topology of the N-terminal domain of the hydrophilic subunit of the mannose transporter of Escherichia coli

A folding topology for the homodimeric N-terminal domain (IIA, 2 × 14 kDa) of the hydrophilic subunit (IIAB man) of the mannose transporter of E. coli is proposed. The prediction is based on (i) tertiary structure prediction methods, and (ii) functional properties of site-directed mutants in correlation with NMR-derived α/β secondary structure data. The 3D structure profile suggested that the overall fold of IIA is similar to that of the unrelated protein, flavodoxin, which is an open-stranded parallel β-sheet with a strand order of 5 4 3 1 2. The 3D model of IIA, constructed using the known atomic structure of flavodoxin, is consistent with the results from site-directed mutagenesis. Recently NMR results confirmed the open parallel β-sheet with a strand order of 4 3 12 (residues 1-120) of our model whereas β-strand 5 (residues 127–130) was shown to be antiparallel to β-strand 4. The correctly predicted fold includes 90% of the monomeric subunit sequence and contains all functional sites of the IIA domain.

The mannose transporter of Escherichia coli. Structure and function of the IIABMan subunit.

The mannose transporter of the bacterial phosphotransferase system consists of two transmembrane subunits (IICMan and IIDMan) and a hydrophilic subunit (IIABMan). IIABMan has two flexibly linked domains containing one phosphorylation site each and occurs as a dimer. Substrate transport is coupled to phosphorylation. The phosphoryl group is transferred from a phosphoryl carrier protein to His10 on IIA, hence to His175 on IIB and finally to the substrate. IIABMan mutants were analyzed in vitro for complementation, negative dominance, cysteine cross-linking and reactivity. (i) His10, Trp12, Lys48, and Ser72 form a functional unit (phosphorylation site 1); (ii) His86 on the IIA domain and His175 on the IIB domain of the same subunit form a functional unit (phosphorylation site 2); (iii) phosphoryl transfer can occur between His10 and His175 of the same as well as of different subunits and His86 is necessary for this transfer; (iv) the subunits in the dimer are interdependent; (v) The phosphorylation site mutant H175C is highly reactive toward thiol reagents and it forms extensive homo- and heterocross-links with other surface-exposed cysteines. The phosphorylation site mutant H10C is 1000-fold less reactive. The two residues might be in complementary locations, His10 buried in a concave, His175 exposed on a convex surface.

Membrane topology of the glucose transporter of Escherichia coli

The glucose transporter of the bacterial phosphotransferase system couples translocation with phosphorylation of the substrate in a 1:1 stoichiometry. It consists of a transmembrane subunit (IIBCGlc) and a hydrophilic subunit (IIAGlc). Both subunits are transiently phosphorylated. The IIBCGlc subunit is 477 residues long and consists of two domains. The amino-terminal hydrophobic domain is involved in glucose binding and translocation, the carboxyl-terminal domain contains the phosphorylation site (Cys421). Protein fusions between IIBCGlc and beta-galactosidase (LacZ) as well as alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the IIBCGlc subunit. The protein fusions were generated by progressively deleting ptsG from its 3' end and ligating the truncated gene to lacZ and 'phoA lacking promoter and leader sequences. LacZ fusions of high activity (32 out of 54) occur at the amino and carboxyl termini and three internal clusters, and 41 active PhoA fusions occur in four internal clusters. Accordingly the hydrophobic domain of IICGlc (residues 19-336) is suggested to contains eight membrane-spanning segments, with the amino terminus and the COOH-terminal hydrophilic domain (IIBGlc) located on the cytoplasmic face of the membrane. A sequence comparison of IIBCGlc with three related proteins indicates that the periplasmic loops differ in size and sequence while the cytoplasmic loops are better conserved.

Resolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centers of the enzyme.

NADH:ubiquinone oxidoreductase (complex I) was purified from bovine heart mitochondria by solubilization with n-dodecyl beta-D-maltoside (lauryl maltoside), ammonium sulfate fractionation, and chromatography on Mono Q in the presence of the detergent. Its subunit composition was very similar to complex I purified by conventional means. Complex I was dissociated in the presence of N,N-dimethyldodecylamine N-oxide and beta-mercaptoethanol, and two subcomplexes, I alpha and I beta, were isolated by chromatography. Subcomplex I alpha catalyzes electron transfer from NADH to ubiquinone-1. It is composed of about 22 different and mostly hydrophilic subunits and contains 2.0 nmol of FMN/mg of protein. Among its subunits is the 51-kDa subunit, which binds FMN and NADH and probably contains a [4Fe-4S] cluster also. Three other potential Fe-S proteins, the 75- and 24-kDa subunits and a 23-kDa subunit (N-terminal sequence TYKY), are also present. All of the Fe-S clusters detectable by EPR in complex I, including cluster 2, are found in subcomplex I alpha. The line shapes of the EPR spectra of the Fe-S clusters are slightly broadened relative to spectra measured on complex I purified by conventional means, and the quinone reductase activity is insensitive to rotenone. Similar changes were found in samples of the intact chromatographically purified complex I, or in complex I prepared by the conventional method and then subjected to chromatography in the presence of lauryl maltoside. Subcomplex I beta contains about 15 different subunits. The sequences of many of them contain hydrophobic segments that could be membrane spanning, including at least two mitochondrial gene products, ND4 and ND5. The role of subcomplex I beta in the intact complex remains to be elucidated.

The relation between the soluble factor associated with H +-transhydrogenase of Rhodospirillum rubrum and the enzyme from mitochondria and Escherichia coli

Although in mitochondria, Escherichia coli and Rhodobacter capsulatus the H +-transhydrogenases are intrinsic membrane proteins, in Rhodospirillum rubrum a water-soluble component (Th 2) and a membrane-bound component are together required for activity. Th s was selectively removed from chromatophore membranes of Rhs. rubrum and was purified to homogeneity by precipitation with (NH 4) 2SO 4 and ion-exchange, affinity dye and gel exclusion chromatography. The latter indicated an M r of approx. 74 000 under non-denaturing conditions but analysis of the pure proteins by SDS PAGE revealed a single polypeptide, M r 43 000. Antibodies against this polypeptide inhibited transhydrogenase activity of chromatographores and decreased the capacity of Th s to restore activity to depleted membranes. They reacted with a polypeptide of M r 43 000 in crude cell extract, chromatophore membranes and chromatophore washing but not with transhydrogenase polypeptides from the membranes of E. coli, Rb. capsulatus or animal mitochondria. The N-terminal amino acid sequence of the 43 000 polypeptide was strongly homologous with the reported N-terminal regions of mitochondrial transhydrogenase and the α subunit of the E. coli protein. The break between the α and β polypeptides of E. coli transhydrogenase is such that both components are membrane associated. In contrast, these results suggest that in the Rhs. rubrum enzyme Th s has been formed by a break closer to the N-terminus, thus avoiding the putative trans-membrane helical segments and yielding a relatively hydrophilic subunit, which is water-soluble. There is a predicted similarity between Th s and the reported sequence of alanine dehydrogenase from Bacillus but Th s did not have any alanine dehydrogenase activity.