Roles of the narJ and narI gene products in the expression of nitrate reductase in Escherichia coli

Nitrate reductase, released and purified from membrane fractions of Escherichia coli, is composed of three subunits. Formation of the enzyme depends on induction of the nar operon, narGHJI, which is composed of four open reading frames (ORF). Previous studies established that the first two genes in the operon narG and narH encode the alpha and beta subunits, respectively, while formation of the gamma subunit, cytochrome bNR, depends on expression of the promoter distal genes. The narJ and narI genes were subcloned separately into plasmids where each was under the control of the nar promoter. Expression of these plasmids in a mutant which forms only alpha and beta subunits revealed that expression of the narI gene is sufficient to restore normal levels of cytochrome bNR, but expression of both genes is required for assembly of fully active, membrane-bound nitrate reductase. The amino acid composition, the N-terminal sequence, and the sequence of cyanogen bromide fragments derived from the isolated gamma subunit corresponds to that expected for a protein produced by the narI ORF. A protein corresponding to the narJ ORF did not appear to be associated with the purified nitrate reductase complex or with the complex immunoprecipitated from Triton X-100-solubilized membrane preparations. We conclude that narI encodes the gamma subunit (cytochrome bNR) and that while the product of the narJ gene is required for assembly of fully active membrane-bound enzyme it is not tightly associated with the active enzyme.     

NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli

The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain. Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro . NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.     

Construction in vitro of a cloned nar operon from Escherichia coli.

To clone the nar operon of Escherichia coli without an effective selection procedure for the nar+ phenotype, a strategy utilizing nar::Tn5 mutants was employed. Partial segments of the nar operon containing Tn5 insertions were cloned into plasmid pBR322 by using the transposon resistance character for selection. A hybrid plasmid was constructed in vitro from two of these plasmids and isolated by a procedure that involved screening a population of transformed nar(Ts) mutant TS9A for expression of thermal stable nitrate reductase activity. A detailed restriction site map of the resulting plasmid, pSR95, corresponded closely to the composite restriction endonuclease map deduced for the nar region from maps of the cloned nar::Tn5 fragments. When transformed with pSR95, wild-type strain PK27 overproduced the alpha, beta, and gamma subunits of nitrate reductase, although nitrate reductase activity was only slightly increased. The alpha and beta subunits were overproduced about 5- to 10-fold and accumulated mostly as an inactive aggregate in the cytoplasm; the gamma subunit overproduction was detected as a threefold increase in the specific content of cytochrome b555 in the membrane fraction. Functional nitrate reductase and the cytochrome spectrum associated with functional nitrate reductase were restored in the nar::Tn5 mutant EE1 after transformation with pSR95. Although the specific activity of nitrate reductase in this case was less than that of the wild type, both the alpha and beta subunits appeared to be overproduced in an inactive form. In both strains PK27(pSR95) and EE1(pSR95), the formation of nitrate reductase activity and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the demonstration that pSR95 contains a functional nor operon that encodes the alpha, beta, gamma subunits of nitrate reductase.     

Biogenesis of a respiratory complex is orchestrated by a single accessory protein

The biogenesis of respiratory complexes is a multistep process that requires finely tuned coordination of subunit assembly, metal cofactor insertion, and membrane-anchoring events. The dissimilatory nitrate reductase of the bacterial anaerobic respiratory chain is a membrane-bound heterotrimeric complex nitrate reductase A (NarGHI) carrying no less than eight redox centers. Here, we identified different stable folding assembly intermediates of the nitrate reductase complex and analyzed their redox cofactor contents using electron paramagnetic resonance spectroscopy. Upon the absence of the accessory protein NarJ, a global defect in metal incorporation was revealed. In addition to the molybdenum cofactor, we show that NarJ is required for specific insertion of the proximal iron-sulfur cluster (FS0) within the soluble nitrate reductase (NarGH) catalytic dimer. Further, we establish that NarJ ensures complete maturation of the b-type cytochrome subunit NarI by a proper timing for membrane anchoring of the NarGH complex. Our findings demonstrate that NarJ has a multifunctional role by orchestrating both the maturation and the assembly steps.     

DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits.

DNA polymerase III holoenzyme has been purified from Escherichia coli HMS-83, using, as an assay, the conversion of coliphage G4 single-stranded DNA to the duplex replicative form. The holoenzyme consists of at least four different subunits: alpha, beta, gamma, and delta of 140,000, 40,000, 52,000, and 32,000 daltons, respectively. The alpha subunit is DNA polymerase III, the dnaE gene product. The holoenzyme has been resolved by phosphocellulose chromatography into an alpha - gamma - delta complex and a subunit beta (copolymerase III*); neither possesses detectable activity in the G4 system but together reconstitute holoenzyme-like activity. The alpha - gamma - delta complex has been further resolved to yield a gamma - delta complex which reconstitutes alpha - gamma - delta activity when added to DNA polymerase III. The gamma - delta complex contains a product of the dnaZ gene and has been purified from a strain which contains a ColE1-dnaZ hybrid plasmid.     

Defective gamma subunit of ATP synthase (F1F0) from Escherichia coli leads to resistance to aminoglycoside antibiotics.

A strain of Escherichia coli which was derived from a gentamicin-resistant clinical isolate was found to be cross-resistant to neomycin and streptomycin. The molecular nature of the genetic defect was found to be an insertion of two GC base pairs in the uncG gene of the mutant. The insertion led to the production of a truncated gamma subunit of 247 amino acids in length instead of the 286 amino acids that are present in the normal gamma subunit. A plasmid which carried the ATP synthase genes from the mutant produced resistance to aminoglycoside antibiotics when it was introduced into a strain with a chromosomal deletion of the ATP synthase genes. Removal of the genes coding for the beta and epsilon subunits abolished antibiotic resistance coded by the mutant plasmid. The relationship between antibiotic resistance and the gamma subunit was investigated by testing the antibiotic resistance of plasmids carrying various combinations of unc genes. The presence of genes for the F0 portion of the ATP synthase in the presence or absence of genes for the gamma subunit was not sufficient to cause antibiotic resistance. alpha, beta, and truncated gamma subunits were detected on washed membranes of the mutant by immunoblotting. The first 247 amino acid residues of the gamma subunit may be sufficient to allow its association with other F1 subunits in such a way that the proton gate of F0 is held open by the mutant F1.     

The napF and narG Nitrate Reductase Operons in Escherichia coli Are Differentially Expressed in Response to Submicromolar Concentrations of Nitrate but Not Nitrite

Escherichia coli synthesizes two biochemically distinct nitrate reductase enzymes, a membrane-bound enzyme encoded by the narGHJI operon and a periplasmic cytochrome c-linked nitrate reductase encoded by the napFDAGHBC operon. To address why the cell makes these two enzymes, continuous cell culture techniques were used to examine napF and narG gene expression in response to different concentrations of nitrate and/or nitrite. Expression of the napF-lacZ and narG-lacZ reporter fusions in strains grown at different steady-state levels of nitrate revealed that the two nitrate reductase operons are differentially expressed in a complementary pattern. The napF operon apparently encodes a "low-substrate-induced" reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the narGHJI operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The narGHJI operon is therefore a "high-substrate-induced" operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Interestingly, nitrite, the end product of each enzyme, had only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, was essential for repression of napF gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion. In light of these findings, prior models for the roles of nitrate and nitrite in control of narG and napF expression must be reconsidered.     

Structure of an ATPase operon of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius

The nucleotide sequence of the operon of the ATPase complex of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, has been determined. In addition to the three previously reported genes for the alpha, beta, and c (proteolipid) subunits of the ATPase complex (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1989) J. Biol. Chem. 264, 7119-7121), the operon contained three other genes encoding hydrophilic proteins with molecular masses 25, 13, and 7 kDa. The 25-kDa protein is the third largest subunit (gamma), the 13-kDa protein is most likely the fourth subunit (delta), and the 7-kDa protein may correspond to an unknown subunit of the ATPase, tentatively named as epsilon subunit. They do not have significant sequence similarity to subunits in F0F1-ATPases and eukaryotic V-type ATPases, whereas the other three subunits, alpha, beta, and c, have homologous counterparts in F0F1- and V-type ATPases. The order of the genes in the operon was delta alpha beta gamma epsilon c. The S. acidocaldarius ATPase operon differed from the eucabacterial F0F1-ATPase operon in that the former contains only one gene for a hydrophobic subunit at the most downstream part of the operon whereas the latter has three hydrophobic F0 genes preceding five hydrophilic F1 genes.     

Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches.

An energy-transducing adenosine triphosphatase (ATPase, EC 3.6.1.3) that contains an extra polypeptide (delta) as well as three intrinsic subunits (alpha, beta, gamma) was purified from Micrococcus lysodeikticus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is alpha 3 beta 3 gamma delta. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its alpha, beta and gamma subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(alpha):3.00(beta:0.17(gamma) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of alpha subunits and absence of gamma subunit are not active. Of the antisera to subunits, only anti-(beta-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(alpha-subunit) or anti-(gamma-subunit) serum increases the effect. The results suggest that beta subunit is required for full ATPase activity, although a minor proportion of alpha and perhaps gamma subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in Micrococcus lysodeikticus ATPase had a molecular weight of 20 000 and was found to be involved in ATPase binding to membranes. This 20 000-dalton component can be equated with the delta subunit of other energy-transducing ATPases and its association with the (alpha, beta, gamma) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The present results do not preclude the possibility that the gamma subunit also plays a role in ATPase binding, in which, however, the major subunits do not seem to play a role.     

A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.     

Subunit arrangement of gamma-aminobutyric acid type A receptors

The GABA(A) receptors are ligand-gated chloride channels. The subunit stoichiometry of the receptors is controversial; four, five, or six subunits per receptor molecule have been proposed for alphabeta receptors, whereas alphabetagamma receptors are assumed to be pentamers. In this study, alpha-beta and beta-alpha tandem cDNAs from the alpha1 and beta2 subunits of the GABA(A) receptor were constructed. We determined the minimal length of the linker that is required between the two subunits for functional channel expression for each of the tandem constructs. 10- and 23-amino acid residues are required for alpha-beta and beta-alpha, respectively. The tandem constructs either alone or in combination with each other failed to express functional channels in Xenopus oocytes. Therefore, we can exclude tetrameric or hexameric alphabeta GABA(A) receptors. We can also exclude proteolysis of the tandem constructs. In addition, the tandem constructs were combined with single alpha, beta, or gamma subunits to allow formation of pentameric arrangements. In contrast to the combination with alpha subunits, the combination with either beta or gamma subunits led to expression of functional channels. Therefore, a pentameric arrangement containing two alpha1 and three beta2 subunits is proposed for the receptor composed of alpha and beta subunits. Our findings also favor an arrangement betaalphagammabetaalpha for the receptor composed of alpha, beta, and gamma subunits.     

Escherichia coli H+-ATPase. Glutamic acid 185 in beta subunit is essential for its structure and assembly

The uncD gene for the beta subunit of Escherichia coli H+-ATPase was cloned downstream of the lac promoter and mutagenized (Glu-185----Gln or Lys) by an oligonucleotide-directed procedure. The recombinant plasmid was introduced into a strain in which the unc operon for subunits of H+-ATPase was deleted. The wild-type or mutant beta subunit synthesized amounted to about 10% total cell protein and was mainly found in the cytoplasmic fraction. These subunits could be purified to almost homogeneity by conventional procedures. The wild-type and two mutant beta subunits had essentially the same Kd values for 8-anilinonaphthalene-1-sulfonate, aurovertin, and ATP, although the fluorescence intensities of 8-anilinonaphthalene-1-sulfonate and aurovertin were significantly less when bound to the two mutant beta subunits than when bound to the wild-type subunit. The three beta subunits showed essentially the same circular dichroism spectra, indicating alpha-helical contents of about 16-18%. Thus, the mutations did not cause marked change of the secondary structure of the subunit. However, measurements of theta 208 during linear increase in temperature suggested that replacement of Glu-185 by Gln or Lys slightly changed the stability of the secondary structure. Only trace amounts of alpha beta gamma complexes could be reconstituted using the two mutant beta subunits. These results suggest that Glu-185 or the region in its vicinity may be essential for subunit assembly. The methods developed in this study should be useful for further studies on the beta subunit.     

G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding alpha subunit and a high-affinity complex of beta and gamma subunits. There is molecular heterogeneity of beta and gamma, but the significance of this diversity is poorly understood. Different G protein beta and gamma subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, beta gamma subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1) is detected only when beta and gamma are expressed concurrently. Of the six combinations of beta gamma tested (beta 1 or beta 2 with gamma 1, gamma 2, or gamma 3), only one, beta 2 gamma 1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using Go alpha-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP-ribosylation of Gi alpha 1; these differences are attributable to the gamma component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate Gs alpha-stimulated type-II adenylylcyclase, recombinant beta 1 gamma 1 and transducin beta gamma are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of gamma are not required for assembly of the beta gamma subunit complex but are indispensable for high affinity interactions of beta gamma with either G protein alpha subunits or adenylylcyclases.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

Intersubunit interactions in proton-translocating adenosine triphosphatase as revealed by hydrogen-exchange kinetics

The rates of hydrogen-deuterium exchange in the peptide groups of the alpha and beta subunits and the alpha-beta subunit complex of proton-translocating adenosine triphosphatase from the thermophilic bacterium PS3 were examined. The exchange was found to be much slower in the isolated beta subunit than in the isolated alpha subunit. This has been taken as indicating that the structure of the beta subunit is tighter than that of the alpha subunit. Adenosine 5'-triphosphate (ATP) caused tightening of a relatively tight portion of the alpha subunit and of a relatively loose portion of the beta subunit. When the alpha and beta subunits are brought into contact, tightening of the alpha subunit, but not the beta subunit, occurs. The effect of ATP on the structure of the beta subunit is more pronounced in the presence of the alpha subunit than in its absence. These findings support the idea proposed previously that the alpha subunit has an allosteric site and the beta subunit a catalytic site and that the conformation of the beta subunit is controlled by the alpha subunit.     

Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello.

The genes encoding the periplasmic [Fe] hydrogenase from Desulfovibrio vulgaris subsp. oxamicus Monticello were cloned by exploiting their homology with the hydAB genes from D. vulgaris subsp. vulgaris Hildenborough, in which this enzyme is present as a heterologous dimer of alpha and beta subunits. Nucleotide sequencing showed that the enzyme is encoded by an operon in which the gene for the 46-kilodalton (kDa) alpha subunit precedes that of the 13.5-kDa beta subunit, exactly as in the Hildenborough strain. The pairs of hydA and hydB genes are highly homologous; both alpha subunits (420 amino acid residues) share 79% sequence identity, while the unprocessed beta subunits (124 and 123 amino acid residues, respectively) share 71% sequence identity. In contrast, there appears to be no sequence homology outside these coding regions, with the exception of a possible promoter element, which was found approximately 90 base pairs upstream from the translational start of the hydA gene. The recently discovered hydC gene, which may code for a 65.8-kDa fusion protein (gamma) of the alpha and beta subunits and is present immediately downstream from the hydAB genes in the Hildenborough strain, was found to be absent from the Monticello strain. The implication of this result for the possible function of the hydC gene product in Desulfovibrio species is discussed.