Wild-type ATPase Activity Research Articles

Mutational analysis demonstrates different functional roles for the two ATP-binding sites in ClpAP protease from Escherichia coli.

ClpA, the regulatory subunit of Clp protease from Escherichia coli, has two ATP-binding sites in non-homologous regions of the protein, referred to as domain I and domain II. We have mutated the invariant lysine in the ATP-binding sites of domain I and domain II and studied the enzymatic properties of the purified mutant ClpA proteins. The domain I mutant, ClpA-K220Q, was unable to form a hexamer in the presence of nucleotide, but the comparable domain II mutant, ClpA-K501Q, associated into a hexamer in the presence of ATP, indicating that nucleotide binding to domain I favors a conformation required to stabilize the quaternary structure of ClpA. ClpA-K220Q was defective in ATPase activity and in the ability to activate protein and peptide degradation by ClpP, but the defects could be partially overcome by formation of hybrid hexamers with wild-type ClpA. Another domain I mutant, ClpA-K220R, readily formed hexamers in the presence of ATP and retained > or = 60% of the wild-type ATPase activity and ability to activate ClpP. These results indicate that hexamer formation is a prerequisite for expression of enzymatic activity. Domain II mutants ClpA-K501Q and ClpA-K501R had very low ATPase activity (< 10% of wild-type) and a severe defect in activation of protein degradation, which requires ATP hydrolysis. Domain II mutants were able to activate ClpP to degrade a peptide whose degradation required nucleotide binding but not hydrolysis. Nucleotide binding to domain II of ClpA is important to form a productive complex with ClpP, and domain II appears to be primarily responsible for an energy-requiring step in the catalytic cycle unique to the degradation of large proteins.

Residues interacting with serine-174 and alanine-295 in the β-subunit of Escherichia coli H +-ATP synthase: Possible ternary structure of the center region of the subunit

The mutation of serine-174 to phenylalanine that causes a defect in the Escherichia coli F 1-ATPase β-subunit is suppressed by further mutations; Gly-149 to Ser, Ala-295 to Thr, Ala-295 to Pro, or Leu-400 to Gln (Miki, J., Fujiwara, K., Tsuda, M., Tsuchiya, T. and Kanazawa, H. (1990) J. Biol. Chem. 265, 21567–21572). We analyzed the effects of these second site mutations and of a newly identified Asn-158 to Tyr mutation on the activities of the ATPase without the original Ser-174 to Phe mutation. The β-subunit with each amino acid replacement was expressed in the mutant strain JP17, which does not have a β-subunit. Cells transformed with the plasmid carrying Ala-295 to Pro mutation alone did not grow on minimal medium agar supplemented with succinate as the sole carbon source, and showed 3% of the wild-type ATPase activity, suggesting that this mutation caused structural alterations affecting the catalytic function of the enzyme. Conversely transformants with other mutations grew well and had higher ATPase activities, suggesting that these mutations did not cause extensive structural alterations. From the transformants with the plasmid carrying the Ala-295 to Pro mutation, seven revertants capable of cell growth on succinate plates were isolated and reversion mutations were identified at residues 140, 159, 166, 171, 172 and 184 of the β-subunits. The results suggested that Ser-174 and Ala-295 do not necessarily interact directly, but that the regions including these suppression mutation sites close to Ser-174, and Ala-295 interact with each other for the proper functioning of the ATPase. The ternary structure of the region surrounded by the residues which were identified as the reversion mutation sites for Ser-174 to Phe and Ala-295 to Pro mutations is important for the catalytic function of this enzyme.

Overexpression of the Yeast ATP Synthase Subunit D in Escherichia coli: Use of Polyclonal Antibodies Directed Against Recombinant Subunit D

The yeast ATP synthase subunit d was over-expressed in E. coli and formed inclusion bodies. It was purified by solubilization in urea and slow removal of the urea by stepwise dialysis in the presence of a non-ionic detergent. The resulting soluble subunit d was used to prepare polyclonal antibodies. Blots of yeast mitochondrial proteins were probed with these antibodies. The strain disrupted in ATP4 gene encoding the subunit 4 displayed only 8% of the wild type subunit d. Antibodies against subunit d did not inhibit the wild type ATPase activity.

A comparison of the rates of reaction and function of UVRB in UVRABC- and UVRAB-mediated anthramycin-N2-guanine-DNA repair.

The repair of anthramycin-DNA adducts by the UVR proteins in Escherichia coli follows two pathways: the adducts may be incised by the combined actions of UVRA, UVRB, and UVRC, or alternatively, the anthramycin may be removed by UVRA and UVRB in the absence of UVRC and with no DNA strand incision. To assess the competition between these two competing pathways, the rate of UVRABC-mediated excision repair of anthramycin-N2-guanine DNA adducts and the rate of UVRAB-mediated removal of the adduct were measured with single end-labeled DNAs under identical reaction conditions. UVR protein concentrations of 15 nM UVRA, 100 nM UVRB, and 10 nM UVRC protein were chosen to mimic in vivo concentrations. With these UVR protein concentrations and anthramycin-DNA concentrations of 1-2 nM the incision reaction and the release reactions are described by first-order kinetics. The rate of the UVRABC reaction, measured as the increase in incised fragments, was six to seven times faster than the rate of the UVRAB reaction, measured as the decrease in incised fragments. The UVRABC incision rate on anthramycin-modified linear DNA was four to five times the incision rate measured on the same DNA irradiated with ultraviolet light. We also investigated the role of the ATPase function of UVRB in UVRAB-mediated anthramycin removal. We found that a UVRB analogue with alanine at arginine 51, which retains near wild type ATPase activity, supported removal of anthramycin in the presence of UVRA, whereas a UVRB analogue with alanine at lysine 45, which abolishes the ATPase activity, did not. UVRB*, a specific proteolytic cleavage product of UVRB which retains the ATPase activity, did support removal of anthramycin in the presence of UVRA.

Altered plasma membrane H(+)-ATPase from the Dio-9-resistant pma1-2 mutant of Schizosaccharomyces pombe.

The pma1-2 mutation affecting the plasma membrane H(+)-ATPase of Schizosaccharomyces pombe has been selected for resistance to the antibiotic Dio-9. In membrane fractions purified from glucose-starved cells, the mutant ATPase activity is reduced by 96%, is insensitive to inhibition by vanadate and has a pH profile displaced in the acidic pH range when compared to the wild type. The maximum velocity of the H(+)-ATPase activity of plasma membranes from glucose-activated pma1-2 cells is activated 20-fold. This is in striking contrast with the wild-type ATPase activity, the maximal velocity of which is not affected by glucose. However, similar to the wild-type enzyme, glucose activation of the pma1-2 mutant H(+)-ATPase reduces the Km for MgATP 9-2 mM and shifts the optimal pH from 4.8 to 6.0-6.5. The pma1-2 mutation modifies Lys250 to a threonine, which is highly conserved in fungal and plant H(+)-ATPases. These results, compared to those reported for mutations of neighbour residues in yeast or mammalian P-type ATPases, suggest that Lys250 could play a significant role, not only in phosphate binding and/or in the E1P-E2P conformational isomerisation, but also in glucose activation of the H(+)-ATPase.

Identification of two nuclear genes (ATP11, ATP12) required for assembly of the yeast F1-ATPase.

Nuclear respiratory-deficient mutants of Saccharomyces cerevisiae (pet mutants) have been screened for defects in the mitochondrial ATPase. Mutants in two complementation groups were found to have 10% or less of wild-type ATPase activity. The two wild-type nuclear genes defined by the mutants have been designated ATP11 and ATP12. The proteins encoded by the two genes are not subunits of the ATPase but rather appear to exercise an important function at a late stage in the synthesis of F1 after transport of the subunits into the internal compartment of mitochondria. Mitochondria of atp11 and atp12 mutants have only marginally reduced levels of the alpha and beta subunits of F1. Both proteins are processed to their mature size but are not part of a native F1 structure or associated with the mitochondrial membrane. The most reasonable explanation for the mutant phenotype is a block in the assembly of the F1 oligomer.

H(+)-ATPase gamma subunit of Escherichia coli. Role of the conserved carboxyl-terminal region.

Cloned uncG genes (wild-type or in vitro mutagenized) for the Escherichia coli gamma subunit were introduced into the uncG mutant Gln-14----end), and the functions of the mutant subunits were studied. The F1's with Ala-283----end and Thr-277----end mutant gamma subunits had 63 and 14% of the wild-type ATPase activity, respectively, and mutants with these subunits showed reduced growth by oxidative phosphorylation, indicating that the 10 residues at the carboxyl terminus (286th residue) are important, but dispensable, for catalysis. On the other hand, F1 with a Gln-269----end gamma subunit was inactive. Replacement of conserved residues (Gln-269, Thr-273, or Glu-275) between Gln-269 and Leu-276 gave enzymes with significantly reduced ATPase activity (2-41% of that of the wild-type) and lower ATP-driven proton conduction. Thus these residues are required for the normal catalytic activity of F1, although they are not absolutely essential. Membranes with amino acid replacements (Thr-277----end, Gln-269----Leu, or Glu-275----Lys) and the frameshift mutation (downstream of Thr-277) had about 15% of the wild-type ATPase activity, but showed different degrees of ATP-dependent H+ translocation and growth yield by oxidative phosphorylation, suggesting that the gamma subunit, especially its carboxyl-terminal region, functions in coupling between catalysis and H+ translocation.

Temperature-sensitive Escherichia coli mutant with an altered initiation codon of the uncG gene for the H+-ATPase gamma subunit.

A mutant of Escherichia coli showing temperature-sensitive growth on succinate was isolated, and its mutation in the initiation codon (ATG to ATA) of the uncG gene (coding for the gamma subunit of H+-ATPase F0F1) was identified. This strain could grow on succinate as the sole carbon source at 25 and 30 degrees C, but not at 37 or 42 degrees C. When this strain was grown at 25 degrees C on succinate or glycerol, its membranes had about 15% of the ATPase activity of wild-type membranes, whereas when it was grown at 42 degrees C, its membranes had about 2% of the wild-type ATPase activity. Membranes of the mutant grown at 25 or 42 degrees C could bind F1 functionally, resulting in about 40% of the specific activity of wild-type membranes. The gamma subunit was identified in an EDTA extract of membranes of the mutant grown at 25 degrees C, but was barely detectable in the same amount of extract from the mutant grown at 42 degrees C. These results indicate that initiation of protein synthesis from the AUA codon is temperature sensitive and that the gamma subunit is essential for assembly of F1 in vivo as shown by in vitro reconstitution experiments (S. D. Dunn and M. Futai, J. Biol. Chem. 255:113-118, 1980).

Membrane bioenergetic parameters in uncoupler-resistant mutants of Bacillus megaterium.

Mutants of Bacillus megaterium displaying malate-driven ATP synthesis resistant to uncouplers of oxidative posphorylation are further characterized. Both the pH gradient and electrical potential generated across the membrane by malate respiration are equally sensitive to uncouplers in the wild type and uncoupler-resistant mutants. The mutants possess 0 to 10% of the wild type ATPase activity which is not activated by pretreatment with heat or trypsin. Despite this inability to measure ATPase activity, the mutants demonstrate acid-pulse-driven ATPase synthesis which is sensitive to uncouplers as well as malate-driven ATP synthesis which becomes uncoupler sensitive at pH 5.5. N,N' -Dicyclohexylcarbodiimide and valinomycin plus potassium inhibition of ATP synthesis is reversed by uncouplers in the mutants but not in the wild type. The data support the existence of a specific site on the ATPase complex for uncoupler binding which, if altered by mutation, affects uncoupler binding to the complex. The retention of malate-driven ATP synthesis in the absence of a significant pH gradient or electrical potential suggests that an alternative intermediate is involved in coupling oxidation to phosphorylation.

Bacillus megaterium mutant deficient in membrane-bound adenosine triphosphatase activity.

An adenosine triphosphatase (ATPase) mutant of Bacillus megaterium was isolated and characterized. This mutant (designated A37) was unable to grow on nonfermentable carbon sources and possessed less than 5% of the wild-type ATPase activity. Oxygen uptake by the mutant was comparable to that in the wild type. Sporulation in the wild type occurred in both glucose- and nitrogen-limiting media; however, A37 sporulated only in the nitrogen-limiting medium. The inability of A37 to sporulate in glucose-limiting medium seemed to be due to insufficient adenosine 5'-triphosphate (ATP) levels during the sporulation stages. Fructose, which can generate ATP via substrate-level phosphorylation, is equally efficient in stimulating ATP synthesis in the wild type and A37. Malate-stimulated ATP synthesis in the wild type was shown to have many characteristics associated with oxidative phosphorylation and was absent in the mutant. These data suggest that the ATPase deficiency results in the loss of oxidative phosphorylation.