Overlapping effects of S3 stalk segment mutations on the affinity of Ca2+-ATPase (SERCA) for thapsigargin and cyclopiazonic acid

Chimeric exchanges and mutations were produced in the Ca(2+)-ATPase (SERCA) to match (in the majority of cases) corresponding sequences of the Na(+),K(+)-ATPase. The effects of these mutations on the concentration dependence of the specific Ca(2+)-ATPase inhibition by thapsigargin (TG) and cyclopiazonic acid (CPA) were then determined. Extensive chimeric mutations on the large cytosolic loop, on the S4 stalk segment, and on the M3 transmembrane segments produced little or no modification of the Ca(2+)-ATPase sensitivity to either inhibitor. On the other hand, the presence of a six amino acid Na(+), K(+)-ATPase sequence within the S3 stalk segment of the Ca(2+)-ATPase raised 60-fold the apparent K(i) for TG and 250-fold the apparent K(i) for CPA. More limited mutations within the same S3 segment, however, affected differently the concentration dependence of the Ca(2+)-ATPase inhibition by TG or CPA. Specifically, single mutation of Phe256 to Val increased 20-fold the apparent K(i) for TG, while having very little effect on the apparent K(i) for CPA. These findings indicate significant overlap of the TG and CPA binding domains within the S3 stalk segment of the Ca(2+)-ATPase, where the contribution of each protein residue is dependent on the structures of the two inhibitors. Saturating concentrations of either or both TG and CPA produce an identical reduction of the affinity of the ATPase for ATP, suggesting that only one inhibitor can bind at any time due to significant overlap of their binding domains. It is suggested that perturbations produced by binding of either inhibitor within the stalk segment interfere with the long-range functional linkage between ATP utilization in the ATPase cytosolic region and Ca(2+) binding in the membrane-bound region.     

Stalk segment 5 of the yeast plasma membrane H+-ATPase: mutational evidence for a role in glucose regulation

In P(2)-type ATPases, a stalk region connects the cytoplasmic part of the molecule, which binds and hydrolyzes ATP, to the membrane-embedded part through which cations are pumped. The present study has used cysteine scanning mutagenesis to examine structure-function relationships within stalk segment 5 (S5) of the yeast plasma-membrane H(+)-ATPase. Of 29 Cys mutants that were made and examined, two (G670C and R682C) were blocked in biogenesis, presumably due to protein misfolding. In addition, one mutant (S681C) had very low ATPase activity, and another (F685C) displayed a 40-fold decrease in sensitivity to orthovanadate, reflecting a shift in equilibrium from the E(2) conformational state toward E(1). By far the most striking group of mutants (F666C, L671C, I674C, A677C, I684C, R687C, and Y689C) were constitutively activated even in the absence of glucose, with rates of ATP hydrolysis and kinetic properties normally seen only in glucose-metabolizing cells. Previous work has suggested that activation of the wild-type H(+)-ATPase results from kinase-mediated phosphorylation in the auto-inhibitory C-terminal region of the 100-kDa polypeptide. The seven residues identified in the present study are located on one face of the S5 alpha-helix, consistent with the idea that mutations along this face serve to release the auto-inhibition.     

Dual requirement for a newly identified phosphorylation site in p70s6k.

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.     

The cadmium transport sites of CadA, the Cd2+-ATPase from Listeria monocytogenes

CadA, the Cd(2+)-ATPase from Listeria monocytogenes, belongs to the Zn(2+)/Cd(2+)/Pb(2+)-ATPase bacterial subfamily of P(1B)-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd(2+), CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd(2+) (Wu, C. C., Bal, N., Pérard, J., Lowe, J., Boscheron, C., Mintz, E., and Catty, P. (2004) Biochem. Biophys. Res. Commun. 324, 1034-1040). This phenotype, which reflects in vivo Cd(2+)-transport activity, was used to select from 33 point mutations, shared out among the eight transmembrane (TM) segments of CadA, those that affect the activity of the protein. Six mutations affecting CadA were found: M149A in TM3; E164A in TM4; C354A, P355A, and C356A in TM6; and D692A in TM8. Functional studies of the six mutants produced in Sf9 cells revealed that Cys(354) and Cys(356) in TM6 as well as Asp(692) in TM8 and Met(149) in TM3 could participate at the Cd(2+)-binding site(s). In the canonical Cys-Pro-Cys motif of P(1B)-ATPases, the two cysteines act at distinct steps in the transport mechanism, Cys(354) being directly involved in Cd(2+) binding, while Cys(356) seems to be required for Cd(2+) occlusion. This confirms an earlier observation that the two equivalent Cys of Ccc2, the yeast Cu(+)-ATPase, also act at different steps. In TM4, Glu(164), which is conserved among P(1B)-ATPases, may be required for Cd(2+) release. Finally, analysis of the role of Cd(2+) in the phosphorylation from ATP and from P(i) of the mutants suggests that two Cd(2+) ions are involved in the reaction cycle of CadA.     

Stalk segment 4 of the yeast plasma membrane H+-ATPase. Mutational evidence for a role in the E1-E2 conformational change

In the P(2)-type ATPases, there is growing evidence that four alpha-helical stalk segments connect the cytoplasmic part of the molecule, responsible for ATP binding and hydrolysis, to the membrane-embedded part that mediates cation transport. The present study has focused on stalk segment 4, which displays a significant degree of sequence conservation among P(2)-ATPases. When site-directed mutants in this region of the yeast plasma membrane H(+)-ATPase were constructed and expressed in secretory vesicles, more than half of the amino acid substitutions led to a severalfold decrease in the rate of ATP hydrolysis, although they had little or no effect on the coupling between hydrolysis and transport. Strikingly, mutant ATPases bearing single substitutions of 13 consecutive residues from Ile-359 through Gly-371 were highly resistant to inorganic orthovanadate, with IC(50) values at least 10-fold above those seen in the wild-type enzyme. Most of the same mutants also displayed a significant reduction in the K(m) for MgATP and an increase in the pH optimum for ATP hydrolysis. Taken together, these changes in kinetic behavior point to a shift in equilibrium from the E(2) conformation of the ATPase toward the E(1) conformation. The residues from Ile-359 through Gly-371 would occupy three full turns of an alpha-helix, suggesting that this portion of stalk segment 4 may provide a conformationally active link between catalytic sites in the cytoplasm and cation-binding sites in the membrane.     

Effects of fluorescent pseudo-ATP and ATP-metal analogs on secondary structure of Na(+)/K(+)-ATPase

The secondary structure of Na(+)/K(+)-ATPase after modification of the ATP-binding sites was analyzed. Consistently with recent reports, we found in trypsin-treated Na(+)/K(+)-ATPase additionally to alpha-helix also beta-sheet structures in the transmembrane segments. However, binding of fluorescein 5'-isothiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not affect the secondary structure of undigested Na(+)/K(+)-ATPase. Consequently, fluorescence intensity changes of FITC-labeled Na(+)/K(+)-ATPase commonly used to observe conformational transitions of the enzyme reflect physiological changes of the native structure. The metal complex analogues of ATP, Cr(H(2)O)(4)ATP and Co(NH(3))(4)ATP, on the other hand, affected the secondary structure of Na(+)/K(+)-ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation.     

Structural determinants for the action of grayanotoxin in D1 S4-S5 and D4 S4-S5 intracellular linkers of sodium channel alpha-subunits

We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletal-muscle (mu 1) Na(+) channel isoforms (segments S4-S5 in domains D1 and D4), using the alanine scanning substitution method. GTX-modification of Na(+) channels, transiently expressed in HEK 293 cells, was evaluated under whole-cell voltage clamp, from the ratio of maximum chord conductance for modified and unmodified Na(+) channels. In mu 1, mutations K237A, L243A, S246A, K248A, K249A, L250A, S251A, or T1463A, caused a moderate, but statistically significant decrease in this ratio. On making corresponding mutations in rH1, only L244A dramatically reduced the ratio. Because in mu 1, the serine at position 251 is the only heterologous residue with respect to rH1 (Ala-252), we made a double mutant L243A&S251A to match the sequence of mu 1 and rH1 in S4-S5 linkers of both domains. This double mutation resulted in a significant decrease in the ratio, to the same extent as L244A substitution in rH1 did, indicating that the site at Leu-244 in rH1 or at Leu-243 in mu 1 is a novel one, exhibiting a synergistic effect of grayanotoxin.     

Stalk segment 5 of the yeast plasma membrane H(+)-ATPase. Labeling with a fluorescent maleimide reveals a conformational change during glucose activation

Glucose is well known to cause a rapid, reversible activation of the yeast plasma membrane H(+)-ATPase, very likely mediated by phosphorylation of two or more Ser/Thr residues near the C terminus. Recent mutagenesis studies have shown that glucose-dependent activation can be mimicked constitutively by amino acid substitutions in stalk segment 5 (S5), an alpha-helical stretch connecting the catalytic part of the ATPase with transmembrane segment 5 (Miranda, M., Allen, K. E., Pardo, J. P., and Slayman, C. W. (2001) J. Biol. Chem. 276, 22485-22490). In the present work, the fluorescent maleimide Alexa-488 has served as a probe for glucose-dependent changes in the conformation of S5. Experiments were carried out in a "3C" version of the ATPase, from which six of nine native cysteines had been removed by site-directed mutagenesis to eliminate background labeling by Alexa-488. In this construct, three of twelve cysteines introduced at various positions along S5 (A668C, S672C, and D676C) reacted with the Alexa dye in a glucose-independent manner, as shown by fluorescent labeling of the 100 kDa Pma1 polypeptide and by isolation and identification of the corresponding tryptic peptides. Especially significant was the fact that three additional cysteines reacted with Alexa-488 more rapidly (Y689C) or only (V665C and L678C) in plasma membranes from glucose-metabolizing cells. The results support a model in which the S5 alpha-helix undergoes a significant change in conformation to expose positions 665, 678, and 689 during glucose-dependent activation of the ATPase.     

Interaction of the S6 Proline Hinge with N-Type and C-Type Inactivation in?Kv1.4 Channels

Several voltage-gated channels share a proline-valine-proline (proline hinge) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels, which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine or alanine: P558A, P558G. These mutations were studied in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N- and C-type inactivation. Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed by more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement play a significant role in inactivation and recovery. Computer modeling suggests that both P558G and P558A mutations modify early voltage-dependent steps and make a final voltage-insensitive step that is rate limiting at positive potentials.     

Site-directed mutagenesis of Asp-376, the catalytic phosphorylation site, and Lys-507, the putative ATP-binding site, of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase

Point mutations of Asp-376 of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase (the site of phosphorylation during the catalytic cycle) to Asn, Glu or Thr led to virtual abolishment of Na+/K(+)-ATPase activity and ouabain-binding capacity. Replacement of Lys-507 of the same subunit (the putative ATP-binding site) by Met resulted in decreases in Na+/K(+)-ATPase activity and ouabain-binding capacity. These results are in agreement with those reported for rabbit sarcoplasmic reticulum Ca2(+)-ATPase (Maruyama, K. and MacLennan, D.H. (1988) Proc. Natl. Acad. Sci. USA 85, 3314-3318).     

Differential temporal and spatial regulation of somatostatin receptor phosphorylation and dephosphorylation

The G(i)-coupled somatostatin 2A receptor (sst2A) mediates many of the neuromodulatory and neuroendocrine actions of somatostatin (SS) and is targeted by the SS analogs used to treat neuroendocrine tumors. As for other G protein-coupled receptors, agonists stimulate sst2A receptor phosphorylation on multiple residues, and phosphorylation at different sites has distinct effects on receptor internalization and uncoupling. To elucidate the spatial and temporal regulation of sst2A receptor phosphorylation, we examined agonist-stimulated phosphorylation of multiple receptor GPCR kinase sites using phospho-site-specific antibodies. SS increased receptor phosphorylation sequentially, first on Ser-341/343 and then on Thr-353/354, followed by receptor internalization. Reversal of receptor phosphorylation was determined by the duration of prior agonist exposure. In acutely stimulated cells, in which most receptors remained on the cell surface, dephosphorylation occurred only on Thr-353/354. In contrast, both Ser-341/343 and Thr-353/354 were rapidly dephosphorylated when cells were stimulated long enough to allow receptor internalization before agonist removal. Consistent with these observations, dephosphorylation of Thr-353/354 was not affected by either hypertonic sucrose or dynasore, which prevent receptor internalization, whereas dephosphorylation of Ser-341/343 was completely blocked. An okadaic acid- and fostriecin-sensitive phosphatase catalyzed the dephosphorylation of Thr-353/354 both intracellularly and at the cell surface. In contrast, dephosphorylation of Ser-341/343 was insensitive to these inhibitors. Our results show that the phosphorylation and dephosphorylation of neighboring GPCR kinase sites in the sst2A receptor are subject to differential spatial and temporal regulation. Thus, the pattern of receptor phosphorylation is determined by the duration of agonist stimulation and compartment-specific enzymatic activity.     

Proximity of periplasmic loops in the metal-Tetracycline/H(+) antiporter of Escherichia coli observed on site-directed chemical cross-linking

Our previous study on second-site suppressor mutations of the Tn10-encoded metal-tetracycline/H(+) antiporter suggested that Leu(30) and Ala(354), located in periplasmic loop 1-2 and 11-12, respectively, are conformationally linked to each other (Kawabe, T., and Yamaguchi, A. (1999) FEBS Lett. 457, 169-173). To determine the spatial proximity of these two residues, cross-linking gel-shift assays of the L30C/A354C double mutant were performed after the mutant had been oxidized with Cu(2+)/o-phenanthroline. The results indicated that Leu(30) and Ala(354) are close to each other but that Gly(62), which is located in cytoplasmic loop 2-3, and Ala(354) are distant from each other, as a negative control. Then, a single Cys residue was introduced into each of the six periplasmic loop regions (P1-P6), and eleven double mutants were constructed. Of these eleven double Cys mutants, the L30C/A354C and L30C/T235C mutants showed a mobility shift on oxidation, indicating that P1 is spatially close to P4 as well as P6. In contrast, the other nine mutants, L30C/S92C, L30C/S156C, L30C/S296C, S92C/S296C, S92C/T235C, S92C/A354C, S156C/T235C, S156C/S296C, and S156C/A354C, showed no mobility shift under oxidized conditions on intramolecular cross-linking. The S92C and S296C mutants showed dimerization on intermolecular cross-linking, indicating that P2 and P5 are located at the periphery of the helix bundle.     

Eight amino acids form the ATP recognition site of Na(+)/K(+)-ATPase

Point mutations of a part of the H(4)-H(5) loop (Leu(354)-Ile(604)) of Na(+)/K(+)-ATPase have been used to study the ATP and TNP-ATP binding affinities. Besides the previously reported amino acid residues Lys(480), Lys(501), Gly(502), and Cys(549), we have found four more amino acid residues, viz., Glu(446), Phe(475), Gln(482), and Phe(548), completing the ATP-binding pocket of Na(+)/K(+)-ATPase. Moreover, mutation of Arg(423) has also resulted in a large decrease in the extent of ATP binding. This residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site, probably due to formation of a hydrogen bond with Glu(472). On the other hand, only some minor effects were caused by mutations of Ile(417), Asn(422), Ser(445), and Glu(505).     

Clathrin-mediated endocytosis of Na+,K+-ATPase in response to parathyroid hormone requires ERK-dependent phosphorylation of Ser-11 within the alpha1-subunit

Parathyroid hormone (PTH) inhibits Na(+),K(+)-ATPase activity through protein kinase C- (PKC) and extracellular signal-regulated kinase- (ERK) dependent pathways and increases serine phosphorylation of the alpha(1)-subunit. To determine whether specific serine phosphorylation sites within the Na(+),K(+)-ATPase alpha(1)-subunit are involved in the Na(+),K(+)-ATPase responses to PTH, we examined the effect of PTH in opossum kidney cells stably transfected with wild type rat Na(+),K(+)-ATPase alpha(1)-subunit (WT), serine 11 to alanine mutant alpha(1)-subunit (S11A), or serine 18 to alanine mutant alpha(1)-subunit (S18A). PTH increased phosphorylation and endocytosis of the Na(+),K(+)-ATPase alpha(1)-subunit into clathrin-coated vesicles in cells transfected with WT and S18A rat Na(+),K(+)-ATPase alpha(1)-subunits. PTH did not increase the level of phosphorylation or stimulate translocation of Na(+),K(+)-ATPase alpha(1)-subunits into clathrin-coated vesicles in cells transfected with the S11A mutant. PTH inhibited ouabain-sensitive (86)Rb uptake and Na(+),K(+)-ATPase activity (ouabain-sensitive ATP hydrolysis) in WT- and S18A-transfected opossum kidney cells but not in S11A-transfected cells. Pretreatment of the cells with the PKC inhibitors and ERK inhibitor blocked PTH inhibition of (86)Rb uptake, Na(+),K(+)-ATPase activity, alpha(1)-subunit phosphorylation, and endocytosis in WT and S18A cells. Consistent with the notion that ERK phosphorylates Na(+),K(+)-ATPase alpha(1)-subunit, ERK was shown to be capable of causing phosphorylation of Na(+),K(+)-ATPase alpha(1)-subunit immunoprecipitated from WT and S18A but not from S11A-transfected cells. These results suggest that PTH regulates Na(+),K(+)-ATPase by PKC and ERK-dependent alpha(1)-subunit phosphorylation and that the phosphorylation requires the expression of a serine at the 11 position of the Na(+),K(+)-ATPase alpha(1)-subunit.     

Structure-function relationships of rat liver CYP3A9 to its human liver orthologs: site-directed active site mutagenesis to a progesterone dihydroxylase

CYP3A9 is an estrogen-inducible ortholog of human liver CYP3A4 with 76.5% sequence identity to CYP3A4. Unlike CYP3A4, it is a very poor testosterone 6beta- and 2beta-hydroxylase, but a relatively better catalyst of progesterone monohydroxylation largely at 6beta, 16alpha, and 21 positions with negligible 6beta, 21-dihydroxylation. We reasoned that such differences in substrate catalyses must be due to differences in the active site architecture of each CYP3A enzyme. Indeed, alignment of CYP3A4 substrate recognition sites (SRSs) with the corresponding regions of CYP3A9 sequence revealed that of the 22 fully divergent residues, 4 reside in SRS regions [P107N (SRS-1), M371G (SRS-5), and L479K and G480Q (SRS-6)]. Accordingly, we substituted these and other divergent CYP3A9 SRS residues with the corresponding residues of CYP3A4 and/or CYP3A5. Our findings of the influence of these site-directed mutations of the CYP3A9 active site on its catalysis of testosterone and three other established but structurally different CYP3A substrates (progesterone, imipramine, and carbamazepine) are described. These findings revealed that some mutations (N107P, N107S, V207T, G371M, and Q480G) not only improved the ability of CYP3A9 to hydroxylate testosterone at the 6beta and 2beta positions, but also converted it into a robust progesterone 6beta, 21-dihydroxylase. The latter in the case of CYP3A9N107P was accompanied by a shift from sigmoidal to hyperbolic enzyme-substrate kinetics. In contrast, the catalytic potential of CYP3A9 mutants K206N, K206S, M240V, and K479L/Q480G was either relatively unchanged or negligible to nonexistent. Together these findings attest to the unique substrate-active site fit of each CYP3A enzyme.     

Phosphate binding and ATP-binding sites coexist in Na+/K(+)-transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4.

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K(+)-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): Co(NH3)4PO4 + E2 Kd in equilibrium E2.Co(NH3)4PO4k2----E'2.Co(NH3)4PO4. The inactivation rate constant k2 for the formation of a stable E'2.Co(NH3)4PO4 at 37 degrees C was 0.057 min-1; the dissociation constant, Kd = 300 microM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[beta, gamma-methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks = 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks = 50 microM, as did phosphate (Ks = 16 mM) and Mg2+ (Ks = 160 microM). It is concluded that the MgPO4 analogue binds to the MgPO4-binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na+ (Ks = 860 microM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 microM, and for the E.Co(NH3)4PO4 complex of 720 microM, were calculated. Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K(+)-ATPase and K(+)-activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na(+)-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P-labelled tetramminecobalt ATP ([gamma-32P]Co(NH3)4ATP) at the low-affinity ATP-binding site, allowed (independent of the purity of the Na+/K(+)-ATPase preparation) a further incorporation of radioactivity from 32P-labelled tetraaquachromium(III) ATP ([gamma-32P]CrATP) to the high-affinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K(+)-ATPase by [gamma-32P]CrATP prevented the binding of Co(NH3)4 32PO4 or [gamma-32P]Co(NH3)4ATP to the enzyme. [gamma-32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (alpha,beta)2-diprotomer, which change their interactions during the Na+/K(+)-pumping process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action.     

Pertussis toxin-catalyzed ADP-ribosylation of G(o) alpha with mutations at the carboxyl terminus.

The guanine nucleotide-binding protein G(o alpha) has been implicated in the regulation of Ca2+ channels in neural tissues. Covalent modification of G(o alpha) by pertussis toxin-catalyzed ADP-ribosylation of a cysteine (position 351) four amino acids from the carboxyl terminus decouples G(o alpha) from receptor. To define the structural requirements for ADP-ribosylation, preparations of recombinant G(o alpha) with mutations within the five amino acids at the carboxyl terminus were evaluated for their ability to serve as pertussis toxin substrates. As expected, the mutant in which cysteine 351 was replaced by glycine (C351G) was not a toxin substrate. Other inactive mutants were G352D and L353 delta/Y354 delta. Mutations that had no significant effect on toxin-catalyzed ADP-ribosylation included G350D, G350R, Y354 delta, and L353V/Y354 delta. Less active mutants were L353G/Y354 delta, L353A/Y354 delta, and L353G. ADP-ribosylation of the active mutants, like that of wild-type G(o alpha), was enhanced by the beta gamma subunits of bovine transducin. It appears that three of the four terminal amino acids critically influence pertussis toxin-catalyzed ADP-ribosylation of G(o alpha).     

Mutations in the peptidyl transferase region of E. coli 23S rRNA affecting translational accuracy.

We have produced mutations in a cloned Escherichia coli 23S rRNA gene at positions G2252 and G2253. These sites are protected in chemical footprinting studies by the 3' terminal CCA of P site-bound tRNA. Three possible base changes were introduced at each position and the mutations produced a range of effects on growth rate and translational accuracy. Growth of cells bearing mutations at 2252 was severely compromised while the only mutation at 2253 causing a marked reduction in growth rate was a G to C transversion. Most of the mutations affected translational accuracy, causing increased readthrough of UGA, UAG and UAA nonsense mutations as well as +1 and -1 frameshifting in a lacZ reporter gene in vivo. C2253 was shown to act as a suppressor of a UGA nonsense mutation at codon 243 of the trpA gene. The C2253 mutation was also found not to interact with alleles of rpsL coding for restrictive forms of ribosomal protein S12. These results provide further evidence that nucleotides localized to the P site in the 50S ribosomal subunit influence the accuracy of decoding in the ribosomal A site.     

NMR studies of the fifth transmembrane segment of Na+,K+-ATPase reveals a non-helical ion-binding region

The structure of a synthetic peptide corresponding to the fifth membrane-spanning segment (M5) in Na(+),K(+)-ATPase in sodium dodecyl sulfate (SDS) micelles was determined using liquid-state nuclear magnetic resonance (NMR) spectroscopy. The spectra reveal that this peptide is substantially less alpha-helical than the corresponding M5 peptide of Ca(2+)-ATPase. A well-defined alpha-helix is shown in the C-terminal half of the peptide. Apart from a short helical stretch at the N-terminus, the N-terminal half contains a non-helical region with two proline residues and sequence similarity to a non-structured transmembrane element of the Ca(2+)-ATPase. Furthermore, this region spans the residues implicated in Na(+) and K(+) transport, where they are likely to offer the flexibility needed to coordinate Na(+) as well as K(+) during active transport.     

A phosphorylation in the c-terminal auto-inhibitory domain of the plant plasma membrane H+-ATPase activates the enzyme with no requirement for regulatory 14-3-3 proteins

The plant plasma membrane H(+)-ATPase is regulated by an auto-inhibitory C-terminal domain that can be displaced by phosphorylation of the penultimate residue, a Thr, and the subsequent binding of 14-3-3 proteins. By mass spectrometric analysis of plasma membrane H(+)-ATPase isoform 2 (PMA2) isolated from Nicotiana tabacum plants and suspension cells, we identified a new phosphorylation site, Thr-889, in a region of the C-terminal domain upstream of the 14-3-3 protein binding site. This residue was mutated into aspartate or alanine, and the mutated H(+)-ATPases expressed in the yeast Saccharomyces cerevisiae. Unlike wild-type PMA2, which could replace the yeast H(+)-ATPases, the PMA2-Thr889Ala mutant did not allow yeast growth, whereas the PMA2-Thr889Asp mutant resulted in improved growth and increased H(+)-ATPase activity despite reduced phosphorylation of the PMA2 penultimate residue and reduced 14-3-3 protein binding. To determine whether the regulation taking place at Thr-889 was independent of phosphorylation of the penultimate residue and 14-3-3 protein binding, we examined the effect of combining the PMA2-Thr889Asp mutation with mutations of other residues that impair phosphorylation of the penultimate residue and/or binding of 14-3-3 proteins. The results showed that in yeast, PMA2 Thr-889 phosphorylation could activate H(+)-ATPase if PMA2 was also phosphorylated at its penultimate residue. However, binding of 14-3-3 proteins was not required, although 14-3-3 binding resulted in further activation. These results were confirmed in N. tabacum suspension cells. These data define a new H(+)-ATPase activation mechanism that can take place without 14-3-3 proteins.