Complementation of the Saccharomyces cerevisiae plasma membrane H+-ATPase by a plant H+-ATPase generates a highly abundant fusicoccin binding site

Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme. The fungal phytotoxin fusicoccin (FC) appears to stabilize this H+-ATPase.14-3-3 complex, thus leading to a persistent activation of the H+-ATPase in vivo. In this study we show that functional replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation of a high affinity fusicoccin binding site that is exceptionally abundant. Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 homologs. The existence of a (plant) PMA2.(yeast)14-3-3 complex was demonstrated using two-dimensional gel systems (native/denaturing). After expression of PMA2 lacking most of its C-terminal region, neither H+-ATPase.14-3-3 complex formation nor FC binding activity could be observed. Furthermore, we obtained direct biochemical evidence for a minimal FC binding complex consisting of the C-terminal PMA2 domain and yeast 14-3-3 homologs. Thus we demonstrated unambiguously the relevance of this regulatory ATPase domain for 14-3-3 interaction as well as its requirement for FC binding.     

Regulation of cellular Mg2+ by Saccharomyces cerevisiae

Regulation of cellular Mg2+ by S. cerevisiae was investigated. The minimal concentration of Mg2+ that results in optimal growth of S. cerevisiae is about 30 microM and a half-maximum growth rate is attained at about 5 microM Mg2+. Since the plasma membrane has an electrical potential greater than 100 mV, passive equilibration of Mg2+ across the plasma membrane would provide sufficient cytosolic Mg2+ (0.1-1 mM). The total cellular Mg2+ of cells grown in synthetic medium containing 1 mM Mg2+ is about 400 nmol/mg protein, most of which is bound to polyphosphate, nucleic acids, and ATP. Total cellular Mg2+ decreases to about 80 nmol/mg protein as the Mg2+ in synthetic growth medium is reduced to 0.02 mM, but remains relatively constant in growth medium containing 1 to 100 mM Mg2+. Cells shifted into Mg(2+)-free medium continue to grow by utilizing the vacuolar Mg2+ stores. Mg(2+)-starved cells replenish vacuolar Mg2+ stores with a halftime of 30 min. following the addition of 1 mM Mg2+ to the growth medium. The data indicate that cytosolic Mg2+ is maintained by the regulation of Mg2+ fluxes across both the vacuolar and plasma membranes.     

Ca2+ regulation of gelsolin activity: binding and severing of F-actin

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.     

Hexokinase 2 from Saccharomyces cerevisiae: regulation of oligomeric structure by in vivo phosphorylation at serine-14

Homodimeric hexokinase 2 from Saccharomyces cerevisiae is known to have two sites of phosphorylation: for serine-14 the modification in vivo increases with glucose exhaustion [Kriegel et al. (1994) Biochemistry 33, 148-152], while for serine-157 it occurs in vitro with ATP in the presence of nonphosphorylateable five-carbon analogues of glucose [Heidrich et al. (1997) Biochemistry 36, 1960-1964]. We show now by site-directed mutagenesis and sedimentation analysis that serine-14 phosphorylation affects the oligomeric state of hexokinase, its substitution by glutamate causing complete dissociation; glutamate exchange for serine-157 does not. Phosphorylation of wild-type hexokinase at serine-14 likewise causes dissociation in vitro. In view of the higher glucose affinity of monomeric hexokinase and the high hexokinase concentration in yeast [Womack, F., and Colowick, S. P. (1978) Arch. Biochem. Biophys. 191, 742-747; Mayes, E. L., Hoggett, J. G., and Kellett, G. L. (1983) Eur. J. Biochem. 133, 127-134], we speculate that the in vivo phosphorylation at serine-14 as transiently occurring in glucose derepression might provide a mechanism to improve glucose utilization from low level and/or that nuclear localization of the monomer might be involved in the signal transduction whereby glucose causes catabolite repression.     

RAS2/PKA pathway activity is involved in the nitrogen regulation of L-leucine uptake in Saccharomyces cerevisiae

The aim of the present work is to study the participation of RAS2/PKA signal pathway in the nitrogen regulation of L-leucine transport in yeast cells. The study was performed on Saccharomyces cerevisiae isogenic strains with the normal RAS2 gene, the RAS2val19 mutant and the disrupted ras2::LEU2. These strains bring about different activities of the RAS2/PKA signal pathway, L-(14C)-Amino acid uptake measurements were determined in cells grown in a rich YPD medium with a mixed nitrogen source or in minimal media containing NH4+ or L-proline as the sole nitrogen source. We report herein that in all strains used, even in those grown in a minimal proline medium, the activity of the general amino acid permease (GAP1) was not detected. L-Leucine uptake in these strains is mediated by two kinetically characterized transport systems. Their KT values are of the same order as those of S1 and S2 L-leucine permeases. Mutation in the RAS2 gene alters initial velocities and Jmax values in both high and low affinity L-leucine transport systems. Activation of the RAS2/PKA signalling pathway by the RAS2val19 mutation, blocks the response to a poor nitrogen source whereas inactivation of RAS2 by gene disruption, results in an increase of the same response.     

Location of a high affinity Zn2+ binding site in the channel of alpha1beta1 gamma-aminobutyric acidA receptors

Zn2+ inhibits currents through gamma-aminobutyric acid (GABA)A receptors. Its affinity depends on the subunit composition; alpha1beta1 receptors are inhibited with high affinity (IC50 = 0.54 micro M). We sought to identify the residues that form this high affinity Zn2+ binding site. beta1His267 aligns with alpha1Ser272, a residue near the extracellular end of the M2 membrane-spanning segment that we previously demonstrated to be exposed in the channel. The Zn2+ affinity of alpha1beta1 H267S was reduced by 300-fold (IC50 = 161 micro M). Addition of a histidine at the aligned position in alpha1 creates a receptor, alpha1S272Hbeta1, that should have five channel-lining histidines; the Zn2+ affinity was increased 20-fold (IC50 = 0.025 micro M). Shifting the position of the histidine from the beta1 subunit to the aligned position in alpha1 with the two mutants alpha1S272Hbeta1H267S reduced the affinity (IC50 = 26 micro M) compared with wild-type. We infer that the high affinity Zn2+ binding site involves beta1His267 from at least two subunits. For two histidines to interact with a Zn2+ ion, the alpha carbons must be separated by <13 A. This limits the separation of the subunits and provides a constraint on the possible quaternary structures of the channel. The ability of a divalent cation to penetrate from the extracellular end of the channel to beta1His267 implies that the charge-selectivity filter, the structure that discriminates between anions and cations, is located at a more cytoplasmic position than beta1His267; this is consistent with our previous work that showed that positively charged sulfhydryl-specific reagents reacted with an engineered cysteine residue as cytoplasmic as alpha1T261C.     

Phosphorylase phosphatase activity in Saccharomyces cerevisiae 257

A phosphatase, active towards phosphorylase a and phosphorylated proteins casein and histone II-A, was isolated from Saccharomyces cerevisiae 257. The enzyme dephosphorylated glycogen phosphorylase from commercial yeast rendering it inactive. The protein phosphatase activity was not influenced by any metal ions. Phosphorylase phosphatase activity was slightly stimulated by p-nitrophenyl phosphate and inhibited by heparin.     

Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 --> Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1. 3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.     

Aggregation behaviour and Zn2+ binding properties of secretin

Dynamic light scattering measurements were carried out on secretin in aqueous solution (2 mM; pH 5.0). The results indicated that the molecule exists as a fairly compact hexamer under these solution conditions. Secondary structural properties of the secretin hexameric complex were evaluated using CD and NMR spectroscopy. Specifically, the spectral properties of secretin in water were examined as a function of peptide concentration. Results from the analyses indicated a 2-fold increase (17-32%) in alpha-helical content within the region Ser11-Arg21 as the peptide concentration was increased from 0.1 to 2 mM. Displacement of the alphaH proton chemical shifts relative to random coil values did not alter significantly with increasing peptide concentration. This observation confirmed that the length of the helical segment is independent of peptide concentration between 0.1 and 2 mM. The nature of the helix was furthermore determined as amphipathic, and thus the potential for a cooperative intermolecular association through the apolar helical face of individual monomers was indicated. These findings suggest that secretin aggregates into symmetric hexamers at millimolar concentrations and, furthermore, that the helical domain is stabilized through this intermolecular association. The potential for secretin to bind divalent cations, including Ca2+ and Zn2+, was also examined by CD1 and NMR spectroscopy. The results revealed that Zn2+ specifically coordinates to the His1 and Asp3 residues of each secretin monomer without disrupting the peptide's helical structure, whereas Ca2+ did not exhibit any interaction with the peptide hormone. It was concluded from these studies that secretin may be stored in a hexameric form within its secretory tissues and that zinc may play a role in the storage of secretin through a specific interaction with the N-terminal histidine and aspartic acid residues.     

Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex

Previous thermodynamic and computational studies have pointed to the important energetic role of aromatic contacts in generating the exceptional binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in these aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the hydrophobic binding contributions, which along with a hydrogen bonding network and a flexible binding loop give rise to tight ligand binding (Ka approximately 10(13) M-1). The crystal structures of ligand-free and biotin-bound mutants, W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 A were determined. Nine data sets for these four different mutants were collected, and structural models were refined to R-values ranging from 0.15 to 0.20. The major question addressed here is how these mutations influence the streptavidin binding site and in particular how they affect the binding mode of biotin in the complex. The overall folding of streptavidin was not significantly altered in any of the tryptophan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the binding pocket was not significantly altered in the mutant complex. Compared with the wild-type streptavidin-biotin complex, there were no additional crystallographic water molecules observed for any of the mutants in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alterations in binding residue composition, rather than a rearrangement of binding site architectures.     

Lysophosphatidylcholine induces Ca2+-independent cellular injury attenuated by d-propranolol in rat cardiomyocytes

In isolated rat cardiomyocytes, exogenous lysophosphatidylcholine (LPC) (15 microM) increased the intracellular Ca2+ concentration (Ca2+]i) from 72 +/- 5 to 3042 +/- 431 nM accompanied by cell injury as indicated by the hypercontracture of the cells and the increase in creatine phosphokinase (CPK) release. In order to understand whether the cell injury induced by LPC was a consequence of the elevation of [Ca2+]i, the effect of LPC was examined in the Ca2+-free solution containing EGTA. Under the Ca2+ -free conditions, LPC did not increase [Ca2+]i, whereas it still inflicted injury on the cells in terms of cell-shape change and CPK release to the same degree as that under the Ca2+-present condition. Addition of ryanodine (10 microM) failed to prevent the changes in cell-shape and CPK release induced by LPC under both Ca2+-free and Ca2+-present conditions. Preincubation of the myocytes with d-propranolol (50 microM) inhibited the LPC-induced changes in cell-shape and CPK release under both Ca2+ -free and Ca2+ -present conditions (p < 0.05). Our study provides clear evidence that the cellular injury induced by LPC could be independent of the increase in [Ca2+]i, and the Ca2+-independent cellular injury induced by LPC could be attenuated by d-propranolol, although the mechanism remains unknown.     

Membrane fusion activity of Semliki Forest virus in a liposomal model system: specific inhibition by Zn2+ ions

A novel, irregular, coccoid-shaped archaeum was isolated from a hydrothermally heated black smoker wall at the TAG site at the Mid Atlantic Ridge (depth 3650 meters). It grew at between 90 degrees C and 113 degrees C (optimum 106 degrees C) and pH 4.0-6.5 (optimum 5.5) and 1%-4% salt (optimum 1.7%). The organism was a facultatively aerobic obligate chemolithoautotroph gaining energy by H2-oxidation. Nitrate, S2O3(2-), and low concentrations of O2 (up to 0.3% v/v) served as electron acceptors, yielding NH4+, H2S, and H2O as end products, respectively. Growth was inhibited by acetate, pyruvate, glucose, starch, or sulfur. The new isolate was able to form colonies on plates (at 102 degrees C) and to grow at a pressure of 25000 kPa (250 bar). Exponentially growing cultures survived a one-hour autoclaving at 121 degrees C. The GC content was 53 mol%. The core lipids consisted of glycerol-dialkyl glycerol tetraethers and traces of 2,3-di-O-phytanyl-sn-glycerol. The cell wall was composed of a surface layer of tetrameric protein complexes arranged on a p4-lattice (center-to-center distance 18.5 nm). By its 16S rRNA sequence, the new isolate belonged to the Pyrodictiaceae. Based on its GC-content, DNA homology, S-layer composition, and metabolism, we describe here a new genus, which we name Pyrolobus (the "fire lobe"). The type species is Pyrolobus fumarii (type strain 1A; DSM 11204).     

Hemiacetal dehydrogenation activity of alcohol dehydrogenases in Saccharomyces cerevisiae

Some methylotrophic yeasts produce methyl formate from methanol and formaldehyde via hemiacetal formation. We investigated Saccharomyces cerevisiae to find whether this yeast has a carboxylate ester producing pathway that proceeds via hemiacetal dehydrogenation. We confirmed that the purified alcohol dehydrogenase (Adh) protein from S. cerevisiae can catalyze the production of esters. High specific activities were observed toward the hemiacetals corresponding to the primary alcohols when ether groups were substituted for methylene groups, resulting in the formation of formate esters. Both ADH and methyl formate synthesizing activities were sharply reduced in the delta adh1 delta adh2 mutant. The ADH1 and ADH2 genes encode the major Adh proteins in S. cerevisiae. Thus, it was concluded that the S. cerevisiae Adh protein catalyzes activities for the production of certain carboxylate esters.     

Characterization of ligands of a high-affinity metal-binding site in the latent chloroplast F1-ATPase by EPR spectroscopy of bound VO2+

Vanadyl, (V = O)2+, is able to substitute for Mg2+ as a cofactor for ATPase activity catalyzed by the chloroplast F1-ATPase (CF1). Mg2+-dependent ATPase activity was also observed with CF1 that contained VO(2+)-ATP bound specifically to the noncatalytic N2 site. Modulation of the Mg(2+)-ATPase activity induced by VO2+ bound at this site indicates that the metal bound to the noncatalytic site affects catalytic activity. When CF1 is depleted of nucleotides from all but the N1 site, a single Mg2+ remains bound at a site designated M1. Addition of VO2+ to the depleted protein gives rise to an EPR spectrum characteristic of a CF1-bound VO2+ species. The binding curve of the VO2+ complex to latent, nucleotide-depleted CF1 was determined by the integrated intensities of the -5/2 parallel peak in the EPR spectrum as calibrated using atomic absorption spectroscopy. Under these conditions, VO2+ binds cooperatively to approximately two sites designated M2 and M3. Three-pulse ESEEM spectra of the CF1-VO2+ complex contain two intense modulations with frequencies and field-dependent behavior that show that they are from a directly coordinated 14N nucleus. Analysis of the bound VO2+ by ENDOR spectroscopy revealed the presence of a single group of protons associated with an equatorial amino or water ligand that is exchangeable with solvent. Using the additivity relation for hyperfine coupling, the most probable set of equatorial ligands to the VO2+ bound to CF1 under these conditions consists of one lysine nitrogen, two carboxyl oxygens from aspartate or glutamate, and one water.     

Insulin signaling in the yeast Saccharomyces cerevisiae. 2. Interaction of human insulin with a putative binding protein

A putative insulin-binding protein (Kd = 0.5 +/- 0.2 microM for human insulin) was partially purified from solubilized plasma membranes of Saccharomyces cerevisiaeby wheat germ agglutinin and insulin affinity chromatographies. The binding affinities of various mutant insulin analogues correlated well with their capacities to activate glycogen synthase and SNF1 kinase in glucose-induced yeast spheroplasts, the ranking of their relative efficacies in yeast and in isolated rat adipocytes being similar. Using a bifunctional cross-linker and two different experimental protocols, a 53-kDa polypeptide contained in the insulin-binding protein preparation was specifically affinity cross-linked to [125I]monoiodo[B26]insulin. The relative rankings of the insulin analogues with respect to inhibition of cross-linking and binding to the partially purified insulin-binding protein were identical. Incubation of intact yeast spheroplasts with [125I]monoiodo[AI4]insulin led to specific and time-dependent association of the radiolabeled insulin with the cell surface followed by its internalization and degradation. These processes were considerably delayed by low temperature and energy depletion of the spheroplasts, suggesting involvement of the ATP-dependent endosomal apparatus. These data provide evidence for the existence of a low-affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae.