Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α–aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-Kg) using NAD+ as an oxidant. The enzyme utilizes a general acid-base mechanism to carry out its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo-form and a closed form of the enzyme with saccharopine and NADH bound have been solved at 2.0 Å and 2.2 Å resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II is observed that closes the active site cleft between the two domains and brings H96 and K77 in close proximity to the substrate binding site. The hydride transfer distance is 3.6 Å, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77 to M, H96 to Q single and the K77M/H96Q double mutant enzymes provide data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/Et and >103 increases in V2/KLysEt and V2/Kα-KgEt (the double mutation gives >105-fold decreases in the second order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V2/KLys. A value of 2.0 was also observed for D(V2/KLys)D2O when the primary deuterium kinetic isotope effect was repeated in D2O, consistent with a rate-limiting hydride transfer step. A viscosity effect of 0.8 was observed on V2/KLys indicating the solvent deuterium isotope effect resulted from stabilization of an enzyme form prior to hydride transfer. A small normal solvent isotope effect is observed on V, which decreases slightly when repeated with NADD, consistent with a contribution from product release to rate limitation. In addition, V2/KLysEt is pH independent consistent with the loss of an acid-base catalyst and perturbation of the pKa of the second catalytic group to higher pH, likely a result of a change in the overall charge in the active site. The primary deuterium kinetic isotope effect for H96Q, measured in H2O or D2O, is within error equal to 1. A solvent deuterium isotope effect of 2.4 is observed with NADH or NADD as the dinucleotide substrate. Data suggest rate-limiting imine formation, consistent with the proposed role of H96 in protonating the leaving hydroxyl as the imine is formed. The pH-rate profile for V2/KLysEt exhibits the pKa for K77, perturbed to a value of about 9, which must be unprotonated in order to accept a proton from the ε-amine of the substrate Lys so that it can act as a nucleophile. Overall, data are consistent with a role for K77 acting as the base that accepts a proton from the ε-amine of the substrate lysine prior to nucleophilic attack on the α-oxo group of α-ketoglutarate, and finally donating a proton to the imine nitrogen as it is reduced to give saccharopine. In addition, data indicate a role for H96 acting as a general acid-base catalyst in formation of the imine between the ε-amine of lysine and the α-oxo group of α-ketoglutarate.

Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to L-lysine and α-ketoglutarate. Lysine 99 is within hydrogen-bond distance to the α-carboxylate of the lysine substrate and D319 is in the vicinity of the carboxamide side chain of NADH. Both are conserved and may be important to the overall reaction. Replacing K99 with M gives decreases of 110-, 80- and 20-fold in the V2/Km values for lysine, α-ketoglutarate and NADH, respectively. Deuterium isotope effects on V and V/KLys increase, while the solvent deuterium isotope effects decrease compared to the C205S mutant enzyme. Data for K99M suggest a decreased affinity for all reactants and a change in the partition ratio of the imine intermediate to favor hydrolysis. A change in the bound conformation of the imine and/or the distance of the imine carbon to C4 of the nicotinamide ring of NADH is also suggested. Changing D319 to A decreases V2/KNADH by 33-fold. Primary deuterium and solvent deuterium isotope effects decrease compared to C205S suggesting a non-isotope sensitive step has become slower. NADH binds to enzyme first, and sets the site for binding of lysine and α-ketoglutarate. The slower step is likely the conformational change generated upon binding of NADH.

H3N2 influenza viruses have now circulated in the human population for 43 years since the pandemic of 1968, accumulating sequence changes in the hemagglutinin (HA) and neuraminidase (NA) that are believed to be predominantly due to selection for escape from antibodies. Examination of mutations that persist and accumulate led to identification of antigenically significant mutations that are contained in five antigenic sites (A–E) mapped on to the H3 HA. In early H3N2 isolates, antigenic site A appeared to be dominant while in the 1990s site B seemed more important. To obtain experimental evidence for dominance of antigenic sites on modern H3 HAs, we have measured antibodies in plasma of human subjects who received the 2006–07 trivalent subunit influenza vaccine (H3 component A/Wisconsin/67/05) or the 2008–09 formulation (H3 component A/Uruguay/716/07). Plasmas were tested against expressed HA of Wisconsin-like influenza A/Oklahoma/309/06 and site-directed mutants in antigenic site A (NNES121-124ITEG, N126T, N133D, TSSS135-138GSNA, K140I, RSNNS142-146PGSG), and antigenic site B (HL156-157KS, KFK158-160GST, NDQI189-192QEQT, A196V). “Native ELISA” analysis and escape mutant selection with two human monoclonal antibodies demonstrated that antibody E05 binds to antigenic site A and 1_C02 binds to site B. We find that most individuals, after vaccination in seasons 2006–07 and/or 2008–09, showed dominance of antigenic site B recognition over antigenic site A. A minority showed dominance of site A in 2006 but these were reduced in 2008 when the vaccine virus had a site A mutation. A better understanding of immunodominance may allow prediction of future antigenic drift and assist in vaccine strain selection.

Summary

The crystal structure of the yeast SLN1 response regulator domain bound to both a phosphoryl analog (BeF3−) and Mg2+ ion in complex with its downstream phosphorelay signaling partner YPD1 has been determined at a resolution of 1.70 Å. Comparisons between the beryllium fluoride-activated complex and the unliganded (or apo) complex determined previously reveal modest but important differences. The SLN1-R1•Mg2+•BeF3− structure from the complex provides evidence for the first time that the mechanism of phosphorylation-induced activation is highly conserved between bacterial response regulator domains and this example from a eukaryotic organism. Residues in and around the active site undergo slight rearrangements in order to form bonds to the essential divalent cation and fluorine atoms of BeF3−. Two conserved switch-like residues (Thr 1173 and Phe 1192) occupy distinctly different positions in the apo- versus BeF3−-bound structures consistent with the “Y-T” coupling mechanism proposed for activation of CheY and other bacterial response regulators. Several loop regions and the α4-β5-α5 surface of the SLN1-R1 domain undergo subtle conformational changes (∼1-3 Å displacements relative to the apo-structure) that lead to significant changes in terms of contacts that are formed with YPD1. Detailed structural comparisons of protein-protein interactions in the apo- and BeF3−-bound complexes suggest at least a two-state equilibrium model for formation of a transient encounter complex, in which phosphorylation of the response regulator promotes the formation of a phosphotransfer-competent complex. In the BeF3−-activated complex, the position of His 64 from YPD1 is within ideal distance and near linear geometry with Asp 1144 from the SLN1-R1 domain for phosphotransfer to occur. The ground state structure presented here suggests that phosphoryl transfer will likely proceed through an associative mechanism involving formation of a pentacoordinate phosphorus intermediate.

The histidine kinase-based phosphorelay has emerged as a common strategy among bacteria, fungi, protozoa, and plants for triggering important stress responses and interpreting developmental cues in response to environmental as well as chemical, nutritional, and hormone signals. The absence of this type of signaling mechanism in animals makes the so-called “two-component” pathway an attractive target for development of antimicrobial agents. The best-studied eukaryotic example of a two-component pathway is the SLN1 pathway in Saccharomyces cerevisiae, which responds to turgor and other physical properties associated with the fungal cell wall. One of the two phosphoreceiver proteins known as response regulators in this pathway is Skn7, a highly conserved stress-responsive transcription factor with a subset of activities that are dependent on SLN1 pathway phosphorylation and another subset that are independent. Interest in Skn7as a determinant in fungal virulence stems primarily from its well-established role in the oxidative stress response; however, the involvement of Skn7 in maintenance of cell wall integrity may also be relevant. Since the cell wall is crucial for fungal survival, structural and biosynthetic proteins affecting wall composition and signaling pathways that respond to wall stress are likely to play key roles in virulence. Here we review the molecular and phenotypic characteristics of different fungal Skn7 proteins and consider how each of these properties may contribute to fungal virulence.

For both prokaryotic and eukaryotic His-Asp phosphorelay signaling pathways, the rates of protein phosphorylation and dephosphorylation determine the stimulus-to-response time frame. Thus, kinetic studies of phosphoryl group transfer between signaling partners are important for gaining a full understanding of how the system is regulated. In many cases, the phosphotransfer reactions are too fast for rates to be determined by manual experimentation. Rapid quench flow techniques thus provide a powerful method for studying rapid reactions that occur in the millisecond time frame. In this chapter, we describe experimental design and procedures for kinetic characterization of the yeast SLN1-YPD1-SSK1 osmoregulatory phosphorelay system using a rapid quench flow kinetic instrument.

The histidine kinase based signal transduction pathway was first uncovered in bacteria and is a prominent form of regulation in prokaryotes. However, this type of signal transduction is not unique to prokaryotes; over the last decade two-component signal transduction pathways have been identified and characterized in diverse eukaryotes, from unicellular yeasts to multicellular land plants. A number of small but important differences have been noted in the architecture and function of eukaryotic pathways. Because of the powerful genetic approaches and facile molecular analysis associated with the yeast system, the SLN1 osmotic response pathway in S. cerevisiae is particularly useful as a eukaryotic pathway model. This chapter provides an overview of genetic and biochemical methods that have been important in elucidating the stimulus-response events that underlie this pathway and in understanding the details of a eukaryotic His-Asp phosphorelay.

The multi-step His-Asp phosphorelay system in Saccharomyces cerevisiae allows cells to adapt to osmotic, oxidative and other environmental stresses. The pathway consists of a hybrid histidine kinase SLN1, a histidine-containing phosphotransfer (HPt) protein YPD1 and two response regulator proteins, SSK1 and SKN7. Under non-osmotic stress conditions, the SLN1 sensor kinase is active and phosphoryl groups are shuttled through YPD1 to SSK1, therefore maintaining the response regulator protein in a constitutively phosphorylated state. The cellular response to hyperosmotic stress involves rapid efflux of water and changes in intracellular ion and osmolyte concentration. In this study, we examined the individual and combined effects of NaCl and glycerol on phosphotransfer rates within the SLN1-YPD1-SSK1 phosphorelay. The results show that the combined effects of glycerol and NaCl on the phosphotransfer reaction rates are different from the individual effects of glycerol and NaCl. The combinatory effect is likely more representative of the in vivo changes that occur during hyperosmotic stress. In addition, the effect of osmolyte concentration on the half-life of the phosphorylated SSK1 receiver domain in the presence/absence of YPD1 was evaluated. Our findings demonstrate that increasing osmolyte concentrations negatively affects the YPD1•SSK1∼P interaction thereby facilitating dephosphorylation of SSK1 and activating the HOG1 MAP kinase cascade. In contrast, at the highest osmolyte concentrations, reflective of the osmoadaptation phase of the signaling pathway, the kinetics of the phosphorelay favor production of SSK1∼P and inhibition of the HOG1 pathway.

Homoisocitrate dehydrogenase (HIcDH) catalyzes the Mg2+- and K+-dependent oxidative decarboxylation of homoisocitrate to α-ketoadipate using NAD as the oxidant. A recent consideration of the structures of enzymes in the same family as HIcDH, including isopropylmalate and isocitrate dehydrogenases, suggests all of the family members utilize a Lys-Tyr pair to catalyze the acid-base chemistry of the reaction (Aktas, D. F., and Cook, P. F. (2009) Biochemistry submitted.). Multiple sequence alignment indicates the active site Lys-Tyr pair consists of lysine-206 and tyrosine-150. Therefore, the K206M and Y150F mutants of HIcDH were prepared and characterized in order to test the potential roles of these residues as acid-base catalysts. The V/Et values of the K206M and Y150F mutant enzymes at pH 7.5 are decreased by about 2400- and 680-fold, respectively, compared to wild type HIcDH; the Km for HIc does not change significantly. V/Et and V/KMgHIc Et for the K206M mutant enzyme are pH independent below pH 6 and decrease to a constant value above pH 7, while V/KNAD Et is independent over the pH range 6.2 to 9.5. In the case of the Y150F mutant enzyme, V/Et and V/KNAD Et are pH independent above pH 9.5 and decrease to a constant value below pH 8. This behavior can be compared to wild type enzyme, where V/Et decreases at high and low pH giving pKa values of about 6.5 and 9.5. Data were interpreted in terms of a group with a pKa of 6.5 that acts as a general base in the hydride transfer step, and a group with a pKa of 9.5 that acts as a general acid to protonate C3 in the tautomerization reaction (Lin, Y., Volkman, J., Nicholas, K. M., Yamamoto, T., Eguchi, T., Nimmo, S. L., West, A. H., Cook, P. F. (2008) Biochemistry 47, 4169–4180.). Solvent deuterium isotope effects on V and V/KMgHIc were near unity for the K206M mutant enzyme, but about 2.2 for the Y150F mutant enzyme. The dramatic decreases in activity, the measured solvent deuterium isotope effects and changes in the pH dependence of kinetic parameters compared to wild type, are consistent with K206 acting as a general base in the hydride transfer step of the wild type enzyme, but as a general acid in the Y150F mutant enzyme, replacing Y150 in the tautomerization reaction. In addition, Y150 acts as a general acid in the tautomerization reaction of the wild type enzyme, and replaces K206 as the general base in the hydride transfer step of the K206M mutant enzyme.

Nitrite is now recognized as a storage pool of bioactive nitric oxide (NO). Hemoglobin (Hb) and myoglobin (Mb) convert, under certain conditions, nitrite to NO. This newly discovered nitrite reductase activity of Hb and Mb provides an attractive alternative to mammalian NO synthesis from the NO synthase pathway that requires dioxygen. We recently reported the X-ray crystal structure of the nitrite adduct of ferric horse heart Mb, and showed that the nitrite ligand binds in an unprecedented O-binding (nitrito) mode to the d5 ferric center in MbIII(ONO) (D. M. Copeland, A. Soares, A. H. West, G. B. Richter-Addo, J. Inorg. Biochem. 100 (2006) 1413-1425). We also showed that the distal pocket in Mb allows for different conformations of the NO ligand (120° and 144°) in MbIINO depending on the mode of preparation of the compound. In this article, we report the crystal structures of the nitrite and NO adducts of manganese-substituted hh Mb (a d4 system) and of the nitrite adduct of cobalt-substituted hh Mb (a d6 system). We show that the distal His64 residue directs the nitrite ligand towards the rare nitrito O-binding mode in MnIIIMb and CoIIIMb. We also report that the distal pocket residues allow a stabilization of an unprecendented bent MnNO moiety in MnIIMbNO. These crystal structural data, when combined with the data for the aquo, methanol, and azide MnMb derivatives, provide information on the role of distal pocket residues in the observed binding modes of nitrite and NO ligands to wild-type and metal-substituted Mb.

Potassium is an activator of the reaction catalyzed by homoisocitrate (HIc) dehydrogenase (HIcDH) from Saccharomyces cerevisiae with either the natural substrate, homoisocitrate, or the slow substrate isocitrate. On the basis of initial velocity studies, the selectivity of the activator site for monovalent ions was determined. Potassium is the best activator, and NH4+ and Rb+ are also activators of the reaction, while Cs+, Li+, and Na+ are not. Chloride inhibits the reaction, while acetate is much less effective. Substitution of potassium acetate for KCl changes the kinetic mechanism of HIcDH from a steady state random to a fully ordered mechanism with the binding of MgHIc followed by K+ and NAD. The change in mechanism likely reflects an apparent increase in the affinity of enzyme for MgHIc as a result of elimination of the inhibitory effect of Cl−. The V/KNAD pH-rate profile in the absence of K+ exhibits a >10-fold decrease in the affinity of enzyme for NAD upon deprotonation of an enzyme side chain with a pKa of about 5.5–6. On the other hand, the affinity for NAD is relatively constant at high pH in the presence of 200 mM KCl. Since the affinity of the dinucleotide decreases as the enzyme group is protonated and the effect is overcome by a monovalent cation, the enzyme residue may be likely a neutral acid, aspartate or glutamate. Data suggest that K+ replaces the proton, and likely binds to the enzyme residue, the pyrophosphoryl moiety of NAD, or both. Viscosity and solvent deuterium isotope effects studies suggest the isomerization of E-MgHIc binary complex limits the rate in the absence of K+.

Saccharopine dehydrogenase (N6-(glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine forming)) catalyzes the final step in the α-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate α-Kg and lysine using NAD+ as an oxidizing agent. Proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate α-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/KNADH, consistent with a steady state random mechanism, and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect of DV2 and D(V2/KLys), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effect, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/KLys suggesting proton transfer exists in at least two sequential transition states.

The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead-end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated as 11 ± 2 mM as measured using Mg2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg2+ and α-ketoadipate. Curvature is observed in the double reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases the inhibition changes to uncompetitive consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO2, α-ketoadipate and NADH. Isocitrate is a slow substrate with a rate of V/Et 216-fold lower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg2+ bind to enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The Keq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M.

A survey of NADH, α-Kg, and lysine analogues has been undertaken to define the substrate specificity of saccharopine dehydrogenase, and to identify functional groups on all substrates and dinucleotides important for substrate binding. A number of NAD analogues, including NADP, 3-acetylpyridine adenine dinucleotide (3-APAD), 3-pyridinealdehyde adenine dinucleotide (3-PAAD), and thionicotinamide adenine dinucleotide (thio-NAD), can serve as a substrate in the oxidative deamination reaction, as can a number of α-keto analogues, including glyoxylate, pyruvate, α-ketobutyrate, α-ketovalerate, α-ketomalonate, and α-ketoadipate. Inhibition studies using nucleotide analogues suggest that the majority of the binding energy of the dinucleotides comes from the AMP portion, and that distinctly different conformations are generated upon binding of the oxidized and reduced dinucleotides. Addition of the 2'-phosphate as in NADPH causes poor binding of subsequent substrates, but has little effect on coenzyme binding and catalysis. In addition, the 10-fold decrease in affinity of 3-APAD in comparison to NAD suggests that the nicotinamide ring binding pocket is hydrophilic. Extensive inhibition studies using aliphatic and aromatic keto acid analogues have been carried out to gain insight into the keto acid binding pocket. Data suggest that a side chain with 3 carbons (from the α-keto group up to and including the side chain carboxylate) is optimal. In addition, the distance between the C1-C2 unit and the C5 carboxylate of the α-keto acid is also important for binding; the α-oxo group contributes a factor of 10 in affinity. The keto acid binding pocket is relatively large and flexible, can accommodate the bulky aromatic ring of a pyridine dicarboxylic acid, and a negative charge at the C3 but not the C4 position. However, the amino acid binding site is hydrophobic and the optimal length of the hydrophobic portion of amino acid carbon side chain is 3 or 4 carbons. In addition, the amino acid binding pocket can accommodate a branch at the γ-carbon, but not at the β-carbon.

Ypd1p, a histidine-containing phosphotransfer protein, plays an important role in a branched His-Asp phosphorelay signal transduction pathway that regulates cellular responses to hyperosmotic stress in Saccharomyces cerevisiae. Ypd1p is required for phosphoryl group transfer from the membrane-bound Sln1p sensor histidine kinase to two downstream response regulator proteins, Ssk1p and Skn7p. To investigate the molecular basis for interaction of Ypd1p with these response regulator domains, we used an approach that coupled alanine-scanning mutagenesis of surface-exposed residues in Ypd1p with a yeast two-hybrid interaction screen. Mutated residues that adversely affected the interaction of Ypd1p with the C-terminal response regulator domain of Ssk1p were identified and found to cluster on or near the αA helix in Ypd1p. Our results, supported by analysis of a modeled complex, identify a binding site on Ypd1p for response regulators that is composed of a cluster of conserved hydrophobic residues surrounded by less conserved polar residues. We propose that molecular interactions involving Ypd1p are mediated primarily through hydrophobic contacts, whereas binding specificity and strength of interaction may be influenced by select polar side chain interactions.

Two-component regulatory systems that utilize a multistep phosphorelay mechanism often involve a histidine-containing phosphotransfer (HPt) domain. These HPt domains serve an essential role as histidine-phosphorylated protein intermediates during phosphoryl transfer from one response regulator domain to another. In Saccharomyces cerevisiae, the YPD1 protein facilitates phosphoryl transfer from a hybrid sensor kinase, SLN1, to two distinct response regulator proteins, SSK1 and SKN7. Because the phosphorylation state largely determines the functional state of response regulator proteins, we have carried out a comparative study of the phosphorylated lifetimes of the three response regulator domains associated with SLN1, SSK1, and SKN7 (R1, R2, and R3, respectively). The isolated regulatory domains exhibited phosphorylated lifetimes within the range previously observed for other response regulator domains (i.e., several minutes to several hours). However, in the presence of YPD1, we found that the half-life of phosphorylated SSK1-R2 was dramatically extended (almost 200-fold longer than in the absence of YPD1). This stabilization effect was specific for SSK1-R2 and was not observed for SLN1-R1 or SKN7-R3. Our findings suggest a mechanism by which SSK1 is maintained in its phosphorylated state under normal physiological conditions and demonstrate an unprecedented regulatory role for an HPt domain in a phosphorelay signaling system.

Osmoregulation in Saccharomyces cerevisiae involves a multistep phosphorelay system requiring three proteins, SLN1, YPD1, and SSK1, that are related to bacterial two-component signaling proteins, in particular, those involved in regulating sporulation in Bacillus subtilis and anaerobic respiration in Escherichia coli. The SLN1-YPD1-SSK1 phosphorelay regulates a downstream mitogen-activated protein kinase cascade which ultimately controls the concentration of glycerol within the cell under hyperosmotic stress conditions. The C-terminal response regulator domains of SLN1 and SSK1 and full-length YPD1 have been overexpressed and purified from E. coli. A heterologous system consisting of acetyl phosphate, the bacterial chemotaxis response regulator CheY, and YPD1 has been developed as an efficient means of phosphorylating SLN1 and SSK1 in vitro. The homologous regulatory domains of SLN1 and SSK1 exhibit remarkably different phosphorylated half-lives, a finding that provides insight into the distinct roles that these phosphorylation-dependent regulatory domains play in the yeast osmosensory signal transduction pathway.