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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 SLN1 two-component signaling pathway of S. cerevisiae is a branched multistep phosphorelay containing a single hybrid histidine kinase, a histidine-containing phosphotransfer protein, and two downstream response regulators (Fig. 1A). Like the well-studied bacterial two-component pathways, the activity of the SLN1 pathway responds to environmental conditions. Phosphorelay from the Sln1 histidine kinase to the cytoplasmic Ssk1 response regulator is important in dampening the activity of the HOG1 MAP kinase pathway under normal osmotic conditions (Maeda et al., 1994; Posas and Saito, 1997; Reiser et al., 2003)while phosphorelay from the Sln1 histidine kinase to the nuclear Skn7 response regulator appears to play an important role in the cellular response to cell wall perturbations (Shankarnarayan et al., 2008).
Like most eukaryotic histidine kinases, Sln1 is a hybrid protein containing both kinase and receiver domains (Ota and Varshavsky, 1993) (Fig. 1B). In this configuration the first phosphotransfer step is between H576, the phoshorylatable histidine within the kinase domain of Sln1 to D1144, the phosphoaccepting aspartate within the receiver domain of Sln1. The second phosphotransfer step is between Sln1 D1144 and histidine H64 in the phosphotransfer protein, Ypd1. The final phosphotransfer steps in the pathway occur between H64 of Ypd1 and the phosphoaccepting aspartate D554 in the receiver domain of the cytoplasmic response regulator, Ssk1 and between H64 of Ypd1 and aspartate D427 in the receiver domain of the nuclear response regulator, Skn7 (Fassler et al., 1997; Li et al., 1998; Posas et al., 1996)(Fig. 1A).
The kinase activity of Sln1 is regulated in response to the environment. Under normal growth conditions, a modest level of kinase activity appears to be crucial for viability (Fig. 1A). Deletion of SLN1 or mutation in any of the phosphorylatable residues in Sln1, Ypd1 or Ssk1 leads to inviability that is suppressible by inactivating mutations in components of the HOG1 MAP Kinase pathway and by over-expression of HOG1 pathway phosphatases (Maeda et al., 1994; Ota and Varshavsky, 1992; Wurgler-Murphy et al., 1997). This type of evidence led to our current understanding of the SLN1-YPD1-SSK1 pathway as important in negatively regulating the activity of the HOG1 pathway in the absence of stress.
SLN1 pathway activity is diminished in the presence of elevated osmotic conditions (Ostrander and Gorman, 1999)leading to decreased turgor (Reiser et al., 2003)(Fig. 1A, C). These conditions lead to the accumulation of SLN1 pathway components in the dephosphorylated form. Dephosphorylated Ssk1 interacts with and activates the kinase activity of the Ssk2 and Ssk22 MAPKKKs (Maeda et al., 1995; Posas and Saito, 1998)of the HOG1 pathway thus setting in motion the signaling cascade that ultimately results in changes in expression of osmotic response genes including those involved in biosynthesis of the compatible osmolyte, glycerol (Albertyn, Hohmann, Thevelein et al., 1994; Albertyn, Hohmann, and Prior, 1994).
SLN1 pathway activity can also be increased (Fig. 1A, C). This was first shown by the isolation of mutations exhibiting elevated expression of SKN7 target genes. These have been dubbed sln1* activating mutants since they increase the activity of the Sln1 phosphorelay (Fassler et al., 1997; Ault et al., 2002; Tao et al., 2002). Both the Skn7 and the Ssk1 response regulators appear to be more highly phosphorylated in these mutants. The elevated phosphorylation of Skn7 is responsible for the increase in expression of Skn7 dependent target genes (Li et al., 1998; Li et al., 2002) and the elevated phosphorylation of Ssk1 causes defects in the cellular response to hyper-osmotic conditions due to changes in the kinetics of Hog1 phosphorylation (Fassler et al., 1997). In principle, sln1 activating mutations leading to increased phosphorylation of the Ssk1 and Skn7 response regulators could be attributable to a variety of mechanisms, including, for example, increased rate of Sln1 autophosphorylation or phosphotransfer activity, or diminished Sln1 directed phosphatase activity. In practice, sln1 activating mutations map to both the Sln1 receiver domain and to the coiled-coil domain located between the second transmembrane domain and the kinase domain (Fassler et al., 1997; Ault et al., 2002; Tao et al., 2002). The sln1* P1148S activating allele is a Pro to Ser mutation of a helix capping proline that is conserved in the receiver domain of most response regulators and is equivalent to P61 of E. coli CheY (Fassler et al., 1997; Ault et al., 2002). In vitro phosphotransfer analysis of the P1148S mutant revealed a shift in the phosphorelay equilibrium from Sln1 to Ypd1 but no change in the rate of hydrolysis of the aspartyl phosphate on the Sln1 receiver domain. Consistent with the increase in expression of SLN1-SKN7 dependent genes observed in the sln1* P1148 mutant, in vitro phosphorelay assays revealed a two-fold increase in accumulation of phosphorylated Skn7 with the mutant Sln1* receiver domain as the phosphodonor compared to the wild type receiver (Fig. 5B) (Ault et al., 2002).
The sln1* T550I activating mutation located just upstream of H576 led to the identification of a functional coiled-coil (CC) domain in the linker region of Sln1 between the extracellular domain and the kinase domain (Fig. 1B). This region was recognizable as a coiled-coil using the Learn Coil algorithm (Singh et al., 1998) that was developed to detect weak coiled-coils thought to be a general feature of histidine kinases occurring just upstream of the H box. The impact of the sln1 T550I mutation and of the coiled-coil domain was studied using a functional kinase domain construct consisting of aa 537–950. Derivatives of that construct that lack aa 537–570 showed no evidence of autophosphorylation at 40 minutes, a period of time sufficient for autophosphorylation of a coiled-coil containing construct to plateau (Tao et al., 2002). At three hours, the mutant was phosphorylated to about 20% the extent of wild type kinase (Ault, 2001). These observations are consistent with the inviable phenotype of the sln1ΔCC mutant (Tao et al., 2002). The activation phenotype of the sln1 T550I mutant correlates with the increased hydrophobicity afforded by the isoleucine residue at position 550. Additional mutations in the region that increased hydrophobicity at the “a” and “d” positions of the helix were likewise activating while mutations at other positions had no effect (Tao et al., 2002). The absence of an effect of CC domain activating mutations on Sln1 homodimerization led to the hypothesis that the rotational flexibility of the coiled-coil in Sln1 is important for normal Sln1 activity (Tao et al., 2002) (Fig. 1B). Consistent with this hypothesis, the coiled-coil domain of the strongly dimerizing mammalian C/EBP transcription factor rescued viability of the inviable sln1ΔCC mutant (Tao et al., 2002), but like sln1* mutants, this chimeric SLN1 allele caused a salt-sensitive phenotype reflecting a defect in the ability of the kinase to be down-regulated in response to osmotic stress (Tao et al., 2002).
Although the isolation of activating mutations in SLN1 suggest that Sln1 kinase activity can be stimulated, it has been difficult to define the environmental conditions that trigger an increase in Sln1 kinase activity. The study of fps1 mutants led to the hypothesis that Sln1 kinase activity and/or signaling is stimulated by osmotic imbalance (Tao et al., 1999). The FPS1 gene encodes the major glycerol channel responsible for glycerol efflux (Tao et al., 1999) and fps1 mutants accumulate intracellular glycerol even in the absence of osmotic stress (Luyten et al., 1995). Interestingly, although SLN1 pathway activity is elevated in the fps1 mutant, transient osmotic imbalance caused, for example, by shifting an FPS1+ strain from high to low osmotic environments does not activate the SLN1 pathway (Li, 2001; Shankarnarayan, 2007). Efforts to clarify the difference in physiology between transient and prolonged hypo-osmotic stress led to the identification of a cell wall protein, encoded by the CCW12 gene. Strains lacking the CCW12 gene exhibit constitutive activation of the Sln1 kinase as do strains in which the Ccw12 protein is enzymatically removed from the wall (Shankarnarayan et al., 2008). Interestingly, the fps1 mutant is deficient in wall-associated Ccw12 protein suggesting that the activity of the Sln1 kinase is regulated by aspects of the environment that trigger specific wall perturbations (Shankarnarayan et al., 2008).
Increased Sln1 kinase activity causes changes in gene expression via changes in the activity of the Skn7 response regulator. Skn7 is a transcription factor that binds to Skn7 response elements regardless of its phosphorylation state, but stimulates expression of SLN1 dependent genes only in response to phosphorylation of D427 in the Skn7 receiver domain. The non-phosphorylatable skn7D427N mutant fails to respond to elevated Sln1 signaling and the constitutive skn7D427E mutant increases expression of SLN1 dependent genes independent of SLN1 (Li et al., 1998; Li et al., 2002).
The branched architecture of the pathway (Fig. 1A) in which a single histidine kinase signals to the Skn7 as well as the Ssk1 response regulator was suggested by genetic analysis and confirmed by biochemical analysis (Li et al., 1998; Ketela et al., 1998). Sln1 and Ypd1 dependent phosphorylation of the Skn7 response regulator was confirmed by reconstitution of the phosphorelay pathway using purified recombinant proteins consisting of single domains of each protein (Li et al., 1998; Ault et al., 2002). The radiolabeled phosphoryl group on the Sln1 kinase domain could be distributed in turn to the Sln1 receiver domain, the Ypd1 phosphorelay protein and to the receiver domain of Skn7. Furthermore, the distribution of the phosphoryl groups to each of these domains was dependent on the presence of domains participating in earlier steps in the pathway and on the phospho-accepting residues in each domain (Li et al., 1998). In vitro reconstitution of the Sln1 phosphorelay pathway allowed for biochemical characterization of sln1 and ypd1 mutants (Ault et al., 2002; Tao et al., 2002; Janiak-Spens et al., 2005; Janiak-Spens et al., 2000; Janiak-Spens and West, 2000)and for measurements of the phosphorylated lifetime of receiver domains (Janiak-Spens et al., 2000; Janiak-Spens et al., 1999). It also paved the way for structural analysis of pathway components (Xu and West, 1999; Xu et al., 2003; Zhao et al., 2008). In this chapter we describe genetic and biochemical methods for analysis of SLN1 pathway activity in response to relevant mutational and environmental factors.
The screen in which the sln1* and fps1 mutants were isolated involved random mutagenesis of the genome using the chemical mutagen, EMS, to identify mutations that elevate expression of a SLN1 dependent lacZ reporter gene (Tao et al., 1999; Yu et al., 1995). EMS mutagenesis is conducted according to the method of Lawrence et al. (Lawrence, 1991)with minor modifications. Grow a 20 ml culture of the starting strain2 to saturation in dropout media to select for the continued presence of the plasmid carrying the lacZ reporter gene. Wash the cells twice with potassium phosphate buffer (0.0195 M KH2PO4, 0.0305 M K2HPO4, pH 7.0) and resuspend them at 1 × 108 cells/ml in the same buffer. Divide the cells into 5 ml aliquots and add 180 μl EMS (Sigma) to each. Incubate the treated cells at 30°C for various times, stopping each reaction by addition of an equal volume of 10% sodium thiosulfate. Collect the cells by centrifugation, wash them twice with water, and resuspend the cells at a concentration of 1 × 108 cells/ml. Perform serial dilutions and spread various dilutions on selective plates to give a projected yield of 300–400 cells/plate.
Select the EMS treatment time resulting in ~ 65% killing and spread these cells on 150–200 selective plates to generate between 50,000 and 100,000 colonies for screening3. Replica-plate colonies to selective media containing X-Gal4. Approximately 1% of colonies screened on X-Gal plates under these conditions exhibit a blue phenotype on X-Galplates consistent with elevated expression of the lacZ reporter gene (Tao et al., 1999). KEX2 mutations account for more than 50% of the mutants in this type of screen (Tao et al., 1999). Mutations in KEX2 (encoding a serine protease) cause a non-specific increase in the color of reporter bearing strains on X-Gal plates. Eliminate kex2 mutants from further analysis by introducing a KEX2 plasmid into the candidates via mass mating with a karyogamy deficient strain (kar1) of the opposite mating type (Georgieva and Rothstein, 2002). Using the recessive drug resistance alleles present in the starting strain, cytoductants retaining the desired nucleus that have acquired the (LEU2 marked) KEX2 plasmid can be selected5 Select only those candidates whose blue phenotype is not complemented by the KEX2 plasmid. Tests of the remaining mutants to ensure that the phenotype is specific for SLN1 dependent reporter genes are described later in this section.
Because mutations in KEX2 represent such a large fraction of the X-Gal positive mutants isolated by mutagenesis, it is useful to repeat the screen by examining individual mutants of the haploid deletion collection (Research Genetics). Conducting both types of screens is especially important when the deletion is lethal or when the deletion phenotype is not the same as the phenotype of point mutants. For example, deletion of the SLN1 gene is lethal and would not be recovered in the deletion screen, but several different sln1* mutations were identified in the EMS screen. The deletion collection is transformed with a SLN1 responsive lacZ reporter plasmid such as POCH1-lacZ plasmid (pZL1320 (Li et al., 2002)) using an adaptation of the micro-scale protocol of the Frozen-EZ yeast transformation kit (Zymo Research). Inoculate wells of a microtiter dish containing 0.25 ml of YPD medium plus 50 mg/liter G418 using a sterile 96 pronged device. Incubate the microtiter dishes at 30°C for 24–48 hours without aeration. Remove the media using a multichannel pipettor being careful not to disturb the cells that have settled to the bottom of the wells. Wash cells in 0.1 ml EZ1 solution and then pellet the cells using a microtiter plate microfuge at 1300 rpm for 5 minutes. Resuspend the pellet then in 10 μl of EZ2 and 0.1 ml of EZ3 to which the plasmid DNA (5–6 μg/well) has been added. Incubate the dishes at 30°C for 90 minutes and spread cells over ¼ of a selective (SC-Ura) plate using a small sterile glass spreader. Incubate the plates several days at30°C until transformants became visible. An alternative approach in which pools of deletion strains are transformed can also be employed. In this approach, individual deletions are grown in the wells of a microtiter dish as before. However, prior to transformation, all the strains in a single microtiter dish are transferred using a sterile 96-prong device into a large petri dish containing YPD. Use the Frozen-EZ yeast transformation kit (Zymo Research) to prepare competent cells and transform them with a SLN1 responsive lacZ reporter plasmid. Spread the transformation mix on selective media.
Replica plate transformants from the deletion collection obtained using either method to X-Gal plates. Compare the color of transformants on X-Gal plates to the parental strain corresponding to the deletion collection in use. For example, transformants of strain BY4742 (Kelly et al., 2001), the parent strain of the MATα haploid deletion collection, carrying the SLN1 responsive lacZ reporter, are white on plates containing X-Gal (50 mg/l). Transformants that are blue on X-Galplates should be chosen for further analysis. Conduct liquid β-galactosidase assays to confirm the phenotype and to establish the magnitude of the effect.
Quantitative β-galactosidase assays are performed using glass bead-lysed extracts. Grow a small culture in synthetic complete dropout media (e.g. SC-uracil to select for the presence of plasmids with a URA3 marker) to saturation. Make a 1:50 dilution into 5 ml of fresh media and grow to a density of 1–2 × 107 cells/ml. Collect the cells by centrifugation and resuspend the pellets in 0.2 ml breaking buffer (0.1 M Tris pH 8.0, 20% glycerol, 1 mM DTT; stored at −20°C) plus 13 μl of 40 mM PMSF (PMSF is prepared in 95% ethanol and stored at −20°C). Transfer the cell suspension to a 1.5 ml microtube and add 200 mg of glass beads. Store frozen at −80°C for at least 15 minutes6. Thaw cells on ice, vortex briefly to mix and then alternate vortexing (15 seconds) with incubation on ice (15 seconds) between 8 and 15 times until cells are broken7. Pellet cell debris by microfugation on high at 4°C for 15 minutes. Keep extracts on ice while measuring protein concentration using a Bradford protein assay (Biorad). The β-gal assay is conducted as described by Miller ((Miller, 1972)p. 352–359) with minor modifications. Add 900–1000 ml of Z buffer (0.06 M Na2HPO4(7H20), 0.04M NaH2PO4(H20), 0.01M KCl, 0.001M MgSO4(7H20) pH 7.0 with 0.05 M βME added fresh for each use) to reaction tubes and add protein extract to generate one ml reaction mixtures. Incubate these tubes at 28°C. Add 200 μl ONPG (4 mg/ml prepared in Z buffer, stored at 4°C and prepared fresh weekly) to each tube and vortex briefly to begin the reaction, noting the time. When the reaction is just visibly yellow8, add 500 μl of 1M Na2CO3 to each tube to stop the reaction. Note the time at which each reaction is stopped. Read the A420. Calculate the activity using the formula: A420 × 378/time (minutes) × volume (ml) × protein (mg/ml). Activities are reported as the average of 4–6 assay values using at least three independent transformants.
The gene of interest in deletion candidates chosen for their blue phenotype from among transformants of pooled deletion strains is identified by sequencing. Prepare genomic DNA and amplify the unique tag sequence built into each deletion cassette using a forward primer complementary to the Up Tag sequence (5′ CTACAGGTGCTCCAGAGA-3′)(http://chemogenomics.stanford.edu/supplements/04tag/protocols.html) and the Kan 250 R reverse primer complementary to sequences within the KanMX4 cassette (5′-CTGCAGCGAGGAGCCGTAAT-3′). Examine the PCR product by agarose gel electrophoresis and purify it using the QIA quick Gel Extraction Kit (Qiagen). Obtain the sequence using the Kan 200 R primer (5′ CGTGCGGCCATCAAAATG-3′). The deletion tag sequence is located just downstream of the forward primer and upstream of the Kan MX4 sequence and is identified using the tag database (http://www-sequence.stanford.edu/group/yeast_deletion_project/downloads.html)
The specificity of the elevated reporter gene expression is examined by comparing the effect of the strain background on the activity of the SLN1-dependent pZL1320 (POCH1-lacZ) reporter versus the activity of control reporters such as pSL1156 in which lacZ is driven by the basal (−154 to −1) OCH1 promoter which lacks SLN1-SKN7 response elements and pZL1369 (Li et al., 2002)which contains a single mutated Skn7 binding site.
In addition to pathway activation in sln1*, fps1 and ccw12 genetic backgrounds, the SLN1pathway can be activated with the cell wall digesting enzyme zymolyase. Zymolyase is a complex of enzymes isolated from Arthrobacterluteus with strong lytic activity for yeast cell walls (U.S. Biological 105 units/g). The complex consists of zymolyase A, β-1,3-glucan laminaripentaohydrolase and zymolyase B, an alkaline protease thought to be required for the action of zymolyase A by changing the structure of the wall. Zymolyase is routinely used at high concentration to digest the walls from yeast cells. At this enzyme concentration cells must be resuspended in sorbitol to provide osmotic support for the fragile spheroplasts that result from removal of the cell wall. However, the very low zymolyase dose of 0.5–1 unit/ml that is sufficient for activation of the SLN1 pathway causes no loss in optical density or viability of cells. Grow cells to saturation in YPD9 and subculture using a 1:50 dilution into fresh YPD medium using a 1:50 dilution. Incubate cells at 30°C with aeration until they reach early log phase (~107cells/ml). Adjust the pH of all cultures by adding Na2HPO4 to a final concentration of 20 mM (Reiser et al., 2003). Add zymolyase to 0.5 or 1 unit/ml and incubate cultures at 30°C for 30–60 minutes. Harvest cells by centrifugation and store pellets at −80°C until needed.
The effect of zymolyase treatment on SLN1 pathway activity can be examined by Northern analysis. Grow small cultures to saturation and make a 1:50 dilution into 10–50 ml of fresh medium. Incubate at 30°C with aeration until early log phase (~107 cells/ml). Harvest cells by centrifugation and store pellets at −80°C. Isolate total RNA using the acid phenol method (Ausubel et al., 1989)with minor modifications. Resuspend the pellet in 0.4 ml TES (10 mM Tris, pH 7.5, 10 mM EDTA, 0.5% SDS) and add 0.4 ml acid phenol. Vortex 10 seconds. Incubate at 65°C for 1 hour, vortexing 5 seconds every 10 minutes. Move the tubes to ice and add 0.1 ml of TE (10 mM Tris, 1 mM EDTA), pH 7.5. Microfuge at high speed for 5 minutes and transfer the upper phase to a fresh microtube on ice. Add 0.4 ml hot acid phenol and vortex 20 seconds. Microfuge for 5 minutes and transfer the upper phase to new tubes on ice. Add 0.4 ml chloroform and vortex for 20 seconds. Microfuge again for 5 minutes and transfer the upper phase to new tubes. Add 1/10 volume of 3 M NaOAc and 1.0ml of cold ethanol and allow the RNA to precipitate at −80°C overnight. Microfuge for 5 minutes, aspirate, wash the pellet in 75% ethanol, repeat the spin and aspirate the ethanol. Dry the pellet and resuspend the RNA in 100 μl RNase free water by incubating at 65°C for 5 minutes and vortexing. Store at −80°C until needed. Thaw the samples on ice. Ensure complete resuspension of the samples by incubating samples at 65°C for 2 minutes. Use 1–2 μl to measure the absorbance at 260 nm. An OD of 1 is equivalent to 40 μg/ml RNA. 10 mls of cells at 1 × 107/ml yields ~ 250 μg of RNA. Check the quality of the RNA by running a sample on a standard (1 ×TAE) 1% agarose gel. Sharp ribosomal RNA bands of 1.8 kb and 2.5 kb and a more diffuse tRNA band at 0.2 kb should be visible. Samples with reduced amounts of one rRNA species relative to the other, general smeariness or an accumulation of smaller molecular weight species should not be used further. Mix 10–20 μg of each total RNA sample with 1.3 × RNA loading buffer (1× MOPS (0.2 M MOPS, 0.5 M sodium acetate, 0.01 M EDTA), 20% formaldehyde, 50% deionized formamide, 0.04 mg/ml ethidium bromide plus xylene cyanol and bromphenol blue loading dyes), heat samples at 65°C for 10 minutes, move to ice and load a 10% (v/v) formaldehyde 1% agarose gel prepared in 1× MOPS buffer. Run the gel at 75V for several hours. Photograph the gel, wash it twice 5 minutes each in water and then set up a capillary blot (Ausubel et al., 1989)to transfer the RNA to a nytran filter. UV crosslink the RNA to the blot by exposing the blot (RNA side up) to a germicidal lamp10. Prepare a 32P labeled probe using random primers and the Prime-It random priming labeling kit (Stratagene) with appropriate PCR fragments as templates11. Store the probe at −20°C. Prehybridize the blot in a small rotisserie bottle (Bellco Glass Inc.) using 4–6 ml of Perfect Hyb (Sigma) hybridization buffer rotating for at least one hour at 68°C. Incubate the probe at 100°C for 10 minutes, and then chill it on ice. Add the probe (~ 10 μl) to the rotisserie bottle and hybridize overnight at 68°C. Wash the blots twice, 5 minutes each, in 30 ml of 2× SSC -0.1% SDS12. Wash the blots twice more in 0.5× SSC -0.1% SDS at 68°C. Remove the blots from the rotisserie bottle, wrap it (RNA side down) in plastic film and place it in a phosphorimager cassette (or X-ray film) for 6–30 hours. Visualize radiolabeled bands with a phosphorimager. Analyze bands using Quantity One software (Biorad). Strip the probe from the blot by incubating the blotin 0.2× SSC -1% SDS at 75°C for 1 hour followed by a rinse in 10mM Tris (pH 7.5). Repeat prehybridization and hybridization procedures using a probe suitable for normalization13.
SLN1 pathway activation leads tocurtailment of HOG1 pathway signaling in response to salt (Fassler et al., 1997). Hence, another assay for SLN1 pathway activation is to examine the effect of osmotic shock on Hog1 phosphorylation over time. To monitor Hog1 phosphorylation, grow cultures selectively overnight, dilute 1:50 into YPD and aerate at 30°C to a cell density of 1 × 107/ml. Use a 4M solution of NaCl prepared in YPD to adjust cultures to a final concentration of 0.4M NaCl. Take ten ml aliquots at 0, 2, 4 and 10 minutes after addition of NaCl. Harvest cells by filtration through a Millipore 0.45 μ HA filter (using full house vacuum). Quickly place filters into a microtube and rapidly freeze in a dry-ice ethanol bath. Store samples at −80°C until preparation of the extract. Remove cells from filter into 1 ml of water and pellet the cells by microfugation. Remove the filter. To the pellet, add 100 μl 16% TCA and ~ 200 mg of glass beads. Vortex approximately 4–5 minutes. Add 400 μl of ice cold 5% TCA and vortex to mix. Transfer liquid to a fresh microtube and microfuge at high speed in the cold for 10 minutes. Aspirate the supernatant and resuspend the pellet in 50–100 μl loading buffer (150 mM Tris Base, 10 mM EDTA, 3% SDS, 5% βME, 15% glycerol, 0.01 mg/ml bromphenol blue). Boil samples for 5 minutes, microfuge samples at high speed for 5 minutes and load (~15 μl) onto two identical 10% SDS PAGE gels (29:1 acrylamide:bisacrylamide, 10% running gel, 6% stacking gel). Transfer proteins to nitrocellulose membranes (0.45 micron; Schleicher & Schuell). Stain with Ponceau S staining solution (0.1% (w/v) Ponceau S in 5% (v/v) acetic acid), wash briefly in distilled water and scan or photograph for a record of relative loading of each lane and the quality of the protein extracts. Block the blot with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) and 4% BSA. Wash twice for 10 minutes in TBST and incubate for 2 hours at 4°C with RC20 recombinant anti-phosphotyrosine antibody conjugated to horseradish peroxidase (Transduction Laboratories) diluted 1:2500 in TBST + 4% BSA. Wash twice with TBST. For Hog1 protein analysis, block the membrane in TBST with 5% skim milk for one hour. Incubate with yC-20 anti-Hog1 antibody (Santa Cruz Biotechnology) diluted 1:2000 in TBST + 5% BSA overnight at4°C. Wash twice in TBST and then incubate with peroxidase conjugated bovine anti-goat IgG (Santacruz Biotech sc2378) diluted 1:1000 in TBST. Wash the blot three times with 10 ml of TBST for 5 minutes each. Treat both blots with Super Signal West Pico chemiluminescent substrate (Pierce) and exposed to film to visualize the proteins.
The Sln1 histidine kinase (HK) domain (aa 537–950) is expressed as a glutathione-S-transferase fusion protein in DH5α cells and is routinely used as the initial phosphodonor to downstream phosphorelay components. This protocol is a slightly modified version of the published procedure (Li et al., 1998; Ault et al., 2002). Grow 1 liter of cells in LB medium containing 100 μg/ml ampicillin at 37°C until late log phase (an optical density at 600 nm of ~0.9). Add IPTG to a final concentration of 0.2 mM and grow cells at a lower temperature (16°C) for an additional 24 hours Pellet cells by centrifuging at 4500 × g for 10 minutes and resuspend in 4–5 ml/g (wet weight of cell pellet) in lysis buffer [50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% βME, 1 mM PMSF] supplemented with a 1X protease inhibitor cocktail14 (0.1 μg/ml chymostatin, 2 μg/ml aprotinin, 1 μg/ml pepstatin, 1.1 μg/ml phosphoramidon, 7.2 μg/mL E-64 protease inhibitor, 0.5 μg/ml leupeptin, 2.5 μg/ml antipain, 100 μM benzamidine, 100 μM sodium metabisulfite). If desired, the cell paste can be stored frozen at −20°C. Lyse (thawed) cells by sonication15 and clarify the cell lysate by centrifugation at 27,200 × g × 30 minutes Apply the supernatant to a 2 ml glutathione-Sepharaose 4B (GE Healthcare) column16 that has been pre-equilibrated in lysis buffer. Wash sequentially with 10 column volumes of lysis buffer, then 5 column volumes of wash buffer [50 mM Tris-HCl pH 8.0, 2 mM DTT, 1 mM EDTA], and finally with 5 column volumes of storage buffer17 (50 mM Tris-HCl pH 8.0, 100 mM KCl, 2 mM DTT, 1 mM EDTA, 10% glycerol). Leave about 2 ml of storage buffer above the resin bed and gently mix the slurry. Bead-bound Sln1-HK protein can be stored in aliquots at −20°C and is fully active for subsequent autophosphorylation and phosphotransfer experiments. By retaining GST-Sln1-HK on the glutathione-Sepharose beads, one can conveniently remove this phosphodonor from phosphorecipient proteins by low speed centrifugation18.
Another plasmid was constructed that expresses the Sln1 HK domain as well as the C-terminal receiver domain, Sln1-HK-Rec (aa 537–1220) (Ault, 2001; Janiak-Spens et al., 2005). Purification is the same as described above for the Sln1-HK domain and is useful in preparing phosphorylated Ypd1 (see section B3. c). [Note: the GST-SLN1-HK-Rec domain protein is susceptible to proteolytic degradation, thus it is important to include protease inhibitor cocktails for all purification and storage conditions.]
Full length Ssk1 and Skn7 are expressed as GST fusions in pGEX vectors (Li et al., 1998; Ault, 2001) in DH5α cells. Grow one liter of culture to an OD600 between 0.6–1.0 in LB medium containing 100 μg/ml ampicillin. Add 0.2 mM IPTG19. Optimal induction of GST-Ssk1 protein occurs after two hours at 37°C and optimal induction of GST-Skn7 protein occurs after 20 hours at 16°C. The low temperature incubation reduces the concentration of inclusion bodies and improves the yield of active protein20. Harvest cells by centrifugation and resuspend the pellet in 5 ml lysis buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 0.1% βME, 1X protease inhibitor cocktail (Sigma) ) and store frozen21. Thaw the cell pellets and add 1 mM PMSF. Lyse cells using a French press or by sonication22. Centrifuge for 10 minutes at 7000 × g and to the supernatant add 3 mls of a 50% slurry of glutathione agarose beads. Incubate for one hour at 4°C. Wash the beads four times in lysis buffer and four times in storage buffer (50 mM Tris pH 7.6, 50 mM KCl, 5 mM MgCl2, 0.1% βME, 50% glycerol). Store bead-bound proteins at −20°C. If desired, proteins can be eluted from the beads by incubation in elution buffer (100 mM glutathione, 50 mM Tris pH7.6, 50 mM KCl, 5 mM MgCl2, 0.1% βME) with shaking at room temperature for one hour23. Remove beads from the eluted protein by brief centrifugation in a microfiltration unit. Protein concentration can be determined based on analysis of band intensities on Coomassie stained SDS-polyacrylamide gels compared to stained gels with BSA concentration standards using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image/).
The C-terminal response regulator domains from Sln1, Ssk1, and Skn7 can be expressed as cleavable tripartite fusion proteins consisting of an N-terminal receiver (Rec) domain, followed by the yeast VMA1 protein-splicing intein domain and a chitin binding domain (CBD) at the C-terminus. The CBD facilitates affinity purification, while the self-splicing activity of the intein domain can be induced at high DTT or βME concentrations to cleave the polypeptide at the junction between the receiver and intein domains. All protein purification steps described below should be carried out at 4°C.
The Sln1 receiver domain (aa 1084–1220) is expressed from an inducible tac promoter (pCYB2 vector, IMPACT system, New England Biolabs)(Janiak-Spens et al., 1999) in DH5α cells. Grow 1 liter of cells in LB medium containing 100 μg/mL ampicillin at 37°C until an optical density at 600 nm reaches 0.6. Add IPTG to a final concentration of 0.4 mM and grow cells an additional 8 hours at room temperature. Harvest cells by centrifugation at 4500 × g for 10 minutes and resuspend the cell pellet in wash buffer (0.1 M sodium phosphate, pH 7.0, 1 mM EDTA). Centrifuge again and resuspend the cells in lysis buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% Triton X-100) at ~5 ml/g wet weight of the cell pellet. Lyse cells by sonication and clarify the cell lysate by centrifugation at 27,200 × g × 30 minutes Load the supernatant onto a 3 ml chitin bead column (New England Biolabs) equilibrated in lysis buffer at 4°C using a peristaltic pump at a flow rate of about 0.5 ml/minutes Wash with 20 column volumes of lysis buffer at a flow rate of about 1 ml/minutes Then wash sequentially with 4–5 column volumes each of cleavage buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.1 mMEDTA), cleavage buffer plus 5 mM ATP, 5 mM MgCl2, then cleavage buffer plus 30 mM βME. Turn off column flow and incubate overnight at 4°C to allow thiol-induced cleavage of the fusion protein. Elute with 3–4 column volumes of cleavage buffer and identify Sln1-Rec containing fractions by 15 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Pool fractions containing Sln1-Rec domain and apply to a Sephadex G50 (Sigma) gel filtration column (300 mL bed volume) equilibrated in 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT. Protein concentration can be determined by absorbance at 280 nm (ε = 4,020 M−1 cm−1). The purified protein can be stored frozen (−80°C) in small aliquots containing 10% glycerol. Typical yields are 0.5 – 1.0 mg pure protein per liter cell culture.
Expression of the Ssk1 receiver domain (aa 495–712) from the pCYB2 vector in DH5α cells was low. Thus, we excised the Ssk1-Rec-intein-CBD fusion gene from the pCYB2 vector and sub-cloned the fragment into pET11a (Novagen) to give an inducible T7-based vector as described (Janiak-Spens et al., 1999). The expression plasmid was transformed into BL21(DE3) cells. Grow 1 liter of cells in LB medium containing 100 μg/ml ampicillin at 37°C until an optical density at 600 nm of about 0.7. Cool the culture to 16°C, then add IPTG to a final concentration of 1 mM and grow cells an additional 16 hours at 16°C. Harvest cells by centrifuging at 4500 × g for 10 minutes and resuspend the cell pellet in wash buffer. Centrifuge again and resuspend the cells in lysis buffer containing 10% glycerol at ~6 ml/g wet weight of the cell pellet. A pinch of RNase (Sigma) and DNase (Sigma) is added, then lyse cells by French press operated at a cell pressure of 14,000 lb/in2. Clarify the cell lysate by centrifugation at 27,200 × g × 30 minutes. Load the supernatant onto a 3 ml chitin bead column (New England Biolabs) equilibrated in lysis buffer containing 10% glycerol. Chitin affinity column chromatography is carried out as described above for the Sln1-Rec domain except that 10% glycerol is included in the cleavage buffer and Sephadex G75 resin (Sigma; 300 ml bed volume) equilibrated in 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM βME is used for the gel filtration chromatography step. Protein concentration can be determined by absorbance at 280 nm (ε = 25,440 M−1 cm−1). The purified protein can be stored frozen (−20°C) in small aliquots containing 10% glycerol. Typical yields are 1.0 – 1.5 mg pure protein per liter cell culture.
The vector expressing the Skn7 receiver domain (aa 361–622) was prepared as described above for the Ssk1-Rec domain (Janiak-Spens and West, 2000)and transformed into BL21(DE3) cells. Purification of the Skn7-Rec domain is similar to the Sln1-Rec domain as described above except an S-200 gel filtration column (300 mL bed volume; GE Healthcare) is used, equilibrated in 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM βME. Protein concentration can be determined by absorbance at 280 nm (ε = 6580 M−1 cm−1). The purified protein can be stored frozen (−20°C) in small aliquots containing 10% glycerol. Typical yields are 1–3 mg pure protein per liter cell culture.
The full length Ypd1 protein (aa1–167) can be constitutively expressed from a pUC12-derived vector in DH5α cells (Xu et al., 1999). Grow a 1 liter culture to saturation overnight at 37°C in LB medium supplemented with 50 μg/ml ampicillin. Harvest cells by centrifuging at 4500 × g for 10 minutes and resuspend the cell pellet in lysis buffer (0.1 M sodium phosphate pH 7.0, 1 mM EDTA, 1.4 mM βME). Centrifuge again and resuspend the cells in lysis buffer at ~5 ml/gram wet weight of the cell pellet. Lyse cells by sonication and clarify the cell lysate by centrifugation at 27,200 × g × 30 minutes Measure the volume of the supernatant. In a small glass beaker at 4°C on a stir plate, while gently stirring, slowly add saturated ammonium sulfate to a final concentration of 55%. Collect the protein precipitate by centrifuging at 12,000 × g × 10 minutes Resuspend the protein pellet in 6–7 ml of dialysis buffer (20 mM BisTris pH 6.5, 1.4 mM βME) and dialyze versus 2 liters of the same buffer for 24 hr. with one change of buffer to remove the ammonium sulfate. Recover the protein solution from the dialysis bag and pellet any precipitated material. Filter the supernatant through a 0.2 μM syringe filter and apply to a 5 ml HiTrap Q Sepharose Fast Flow (Pharmacia) anion-exchange column pre-equilibrated in 50 mM BisTris pH 6.5. Elute bound protein from the column with a linear salt gradient of 0–500 mM NaCl in 50 mM BisTris pH 6.5. Identify Ypd1-containing fractions by running aliquots (12–15 μl) from fractions on 15% SDS-polyacrylamide gels. Pool fractions containing Ypd1, concentrate using Centricon 10 filter units (Amicon) to a volume ≤10 ml. Apply sample to a Sephadex G50 (Sigma) gel filtration column (350 ml bed volume) equilibrated in 50 mM sodium phosphate pH 7.0, 1 mM EDTA, 1.4 mM βME. Identify fractions containing pure Ypd1 protein by SDS-PAGE and combine. Protein concentration can be determined by absorbance at 280 nm (ε = 15,280 M−1 cm−1). Ypd1 can be stored frozen (−20°C or −80°C) in small aliquots containing 15% glycerol. Yields can be as high as 40 mg pure protein per liter cell culture.
Note: When we embarked on reconstituting the phosphorelay system in vitro, it was desirable to express and purify these domains without His-or other affinity tags that potentially could interfere with phosphotransfer assays. We have since discovered that most of these proteins can in fact be expressed as GST or His-tagged proteins with little or no loss in phosphotransfer activity. The Sln1-Rec domain and full-length Skn7 and YPD1 can also be expressed and purified as GST-fusion proteins using the procedure described in Section 3.1 a (Li et al., 1998; Ault et al., 2002). Full length Skn7, Ssk1-Rec, and Skn7-Rec domains can be also be expressed and purified as His-tagged proteins (Janiak-Spens, F., Sparling, D., Zhao, X., and West, A.H., unpublished) (Dean, 2004; Mulford, 2009).
The phosphotransfer reactions that comprise the full phosphorelay (Sln1-HK to Sln1-Rec to Ypd1 to Ssk1-Rec or Skn7-Rec) occur very rapidly, within the earliest 8 second time point that can be taken manually at the bench top (Janiak-Spens and West, 2000)(Fig. 3B). Thus, a rapid quench flow instrument is required to carry out initial velocity studies and to measure rate constants for the individual steps (Janiak-Spens et al., 2005). This approach is the subject of a separate chapter in this volume (Kaserer, A., Andi, B., Cook, P.F. and West, A.H. “Kinetic measurements for studying phosphorelay signaling pathways”). Here, we provide methods and strategies for obtaining phosphorylated proteins and for in vitro phosphotransfer assays, in which distribution of radiolabeled phosphoryl groups can be monitored over a time course of 5–60 minutes These procedures have been particularly useful for characterizing mutant proteins (Li et al., 1998; Ault et al., 2002; Tao et al., 2002; Ault, 2001; Janiak-Spens et al., 2005; Janiak-Spens and West, 2000)and for examining half-lives of phosphorylated receiver domains (Janiak-Spens et al., 2000; Janiak-Spens etal., 1999).
Incubate approximately 3 μM of bead-bound protein with 7 μM [γ-32P]-ATP (30 Ci/mmol) in 50 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM MgCl2, 2 mM DTT, 20% glycerol for 30–60 minutes at room temperature in a total reaction volume of 100 μl. Gently pellet the resin-bound GST-Sln1-HK~P (100 × g for 1 minutes), wash three times with 100 μl wash buffer (50 mM Tris-HCl pH 8.0, 50 mM KCl, 10 mM MgCl2, 1 mM DTT) to remove unincorporated radiolabel (Fig. 2A). Resuspend final pellet in 100 μl wash buffer. A typical autophosphorylation time course is shown in Fig. 2B. The phosphorylated kinase is stable for 8 hours at room temperature. This preparation can be used immediately for phosphotransfer assays (see for example Fig. 2C) or stored frozen in aliquots at −20°C for about a week. For some applications (e.g. mutant studies of the HK domain itself), it may be useful to elute the GST-Sln1-HK protein from the resin or remove the GST tag using thrombin (Fig. 2A).
To examine levels of autophosphorylation or to evaluate phosphotransfer from component to component in the pathway as described in subsequent sections, radiolabled reaction products are typically separated on 10–15% SDS-polyacrylamide gels24. The wet gel should be wrapped in plastic wrap and exposed to a phosphorimager screen for quantification of radiolabeled band intensity.
Phosphorylation of Sln1-Rec can be achieved by direct phosphotransfer from Sln1-HK~P (Fig. 3A). Incubate 7 μM GST-Sln1-HK bound to glutathione-Sepharose beads (as described in B. 2a) with 7 μM [γ-32P]-ATP (30 Ci/mmol), 60 μM unlabeled ATP25, and 30 μM Sln1-Rec in 50 mM Tris-HCl pH 8.0, 100 mM KCl, 15 mM MgCl2, 2 mM DTT, and 20% glycerol for 30 minutes at room temperature in a total volume of 100 μl. Centrifuge the mixture (1 minute at 100 × g) topellet the resin-bound GST-Sln1-HK and recover P~Sln1-Rec in supernatant. This preparation should be used immediately for half-life studies (see Section B. 3e) or, for example, in vitro phosphotransfer reactions involving Ypd1 (Fig. 3B).
Incubate GST-Sln1-HK-Rec (1 μM) bound to glutathione-Sepharose beads (as described in B. 1a) with 7 μM [γ-32P]-ATP (30 Ci/mmol), 100 μM unlabeled ATP, and 40 μM YPD1 in 50 mM Tris-HCl pH 8.0, 100 mM KCl, 15 mM MgCl2, 2 mM DTT, and 20% glycerol for 90 minutes at room temperature in a total volume of 100 μl (Fig. 4). Centrifuge the mixture (1 minutes at 100 × g) to pellet the resin-bound GST-Sln1-HK-Rec and recover P~Ypd1 in the supernatant. This preparation can then be used immediately for half-life studies or phosphotransfer experiments involving receiver domains (see Ypd1 mutant study, for example, in Fig. 4B).
The most efficient manner of phosphorylating the Ssk1 and Skn7 receiver domains is through the full phosphorelay (Sln1-HK to Sln1-R1 to Ypd1). However, one can phosphorylate Ssk1-Rec and Skn7-Rec directly using bead-bound Sln1-HK~P as a phosphodonor (as described for Sln1-Rec), but a longer incubation time is necessary (up to 90 minutes) (Fig. 3). This route reduces the number of phosphotransfer and intermediary purification steps. The phosphorylated receiver domain(s) should be used immediately due to its short half-life (Janiak-Spens et al., 2000).
Response regulator proteins are activated and deactivated by phosphorylation, and the lifetime of the phosphorylated state often correlates with the duration of the output response. Thus, it is important to measure the half-life of the phosphorylated protein domains and to keep the half-life in mind when setting up in vitro time-dependent phosphotransfer experiments. One can obtain phosphorylated Sln1, Ssk1 or Skn7 receiver domains by incubation with 32P-labeled GST-Sln1-HK as described in Sections B. 3b and B. 3d (Fig. 3A). The phosphodonor should be removed by centrifugation and the isolated phosphorylated receiver domain is then recovered in the supernatant. Incubate approximately 5 μM of P~receiver in 50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1 mM DTT in a total volume of 50 μl at room temperature. Remove aliquots (4.5 μl) at various time points and mix with 4X stop buffer (0.25 M Tris-HCl pH 6.8, 8% SDS, 60 mM EDTA, 40% glycerol, 0.0008% bromophenol blue) to terminate the reaction. Freeze the aliquots at −20°C until SDS-PAGE analysis. Determine the relative amounts of phosphorylated protein remaining at each time point by phosphorimager quantitation. Plot ln(% P~receiver remaining) vs. time. The rate constant (k) for the dephosphorylation reaction is determined by least-squares fitting of the natural logarithm of the data to a linear relationship assuming first-order kinetics. The half-life of the phosphorylated receiver domain26 is calculated accordingly (t1/2 = ln2/k). Phosphorylated Ypd1 can be obtained as described in Section B.3.c and the rate constant for dephosphorylation27 is measured in the same manner as for receiver domains.
To monitor Sln1-Rec to Ypd1 phosphotransfer (see Fig. 3B for example), incubate P~Sln1-Rec with Ypd1 (approximately equimolar amounts) in a total volume of 15 μl in reaction buffer (50 mM Tris pH 8.0, 30 mM KCl, 10 mM MgCl2, 1 mM DTT) for 5 minutes at room temperature. Stop the reaction by adding 5 μl of 4X stop buffer (0.25 M Tris-HCl pH 6.8, 8% SDS, 60 mM EDTA, 40% glycerol, 0.008% bromophenol blue). For time-course experiments, the reactions are prepared similarly except that the initial volume is 100 μl and aliquots (9μl) can be removed at various time points, mixed with 4X stop buffer and keep on ice until gel analysis.
To assay phosphotransfer from P~Ypd1 to Ssk1-Rec, Skn7-Rec (or the reverse phosphotransfer to Sln1-Rec), follow the same procedure as above, except P~Ypd1 (1–2 μM) is typically incubated with slight molar excess of receiver domain (see for example Fig. 4B).
Phosphorelay experiments involving all protein components (Fig. 5A) in the reaction mix are typically carried out by incubating equimolar amounts (~2 μM) of either P~GST-Sln1-HK or P~Sln1-Rec as the initial phosphodonor, and Ypd1 and Ssk1-Rec (or Skn7-Rec) for 5 min in reaction buffer at room temperature. Representative phosphorelay experiments are shown in Fig. 5B and 5C.
We gratefully acknowledge funding from the NIH (GM59311 to AHW, GM068746 to Robert Deschenes and JSF, and GM56719 to JSF), the Oklahoma Center for the Advancement of Science and Technology (AHW), and the Center for Biocatalysis and Bioprocessing at the University of Iowa (JSF)for the research described here.
1Abbreviations used in this chapter include βME, β-mercaptoethanol; BSA, bovine serum albumin; CBD, chitin binding domain; DTT, dithiothreitol; EDTA, ethylenediaminetetracetic acid; EMS, ethyl methane sulfonate; GST, glutathione-S-transferase; HK, histidine kinase; IPTG, isopropyl-β-D-thiogalactopyranoside; MOPS, 3-morpholinopropanesulfonic acid, ONPG, ortho-nitrophenyl-β-galactoside; PMSF, phenylmethanesulfonyl fluoride; Rec, receiver domain of a response regulator; SC, synthetic complete; SC-aa (synthetic complete media lacking specific amino acids); SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCA, trichloroacetic acid; X-Gal, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.
2JF1567, the strain used in Tao et al. (Tao et al., 1999)was transformed with a plasmid bearing a SLN1 responsive lacZ reporter gene. JF1567 has recessive canavanine and cycloheximide resistance mutations (canR and cyhR) useful in the later screening steps of this protocol.
3A final plating density of between 300 and 400 colonies per plate is best for subsequent screening on X-Gal plates.
4X-Gal plates are prepared by addition of 1 × M9 salts (Miller, 1972)(Miller, 1972)and 50 mg/ml X-Gal dissolved in N, N-dimethylformamide to standard synthetic complete (SC or SC -aa) media. X-Gal is added to media cooled to 65°C immediately prior to pouring. The X-Gal concentration can be increased or decreased but should be titrated such that the starting strain is white or very pale blue.
5Selection is on SC media plates lacking the amino acid encoded by the KEX2 plasmid and containing the appropriate drugs.
6Incubation for 15 minutes at −80°C facilitates breakage of the cells.
7The exact number of vortexing cycles will depend on the vortex and the strain. Cell breakage can be monitored by phase contrast microscopy. Ideally, cell breakage should exceed 80%.
8The yellow color should be monitored closely and the reaction stopped promptly so that the A420 reading remains within the linear range of the spectrophotometer. Reaction times should not be less than 5 minutes, as these times will tend to be inaccurate. If necessary, repeat the reaction with less extract.
9Where plasmid retention is an issue, grow cultures in selective medium overnight and then subculture (1:50) into YPD for two generations prior to harvesting at log phase.
10The appropriate time of exposure for optimal retention of RNA on the blot will depend on the age of the bulb and the distance from the bulb to the blot and should be determined empirically.
11Expression of the SLN1 responsive NCA3 gene is frequently used to monitor SLN1-SKN7 pathway expression because it is more abundant than OCH1. The NCA3 probe is 389 bp PCR product generated using NCA3 9F (5′-TTCCGCAGCTTTAATATTGTCTTCC-3′) and NCA3 398R (5′-TCACATGCATAAGAACAGTAGTAGCC-3′).
12SSC is diluted from a 20× stock (3M NaCl, 0.3 M sodium citrate pH 7).
13Normalization in zymolyase treatment experiments is performed using a probe corresponding to CDC33. The CDC33 probe is a 636 bp PCR product generated using CDC33 3F (5′-GTCCGTTGAAGAAGTTAGCAAG-3′) and CDC33 639R (5′-CAAGGTGATTGATGGTTGAGGG-3′).
14Any commercial PIC cocktail appropriate for bacterial cells can be substituted here.
15Lysis can be achieved using a French press in place of sonication.
16Alternatively, batch purification method can work as well using a 50% slurry as described for full-length Skn7 (Section B.1b)
17GST-Sln1-HK can also be stored in reaction buffer (50 mM Tris-HCl, pH 8.0, 100 mM KCl, 10 mM MgCl2, 2 mM DTT with the addition of 10–50% glycerol).
18For experiments in which the presence of GST might complicate analysis, use thrombin to cleave the protein from the GST moiety. Wash the bead bound protein four times in thrombin cleavage buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2), resuspend in the same buffer and add 5 units of biotinylated thrombin. Incubate at 25°C for times from 1–6 hours depending on the protein. Add 25 μl of streptavidin beads during the final 15 minutes of the reaction. Recover the protein by transferring the reactions to microfuge filtration units and centrifuging briefly.
19This relatively low concentration of IPTG is important in minimizing the number of truncation products in GST-Skn7 cultures. Since D427 is at the C terminus of the protein and the GST tag at the N terminus, truncation products are inactive.
20Induction of GST-Skn7 and GST-Ssk1 at 37°C results in a low yield of full-length protein with many truncation products. GST-Skn7 that is induced at 37°C is inactive in phosphotransfer assays. Performing the induction at 16°C does not improve the yield of full-length protein but does improve the activity of the protein in the phosphotransfer assay (Ault et al., 2002)
21Freezer storage can be at −20°C or at −80°C.
22Add 1.25 mg lysozyme, 50 units of DNase (Boehringer Mannheim) and 10 μg of RNAse to lysis buffer prior to sonication. Set tubes in an ice water bath and do multiple rounds of sonication for 30 seconds each at 60 seconds intervals
23A higher concentration of Skn7 is achieved by replacing batch-wise elution of GST-Skn7 with elution of the protein in a chromatography column using 50 mM glutathione. For a column consisting of three ml bead bound GST-Skn7, discard one ml of void volume and collect 0.5 ml aliquots for analysis by SDS-PAGE. The GST-Skn7 protein is concentrated in aliquots 3 and 4 (Ault et al., 2002).
24Do not boil samples prior to gel loading as this increases the rate of phosphate hydrolysis.
25Addition of unlabeled ATP is included to drive stoichiometric labeling of Sln1-Rec.
26The half-lives of P~Sln1-Rec, P~Ssk1-Rec and P~Skn7-Rec are 13 ± 2 minutes, 13 ± 3 minutes, and 144 ± 6 minutes, respectively (Janiak-Spens et al., 2000). However, in the presence of Ypd1, the lifetime of P~Ssk1-Rec is dramatically extended to 2280 ±240 minutes presumably through complex formation that shields the phospho-aspartate from hydrolysis. This stabilization effect is specific to theSsk1-Rec domain and is not observed for Sln1-Rec or Skn7-Rec.
27The half-life for P~Ypd1 is 4.1 ± 0.8 hr (Janiak-Spens and West, 2000).
Jan S. Fassler, Department of Biology, University of Iowa, Iowa City, IA 52242.
Ann H. West, Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Normal, OK 73109.