Sml1 is a small protein in Saccharomyces cerevisiae which inhibits the activity of ribonucleotide reductase (RNR). RNR catalyzes the rate-limiting step of de novo dNTP synthesis. Sml1 is a downstream effector of the Mec1/Rad53 cell cycle checkpoint pathway. The phosphorylation by Dun1 kinase during S phase or in response to DNA damage leads to diminished levels of Sml1. Removal of Sml1 increases the population of active RNR, which raises cellular dNTP levels. In this study using mass spectrometry and site-directed mutagenesis, we have identified the region of Sml1 phosphorylation to be between residues 52 and 64 containing the sequence GSSASASASSLEM. This is the first identification of a phosphorylation sequence of a Dun1 biological substrate. This sequence is quite different from the consensus Dun1 phosphorylation sequence reported previously from peptide library studies. The specific phosphoserines were identified to be Ser56, Ser58, and Ser60 by chemical modification of these residues to S-ethylcysteines followed by collision activated dissociation. To investigate further Sml1 phosphorylation, we constructed the single mutants S56A, S58A, S60A, and the triple mutant S56A/S58A/S60A and compared their degrees of phosphorylation with that of wild type Sml1. We observed a 90% decrease in the relative phosphorylation of S60A compared with that of wild type, a 25% decrease in S58A, and little or no decrease in the S56A mutant. There was no observed phosphate incorporation in the triple mutant, suggesting that Ser56, Ser58, and Ser60 in Sml1 are the sites of phosphorylation. Further mutagenesis studies reveal that Dun1 kinase requires an acidic residue at the +3 position, and there is cooperativity between the phosphorylation sites. These results show that Dun1 has a unique phosphorylation motif. Sml1 is a small protein in Saccharomyces cerevisiae which inhibits the activity of ribonucleotide reductase (RNR). RNR catalyzes the rate-limiting step of de novo dNTP synthesis. Sml1 is a downstream effector of the Mec1/Rad53 cell cycle checkpoint pathway. The phosphorylation by Dun1 kinase during S phase or in response to DNA damage leads to diminished levels of Sml1. Removal of Sml1 increases the population of active RNR, which raises cellular dNTP levels. In this study using mass spectrometry and site-directed mutagenesis, we have identified the region of Sml1 phosphorylation to be between residues 52 and 64 containing the sequence GSSASASASSLEM. This is the first identification of a phosphorylation sequence of a Dun1 biological substrate. This sequence is quite different from the consensus Dun1 phosphorylation sequence reported previously from peptide library studies. The specific phosphoserines were identified to be Ser56, Ser58, and Ser60 by chemical modification of these residues to S-ethylcysteines followed by collision activated dissociation. To investigate further Sml1 phosphorylation, we constructed the single mutants S56A, S58A, S60A, and the triple mutant S56A/S58A/S60A and compared their degrees of phosphorylation with that of wild type Sml1. We observed a 90% decrease in the relative phosphorylation of S60A compared with that of wild type, a 25% decrease in S58A, and little or no decrease in the S56A mutant. There was no observed phosphate incorporation in the triple mutant, suggesting that Ser56, Ser58, and Ser60 in Sml1 are the sites of phosphorylation. Further mutagenesis studies reveal that Dun1 kinase requires an acidic residue at the +3 position, and there is cooperativity between the phosphorylation sites. These results show that Dun1 has a unique phosphorylation motif. In the yeast Saccharomyces cerevisiae, Mec1 and Rad53 kinases are transducers of all known cell cycle checkpoint pathways (1Elledge S.J. Science. 1996; 274: 1664-1672Crossref PubMed Scopus (1751) Google Scholar). In response to DNA damage, cell cycle progression is arrested at certain phases known as checkpoints, and simultaneously cells increase their capacity to repair DNA damage. A downstream effector of the pathway modulated by Mec1 and Rad53 is ribonucleotide reductase (RNR). 1The abbreviations used are: RNR, ribonucleotide reductase; CAD, collision-activated dissociation; ESI, electrospray ionization; FTICR, Fourier transform ion cyclotron resonance; GST, glutathione S-transferase; IMAC, immobilized metal affinity chromatography; MS, mass spectrometry; SORI, sustained off-resonance irradiation; cAPK, cylic AMP-dependent protein kinase. 1The abbreviations used are: RNR, ribonucleotide reductase; CAD, collision-activated dissociation; ESI, electrospray ionization; FTICR, Fourier transform ion cyclotron resonance; GST, glutathione S-transferase; IMAC, immobilized metal affinity chromatography; MS, mass spectrometry; SORI, sustained off-resonance irradiation; cAPK, cylic AMP-dependent protein kinase. RNR catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates, which is the rate-limiting step for dNTP synthesis. Overexpression of RNR genes rescues lethality caused by deletion of MEC1 and RAD53 genes (2Zhao X. Muller E.G. Rothstein R. Mol. Cell. 1998; 2: 329-340Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). During DNA damage and at S phase, levels of dNTP pools are increased to enhance the capacity of DNA repair (2Zhao X. Muller E.G. Rothstein R. Mol. Cell. 1998; 2: 329-340Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). This is achieved by transcriptional activation of RNR genes in a Mec1/Rad53-dependent manner (3Zhou Z. Elledge S.J. Cell. 1993; 75: 1119-1127Abstract Full Text PDF PubMed Scopus (293) Google Scholar, 4Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). The activity of RNR in yeast is also regulated by the small regulatory protein Sml1 (2Zhao X. Muller E.G. Rothstein R. Mol. Cell. 1998; 2: 329-340Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). Sml1 inhibits RNR activity through an interaction with the large subunit of the RNR complex, Rnr1 (2Zhao X. Muller E.G. Rothstein R. Mol. Cell. 1998; 2: 329-340Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 5Chabes A. Domkin V. Thelander L. J. Biol. Chem. 1999; 274: 36679-36683Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In response to DNA damage or at S phase, the intracellular level of Sml1 is reduced significantly (6Zhao X. Chabes A. Domkin V. Thelander L. Rothstein R. EMBO J. 2001; 20: 3544-3553Crossref PubMed Scopus (225) Google Scholar), resulting in the activation of RNR. Zhao and Rothstein (7Zhao X. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3746-3751Crossref PubMed Scopus (209) Google Scholar) demonstrated that the removal of Sml1 is triggered through its phosphorylation by Dun1 kinase. Evidence for this comes from the following observations: 1) deletion of the SML1 gene suppresses several phenotypes of the DUN1 null mutant, including its prolonged S phase; 2) in the null mutant of DUN1, both phosphorylation and degradation of Sml1 in response to DNA damage are diminished significantly; 3) Sml1 and Dun1 physically interact in vivo; and 4) Sml1 is phosphorylated by Dun1 in vitro. In S. cerevisiae, Dun1 is a particularly important serine/threonine kinase in that it acts upstream of multiple pathways such as transcriptional activation of genes required for DNA synthesis (3Zhou Z. Elledge S.J. Cell. 1993; 75: 1119-1127Abstract Full Text PDF PubMed Scopus (293) Google Scholar) and the cell cycle arrest at G2/M phase (8Gardner R. Putnam C.W. Weinert T. EMBO J. 1999; 18: 3173-3185Crossref PubMed Scopus (144) Google Scholar, 9Pati D. Keller C. Groudine M. Plon S.E. Mol. Cell. Biol. 1997; 17: 3037-3046Crossref PubMed Scopus (87) Google Scholar). In addition, more than 20 different proteins physically interact with Dun1, suggesting that Dun1 may be involved in multiple pathways (10Ho Y. Gruhler A. Heilbut A. Bader G.D. Moore L. Adams S.L. Millar A. Taylor P. Bennett K. Boutilier K. Yang L. Wolting C. Donaldson I. Schandorff S. Shewnarane J. Vo M. Taggart J. Goudreault M. Muskat B. Alfarano C. Dewar D. Lin Z. Michalickova K. Willems A.R. Sassi H. Nielsen P.A. Rasmussen K.J. Andersen J.R. Johansen L.E. Hansen L.H. Jespersen H. Podtelejnikov A. Nielsen E. Crawford J. Poulsen V. Sorensen B.D. Matthiesen J. Hendrickson R.C. Gleeson F. Pawson T. Moran M.F. Durocher D. Mann M. Hogue C.W. Figeys D. Tyers M. Nature. 2002; 415: 180-183Crossref PubMed Scopus (3035) Google Scholar). Although the mechanism by which Dun1 transmits signals during cell cycle checkpoints to its downstream effectors is not well understood, its kinase activity is crucial for its biological function. For instance, the kinase-deficient mutants D328A and K229R, unlike wild type Dun1, cannot respond to DNA damage and do not induce the expression of the RNR3 gene (3Zhou Z. Elledge S.J. Cell. 1993; 75: 1119-1127Abstract Full Text PDF PubMed Scopus (293) Google Scholar). In addition, phosphorylation of cell cycle checkpoint proteins such as the Crt1 repressor (11Huang M. Elledge S.J. Mol. Cell. Biol. 1997; 17: 6105-6113Crossref PubMed Scopus (154) Google Scholar) and the DNA repair protein Rad55 (12Bashkirov V.I. Bashkirova E.V. Haghnazari E. Heyer W.D. Mol. Cell. Biol. 2003; 23: 1441-1452Crossref PubMed Scopus (68) Google Scholar, 13Bashkirov V.I. King J.S. Bashkirova E.V. Schmuckli-Maurer J. Heyer W.D. Mol. Cell. Biol. 2000; 20: 4393-4404Crossref PubMed Scopus (130) Google Scholar) depends on Dun1, and the pathways in which these proteins are involved are independent of Sml1. Therefore, it is likely that there are other unidentified substrates of Dun1. So far, Sml1 is the only known natural substrate of Dun1 kinase. Prior to this study, the sites of Sml1 phosphorylated by Dun1 kinase had not been identified. By using an in vitro screen with a combinatorial library of 70 synthetic peptides that were known to be substrates of other serine/threonine kinases, Sanchez et al. (14Sanchez Y. Zhou Z. Huang M. Kemp B.E. Elledge S.J. Methods Enzymol. 1997; 283: 398-410PubMed Google Scholar) showed that Dun1 phosphopeptides have a basic residue at the -3 position from the phospho-Ser/Thr site. Furthermore, they found that Dun1 phosphorylates the consensus cyclic AMP-dependent protein kinase (cAPK) recognition sequence and that Dun1 and cAPK have similar substrate specificity. Nevertheless, to determine the Dun1 recognition sequence, it is necessary to identify phosphorylation sites in a natural substrate of Dun1. In this study, we identified the in vitro phosphorylation sites of Sml1 by Dun1 kinase using electrospray mass spectrometry and site-directed mutagenesis. Mass spectrometry (MS) has become a powerful technique for characterizing native and modified proteins. In particular, MS has been used extensively for phosphorylation mapping experiments by virtue of the capability of this technique to measure phosphorylated species at the molecular level (15Vacratsis P.O. Phinney B.S. Gage D.A. Gallo K.A. Biochemistry. 2002; 41: 5613-5624Crossref PubMed Scopus (33) Google Scholar, 16Cleverley K.E. Betts J.C. Blackstock W.P. Gallo J.M. Anderton B.H. Biochemistry. 1998; 37: 3917-3930Crossref PubMed Scopus (39) Google Scholar, 17Lapko V.N. Jiang X.Y. Smith D.L. Song P.S. Biochemistry. 1997; 36: 10595-10599Crossref PubMed Scopus (61) Google Scholar, 18Watts J.D. Affolter M. Krebs D.L. Wange R.L. Samelson L.E. Aebersold R. J. Biol. Chem. 1994; 269: 29520-29529Abstract Full Text PDF PubMed Google Scholar, 19Resing K.A. Ahn N.G. Methods Enzymol. 1997; 283: 29-44Crossref PubMed Scopus (55) Google Scholar). The ability to conduct high resolution MS experiments on the phosphorylated version of Sml1 proved to be essential for unraveling the complex phosphorylation signature of this important protein. In our study, MS analysis combined with β-elimination of the Sml1 phosphopeptide greatly aided identification of the phosphorylation sites. Unlike the study with synthetic peptide libraries, the amino acid sequence spanning the phosphorylation sites of Sml1 did not show any similarity to the cAPK recognition sequence. Furthermore, site-directed mutagenesis was used to identify unambiguously the Sml1 sites of phosphorylation and for the identification of crucial residues required for Dun1 recognition. Yeast Strain and Plasmids—GST-Dun1 expression plasmid (pWJ772-11), 2V. Gupta, C. Peterson, L. Dice-Turner, T. Uchiki, R. Hettich, J.-T. Guo, and X. Ying, X., submitted for publication. wild type Sml1 expression plasmid (pWJ-750-2) (22Posewitz M.C. Tempst P. Anal. Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (775) Google Scholar), and the S. cerevisiae strain U952-B (MATa, sml1Δ::HIS3. RAD5 in W303) (20Thomas B.J. Rothstein R. Genetics. 1989; 123: 725-738Crossref PubMed Google Scholar) were kindly provided by Dr. Rodney Rothstein at Columbia University (New York). The procedure for constructing the C14S Sml1 mutant is described elsewhere. 2V. Gupta, C. Peterson, L. Dice-Turner, T. Uchiki, R. Hettich, J.-T. Guo, and X. Ying, X., submitted for publication. The wild type Sml1 expression plasmid was used as a template to create S56A, S58A, S60A, S61A, E63Q, and the S56A/S58A/S60A Sml1 mutants using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Expression and Purification of Bacterially Expressed Sml1—An overnight culture of BL21(DE3)pLysS transformed by Sml1 expression plasmid was grown in Terrific Broth medium containing 100 mg/liter ampicillin and 34 mg/liter chloramphenicol at 37 °C. The culture was diluted 100-fold in fresh Terrific Broth medium and grown to an A600 of 0.6. Expression of the protein was induced by the addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 0.5 mm followed by a 3-h incubation in the shaker. The cells were harvested by centrifugation and resuspended in buffer A (50 mm Tris-HCl, 1 mm EDTA, 5 mm dithiothreitol, containing 1× Complete™ protease inhibitor mixture (Roche Applied Science) pH 7.4) and were frozen in liquid nitrogen. The following procedures were all carried out at 4 °C. After thawing the frozen cell suspension, the lysate was centrifuged at 150,000 × g for 1 h. The protein in the supernatant fraction was precipitated by adding ammonium sulfate to 25% saturation. The precipitate was harvested by centrifugation at 12,000 × g for 30 min and dissolved in buffer A before applying to a HiLoad 26/60 Superdex 75 gel filtration chromatography column (26-mm inner diameter and 60-cm height; Amersham Biosciences), which was preequilibrated with buffer B (50 mm Tris, pH 7.5, 10 mm MgCl2,1mm dithiothreitol, 0.1 mm NaVO4). The eluted fractions containing Sml1 were identified by SDS-PAGE using a 15% acrylamide gel followed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) MS as described below. These fractions were pooled and used for phosphorylation assays. The concentration of Sml1 was determined by a Coomassie Protein Assay Kit (Pierce) using bovine serum albumin as a standard. ADR1 G233 Peptide—The ADR1 G233 peptide (LKKLTRRASFSGQ) was custom synthesized at the Yale University W. M. Keck biotechnology resource center. This peptide was purified further by C18 reverse phase high performance liquid chromatography, and its purity was checked by ESI-FTICR-MS. The purified peptide was dissolved in water, and its concentration was determined by 2,4,6-trinitrobenzene sulfonic acid (Pierce) using tryptophan dissolved in water as a standard. Expression of GST-Dun1 and Kinase Reaction—Expression of GST-Dun1 was carried out as described by Zhao and Rothstein (7Zhao X. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3746-3751Crossref PubMed Scopus (209) Google Scholar). Briefly, the yeast cells (U952-B) transformed by the GST-Dun1 expression plasmid (pWJ772-11) were grown in SC-URA raffinose, and the expression of Dun1 was induced at mid-log phase (5∼6 × 107 cells/ml) by the addition of galactose to a final concentration of 2% (w/v). After two doubling times, the cells were harvested, washed with water, and stored at -80 °C. The purification of GST-Dun1 was carried out as described by Zhao and Rothstein (7Zhao X. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3746-3751Crossref PubMed Scopus (209) Google Scholar) and Sanchez et al. (14Sanchez Y. Zhou Z. Huang M. Kemp B.E. Elledge S.J. Methods Enzymol. 1997; 283: 398-410PubMed Google Scholar). Briefly, cells were lysed in buffer C (50 mm Tris-HCl, 150 mm NaCl, 50 mm KCl, 5 mm MgCl2, 1% (v/v) Igepal 630, 10% (v/v) glycerol, 10 mm dithiothreitol, 0.1 mm NaVO4, 30 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1× Complete™ protease inhibitor mixture, 2 μm pepstatin, pH 8.0) by glass bead disruption. The supernatant containing Dun1 was incubated with glutathione beads (glutathione-4-Sepharose Superflow: Amersham Biosciences) for 50 min and washed with buffer C. Examination of the glutathione beads by SDS-PAGE revealed pure GST-Dun1. The resin-bound GST-Dun1 was incubated with 2 bed volumes of buffer B containing 250 μm ATP, 8.3 μm Sml1 at 30 °C for 2 h. At the completion of the reaction, glutathione beads were harvested by centrifugation, and the supernatant was collected for MS analysis. For a quantitative kinase assay, 0.06 μCi/μl(1Ci = 37 GBq) [γ-32P]ATP (4,500 mCi/mmol; ICN, Costa Mesa, CA) was included in the buffer B. To compare the degree of phosphorylation in wild type Sml1 and ADR1 G233 peptide, the kinase assay was conducted with substrate concentrations of 8.3 μm and 67 μm for 30 min. Immediately after the reaction, 20 μl of supernatant was spotted on a 1-cm2 piece of phosphocellulose membrane. The membrane was washed in 10 mm phosphoric acid first and then with 95% (v/v) ethanol. The radioactivity on the membrane was measured by a liquid scintillation counter (LS3801; Beckman, Fullerton, CA). For autoradiography, 10 μl of the supernatant was mixed with SDS-gel loading buffer (62.5 mm Tris, pH 6.8, 2% SDS, 10% (v/v), 5% (v/v) β-mercaptoethanol, and 0.025 bromphenol blue) and was separated on a 15% polyacrylamide gel. Cerenkov radiation on the gel was detected by an electric autoradiography imager (Instant Imager; A Packard Bioscience Company, Ontario, Canada). Enzymatic Digestion and Immobilized Metal Affinity Chromatography (IMAC)—For tryptic digestion, a sample containing ∼10 μg (83 pmol) of Sml1 was incubated with 0.2 μg of sequencing grade trypsin (Promega, Madison, WI) at 37 °C for 10 h. To quench the reaction, acetic acid was added to a final concentration of 0.1% (v/v). For cyanogen bromide (CNBr) digestion, a sample containing ∼10 μg of Sml1 was dried using a speed vacuum apparatus. 10 μl of 10 mg/ml CNBr (Sigma) in 70% (v/v) formic acid was added to the sample and incubated for 24 h. To quench the reaction, 50 μl of water was added, and the sample was dried using a speed vacuum apparatus. This step was repeated five times. A 20-μl bed volume of Ga(III) IMAC column with Poros MC resin (PerSeptive Biosystems, Framingham, MA) was prepared as described previously (22Posewitz M.C. Tempst P. Anal. Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (775) Google Scholar). The CNBr-digested samples were dissolved in 10 μl of 10% (v/v) of acetic acid and manually loaded onto the column preequilibrated with 1% (v/v) acetic acid. The column was washed with 6 bed volumes of 1% (v/v) acetic acid, 6 bed volumes of a mixture consisting of water, acetonitrile, and acetic acid in a 70:30:1 respective ratio, followed by 6 bed volumes of 1% (v/v) acetic acid in water. The bound peptide was eluted with 3 bed volumes of 200 mm sodium phosphate, pH 8.5. Derivatization of Phosphoserine—The phosphopeptides enriched by Ga(III) IMAC were desalted once with C18 reverse phase ZipTip columns (Millipore, Bedford, MA), and the sample volume was reduced to less than 5 μl using a speed vacuum apparatus. The phosphoserine residues of the peptides were chemically modified to S-ethylcysteine by a β-elimination reaction in the presence of ethanethiol as described previously (23Meyer H.E. Hoffmann-Posorske E. Heilmeyer Jr., L.M. Methods Enzymol. 1991; 201: 169-185Crossref PubMed Scopus (92) Google Scholar, 24Oda Y. Nagasu T. Chait B.T. Nat. Biotechnol. 2001; 19: 379-382Crossref PubMed Scopus (749) Google Scholar). Briefly, 50 μl of H2O, 4 m LiOH, acetonitrile, ethanol, ethanethiol mixed in a ratio of (5:14:5:5:2) was added to the sample and incubated in 37 °C for 1 h. The reaction was quenched by the addition of 25 μl of acetic acid. Because Sml1 contains only one cysteine residue (Cys14), we omitted the oxidation step, which is normally employed prior to the derivatization. ESI-FTICR-MS—All samples were desalted with C18 reverse phase ZipTips prior to MS analysis. For positive ion analysis, samples were prepared in a 50:50 mixture of water and acetonitrile containing 0.1% (v/v) acetic acid. For negative ion analysis samples were prepared in a 40:60 water:acetonitrile mixture containing 20 mm piperidine. All mass spectra were acquired with an IonSpec (Lake Forest, CA) 9.4-tesla HiRes ESI-FTICR-MS, as described previously for the Sml1-His tag species (25Uchiki T. Hettich R. Gupta V. Dealwis C. Anal. Biochem. 2002; 301: 35-48Crossref PubMed Scopus (10) Google Scholar). The MS experiment consisted of the four following steps: 1) ions were generated in the electrospray source; 2) the ions were accumulated in an external hexapole; 3) the ions were transferred into the high vacuum region with a quadrupole lens system; and 4) the ions were detected in the cylindrical analyzer cell of the mass spectrometer. To enhance ion trapping, nitrogen gas was pulsed into the mass analyzer to cool the ion packet prior to detection. Ions were measured under broadband conditions with resolutions ranging from 50,000 to 150,000 (full width at half-maximal height). External calibration was accomplished using the various charge states of bovine ubiquitin. The high resolution mass measurement enables isotopic resolution of multiply charged ions. Thus, the charge state of multiply charged ions can be determined solely by its isotopic spacing (26Horn D.M. Zubarev R.A. McLafferty F.W. J. Am. Soc. Mass Spectrom. 2000; 11: 320-332Crossref PubMed Scopus (471) Google Scholar). The deconvoluted molecular mass spectra were generated with the IonSpec software from the ESI-MS by multiplying the masses of the electrospray ions by their respective charge and then subtracting the masses of the protons added. This "unfolds" the multiply charged ion mass spectrum into a more easily interpreted molecular mass spectrum. Errors in the mass measurement for the multiply charged ions will be scaled proportional to the charge in the calculation of the molecular masses in the deconvoluted mass spectra. By calibrating on the calculated values of the most abundant isotopic peaks of six different charge states (7+ to 12+) of bovine ubiquitin, the deconvoluted molecular mass spectrum yielded a measured molecular mass for ubiquitin which was within 0.030 Da of the calculated value. Ion collision dissociation was conducted by isolating an ion of interest within the analyzer cell of the mass spectrometer and then accelerating the ion into nitrogen target gas under sustained off-resonance irradiation collision-activated dissociation, or SORI-CAD (27Gauthier J.W. Trauman T.R. Jacobson D.B. Anal. Chem. Acta. 1991; 246: 211-225Crossref Scopus (647) Google Scholar). For the SORI-CAD experiments, the ion excitation was accomplished with an RF pulse (∼1 KHz lower in frequency than the ion cyclotron frequency) applied for a 2-s duration and with an amplitude in the range of 1–4 volts (peak-to-peak). A pulsed valve was used to admit the nitrogen collision gas into the high vacuum region to a maximum pressure of about 5 × 10-6 torr during the ion excitation step. A base pressure of about 1 × 10-9 torr was reestablished prior to ion detection. Results from the in vitro phosphorylation of Sml1 by Dun1 where 32P is incorporated are shown in Fig. 1. Sml1 is specifically phosphorylated by Dun1 with 32P incorporation at 40–50 times greater than that of the negative control samples (Fig. 1). These results show that phosphorylation of Sml1 is solely the result of GST-Dun1 and that the kinase activity of GST-Dun1 is specific to Sml1. The number of phosphoryl groups attached to Sml1 was determined by a combination of MS and site-directed mutagenesis. To estimate the minimum number of phosphorylation sites, intact molecules of Sml1 were first analyzed by ESI-FTICR. The attachment of a phosphate to a hydroxyl group of serine, threonine, or tyrosine results in mass increases of the proteins or peptides by 79.966 Da in monoisotopic mass, and it was possible to estimate the number of phosphates on a protein by analyzing the mass shift. We observed monophosphorylated, diphosphorylated, triphosphorylated, and unphosphorylated Sml1. These observations indicated that at least three sites of Sml1 can be phosphorylated (Fig. 2). To find the region of Sml1 containing the phosphorylation sites, the protein was digested by trypsin or CNBr and then analyzed by ESI-FTICR. In the positive ion analysis of the tryptic digest, we detected peptides that covered 75% of the Sml1 sequence spanning residues 1–67 and 73–84. In the negative ion analysis, we detected peptides constituting 89% of the Sml1 sequence, which almost covered the entire Sml1 sequence with the exception of residues 16–26 (Fig. 3A). In both positive and negative ion analysis, the spectrum for the samples taken from the in vitro Dun1 phosphorylation assays showed that peptides spanning residues 33–67 (3,549.720 Da) and 34–67 (3,705.857 Da) are associated with their singly phosphorylated forms of 3,629.695 and 3,785.829 Da, respectively. In the positive ion analysis of CNBr digests, we detected four peptides that cover 34% of the Sml1 sequence spanning residues 29–39 and 81–104. However, none of these peptides was phosphorylated. In the negative ion analysis of CNBr digests, 7 or 8 peptides that cover 71% of Sml1 sequence (residues 31–106) were observed (Fig. 3B). These results showed that the peptide spanning residues 52–64 consisting of GSSASASASSLEM (1,153.539 Da) was associated with its singly phosphorylated form (Fig. 4, A and B). The phosphopeptides of Sml1 identified by both CNBr and tryptic digest are entirely consistent. The combined data generated from the CNBr and tryptic digest gave us 100% sequence coverage of Sml1.Fig. 4Negative ion analysis of Sml1 CNBr digest. A, the deconvoluted mass spectrum of the negative control of the kinase reaction subjected to CNBr digestion in which Sml1 was incubated in the presence of 250 μm ATP without GST-Dun1. As expected, no phosphorylated species were identified (see inset). In general, residues at the C terminus of peptides generated by CNBr digestion are homolactones, which can be further converted to homoserine by intramolecular hydrolysis. In this spectrum, residues at the C terminus of most of the peptides were homoserine. B, the deconvoluted mass spectrum of phosphorylated Sml1 subjected to CNBr digestion in which Sml1 was incubated with GST-Dun1 in the presence of 250 μm ATP. The peak (1,233.535) corresponds to a singly phosphorylated peptide consisting of residues 52–64 (see inset). Inthis spectrum, residues at the C terminus of the majority of the peptides were homoserine. C, phosphopeptides enriched by Ga(III) IMAC show singly, doubly, and triply phosphorylated species. In this spectrum, residues at the C terminus of these peptides were either homolactone or homoserine (denoted as C-homoserine).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To enrich phosphopeptides, CNBr digest samples were subjected to Ga(III) IMAC chromatography and analyzed in the negative ion mode. Singly, doubly, and triply phosphorylated forms of residues 52–64 were observed (Fig. 4C), which were absent in the negative control, which contained CNBr peptides generated from only unphosphorylated Sml1. In most of the samples, peak intensities of doubly or triply phosphorylated species were stronger than the singly phosphorylated species. Singly phosphorylated species could only be observed prior to IMAC chromatography. To confirm that the observed mass shift was the result of phosphorylation, an ion of the doubly phosphorylated species (M-H-1 = 1,294.452 Da) was subjected to CAD. In this experiment, the species of interest was isolated in the analyzer region of the mass spectrometer, accelerated with a RF voltage and bombarded into nitrogen gas. Depending on RF voltage used to accelerate the ion, we observed fragment ions of 1,196.489 and 1,098.466, which corresponded to the loss of one or two phosphoric acids (data not shown). We could not obtain a reasonable signal in the CAD experiment for singly and triply phosphorylated species, which was possibly because of the low abundance of the parent ions. To confirm our findings further, we repeated the same experiment with the C14S mutant form of Sml1 and its proteolytic fragment (29Uchiki T. Gupta V. Dealwis C. Hettich R.L. 50th ASMS Conference on Mass Spectrometry and Allied Topics. Orlando, FL2002Google Scholar). During purification of C14S Sml1 by gel filtration chromatography, the intact molecule was separated from a smaller degraded C14S Sml1 fragment corresponding to a mass of 8,147.073 Da. Mass spectrometric analysis of the latter revealed that the degraded peptide was an N-terminal fragment of C14S Sml1 consisting of residue 1–71 (29Uchiki T. Gupta V. Dealwis C. Hettich R.L. 50th ASMS Conference on Mass Spectrometry and Allied Topics. Orlando, FL2002G
Sml1p is a small 104-amino acid protein from Saccharomyces cerevisiae that binds to the large subunit (Rnr1p) of the ribonucleotide reductase complex (RNR) and inhibits its activity. During DNA damage, S phase, or both, RNR activity must be tightly regulated, since failure to control the cellular level of dNTP pools may lead to genetic abnormalities, such as genome rearrangements, or even cell death. Structural characterization of Sml1p is an important step in understanding the regulation of RNR. Until now the oligomeric state of Sml1p was unknown. Mass spectrometric analysis of wild-type Sml1p revealed an intermolecular disulfide bond involving the cysteine residue at position 14 of the primary sequence. To determine whether disulfide bonding is essential for Sml1p oligomerization, we mutated the Cys14 to serine. Sedimentation equilibrium measurements in the analytical ultracentrifuge show that both wild-type and C14S Sml1p exist as dimers in solution, indicating that the dimerization is not a result of a disulfide bond. Further studies of several truncated Sml1p mutants revealed that the N-terminal 8−20 residues are responsible for dimerization. Unfolding/refolding studies of wild-type and C14S Sml1p reveal that both proteins refold reversibly and have almost identical unfolding/refolding profiles. It appears that Sml1p is a two-domain protein where the N-terminus is responsible for dimerization and the C-terminus for binding and inhibiting Rnr1p activity.
Zymomonas mobilis is an excellent ethanologenic bacterium. Biomass pretreatment and saccharification provides access to simple sugars, but also produces inhibitors such as acetate and furfural. Our previous work has identified and confirmed the genetic change of a 1.5-kb deletion in the sodium acetate tolerant Z. mobilis mutant (AcR) leading to constitutively elevated expression of a sodium proton antiporter encoding gene nhaA, which contributes to the sodium acetate tolerance of AcR mutant. In this study, we further investigated the responses of AcR and wild-type ZM4 to sodium acetate stress in minimum media using both transcriptomics and a metabolic labeling approach for quantitative proteomics the first time. Proteomic measurements at two time points identified about eight hundreds proteins, or about half of the predicted proteome. Extracellular metabolite analysis indicated AcR overcame the acetate stress quicker than ZM4 with a concomitant earlier ethanol production in AcR mutant, although the final ethanol yields and cell densities were similar between two strains. Transcriptomic samples were analyzed for four time points and revealed that the response of Z. mobilis to sodium acetate stress is dynamic, complex and involved about one-fifth of the total predicted genes from all different functional categories. The modest correlations between proteomic and transcriptomic data may suggest the involvement of posttranscriptional control. In addition, the transcriptomic data of forty-four microarrays from four experiments for ZM4 and AcR under different conditions were combined to identify strain-specific, media-responsive, growth phase-dependent, and treatment-responsive gene expression profiles. Together this study indicates that minimal medium has the most dramatic effect on gene expression compared to rich medium followed by growth phase, inhibitor, and strain background. Genes involved in protein biosynthesis, glycolysis and fermentation as well as ATP synthesis
Amyloid aggregates of the amyloid-beta (Abeta) peptide are implicated in the pathology of Alzheimer's disease. Anti-Abeta monoclonal antibodies (mAbs) have been shown to reduce amyloid plaques in vitro and in animal studies. Consequently, passive immunization is being considered for treating Alzheimer's, and anti-Abeta mAbs are now in phase II trials. We report the isolation of two mAbs (PFA1 and PFA2) that recognize Abeta monomers, protofibrils, and fibrils and the structures of their antigen binding fragments (Fabs) in complex with the Abeta(1-8) peptide DAEFRHDS. The immunodominant EFRHD sequence forms salt bridges, hydrogen bonds, and hydrophobic contacts, including interactions with a striking WWDDD motif of the antigen binding fragments. We also show that a similar sequence (AKFRHD) derived from the human protein GRIP1 is able to cross-react with both PFA1 and PFA2 and, when cocrystallized with PFA1, binds in an identical conformation to Abeta(1-8). Because such cross-reactivity has implications for potential side effects of immunotherapy, our structures provide a template for designing derivative mAbs that target Abeta with improved specificity and higher affinity.
A primary focus of the rapidly growing field of plant synthetic biology is to develop technologies to precisely regulate gene expression and engineer complex genetic circuits into plant chassis. At present, there are few orthogonal tools available for effectively controlling gene expression in plants, with most researchers instead using a limited set of viral elements or truncated native promoters. A powerful repressible-and engineerable-binary system that has been repurposed in a variety of eukaryotic systems is the Q-system from Neurospora crassa. Here, we demonstrate the functionality of the Q-system in plants through transient expression in soybean (Glycine max) protoplasts and agroinfiltration in Nicotiana benthamiana leaves. Further, using functional variants of the QF transcriptional activator, it was possible to modulate the expression of reporter genes and to fully suppress the system through expression of the QS repressor. As a potential application for plant-based biosensors (phytosensors), we demonstrated the ability of the Q-system to amplify the signal from a weak promoter, enabling remote detection of a fluorescent reporter that was previously undetectable. In addition, we demonstrated that it was possible to coordinate the expression of multiple genes through the expression of a single QF activator. Based on the results from this study, the Q-system represents a powerful orthogonal tool for precise control of gene expression in plants, with envisioned applications in metabolic engineering, phytosensors, and biotic and abiotic stress tolerance.
Abstract Background The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. It is capable of both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Intolerance to stresses routinely encountered during industrial fermentations may hinder the commercial development of this organism. A previous C. thermocellum ethanol stress study showed that the largest transcriptomic response was in genes and proteins related to nitrogen uptake and metabolism. Results In this study, C. thermocellum was grown to mid-exponential phase and treated with furfural or heat to a final concentration of 3 g.L -1 or 68°C respectively to investigate general and specific physiological and regulatory stress responses. Samples were taken at 10, 30, 60 and 120 min post-shock, and from untreated control fermentations, for transcriptomic analyses and fermentation product determinations and compared to a published dataset from an ethanol stress study. Urea uptake genes were induced following furfural stress, but not to the same extent as ethanol stress and transcription from these genes was largely unaffected by heat stress. The largest transcriptomic response to furfural stress was genes for sulfate transporter subunits and enzymes in the sulfate assimilatory pathway, although these genes were also affected late in the heat and ethanol stress responses. Lactate production was higher in furfural treated culture, although the lactate dehydrogenase gene was not differentially expressed under this condition. Other redox related genes such as a copy of the rex gene, a bifunctional acetaldehyde-CoA/alcohol dehydrogenase and adjacent genes did show lower expression after furfural stress compared to the control, heat and ethanol fermentation profiles. Heat stress induced expression from chaperone related genes and overlap was observed with the responses to the other stresses. This study suggests the involvement of C. thermocellum genes with functions in oxidative stress protection, electron transfer, detoxification, sulfur and nitrogen acquisition, and DNA repair mechanisms in its stress responses and the use of different regulatory networks to coordinate and control adaptation. Conclusions This study has identified C. thermocellum gene regulatory motifs and aspects of physiology and gene regulation for further study. The nexus between future systems biology studies and recently developed genetic tools for C. thermocellum offers the potential for more rapid strain development and for broader insights into this organism’s physiology and regulation.
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