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• W2048885488 abstract "The Saccharomyces cerevisiae N-terminal acetyltransferase NatB consists of the subunits Nat3p and Mdm20p. We found by two-dimensional PAGE analysis that nat3Δ exhibited protein expression during growth in basal medium resembling protein expression in salt-adapted wild-type cells. The stress-induced carboxypeptidase Y (CPY) inhibitor and phosphatidylethanolamine-binding protein family member Tfs1p was identified as a novel NatB substrate. The N-terminal acetylation status of Tfs1p, Act1p, and Rnr4p in both wild type and nat3Δ was confirmed by tandem mass spectrometry. Furthermore it was found that unacetylated Tfs1p expressed in nat3Δ showed an ∼100-fold decrease in CPY inhibition compared with the acetylated form, indicating that the N-terminal acetyl group is essential for CPY inhibition by Tfs1p. Phosphatidylethanolamine-binding proteins in other organisms have been reported to be involved in the regulation of cell signaling. Here we report that a number of proteins, whose expression has been shown previously to be dependent on the activity in the protein kinase A (PKA) signaling pathway, was found to be regulated in line with low PKA activity in the nat3Δ strain. The involvement of Nat3p and Tfs1p in PKA signaling was supported by caffeine growth inhibition studies. First, growth inhibition by caffeine addition (resulting in enhanced cAMP levels) was suppressed in tfs1Δ. Second, this suppression by tfs1Δ was abolished in the nat3Δ background, indicating that Tfs1p was not functional in the nat3Δ strain possibly because of a lack of N-terminal acetylation. We conclude that the NatB-dependent acetylation of Tfs1p appears to be essential for its inhibitory activity on CPY as well its role in regulating the PKA pathway. The Saccharomyces cerevisiae N-terminal acetyltransferase NatB consists of the subunits Nat3p and Mdm20p. We found by two-dimensional PAGE analysis that nat3Δ exhibited protein expression during growth in basal medium resembling protein expression in salt-adapted wild-type cells. The stress-induced carboxypeptidase Y (CPY) inhibitor and phosphatidylethanolamine-binding protein family member Tfs1p was identified as a novel NatB substrate. The N-terminal acetylation status of Tfs1p, Act1p, and Rnr4p in both wild type and nat3Δ was confirmed by tandem mass spectrometry. Furthermore it was found that unacetylated Tfs1p expressed in nat3Δ showed an ∼100-fold decrease in CPY inhibition compared with the acetylated form, indicating that the N-terminal acetyl group is essential for CPY inhibition by Tfs1p. Phosphatidylethanolamine-binding proteins in other organisms have been reported to be involved in the regulation of cell signaling. Here we report that a number of proteins, whose expression has been shown previously to be dependent on the activity in the protein kinase A (PKA) signaling pathway, was found to be regulated in line with low PKA activity in the nat3Δ strain. The involvement of Nat3p and Tfs1p in PKA signaling was supported by caffeine growth inhibition studies. First, growth inhibition by caffeine addition (resulting in enhanced cAMP levels) was suppressed in tfs1Δ. Second, this suppression by tfs1Δ was abolished in the nat3Δ background, indicating that Tfs1p was not functional in the nat3Δ strain possibly because of a lack of N-terminal acetylation. We conclude that the NatB-dependent acetylation of Tfs1p appears to be essential for its inhibitory activity on CPY as well its role in regulating the PKA pathway. N-terminal acetylation, together with N-terminal methionine cleavage, is the most common protein modification in eukaryotic cells. Over 40% of all yeast proteins and almost 90% of mammalian proteins are estimated to be N-terminally acetylated (1Polevoda B. Sherman F. J. Mol. Biol. 2003; 325: 595-622Crossref PubMed Scopus (355) Google Scholar). N-terminal acetylation occurs co-translationally when nascent peptides are between 20 and 50 amino acids long (2Driessen H.P. de Jong W.W. Tesser G.I. Bloemendal H. CRC Crit. Rev. Biochem. 1985; 18: 281-325Crossref PubMed Scopus (180) Google Scholar, 3Bradshaw R.A. Brickey W.W. Walker K.W. Trends Biochem. Sci. 1998; 23: 263-267Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). The addition of acetyl groups is catalyzed by N-terminal acetyl transferases (NATs). 1The abbreviations used are: NAT, N-terminal acetyl transferase; CPY, carboxypeptidase Y; PEBP, phosphatidylethanolamine-binding protein; PKA, protein kinase A; SD, synthetic dropout; MS, mass spectrometry; MS/MS, tandem MS; ESI, electrospray ionization; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MES, 4-morpholineethanesulfonic acid; FA, N-(3-[2-furyl]acryloyl); TRITC, tetramethylrhodamine isothiocyanate; LPI, logarithmic phenotypic index; LSC, logarithmic strain coefficient; RKIP, Raf-1 kinase inhibitor protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. In Saccharomyces cerevisiae there are three known NATs: NatA, NatB, and NatC (1Polevoda B. Sherman F. J. Mol. Biol. 2003; 325: 595-622Crossref PubMed Scopus (355) Google Scholar). NatA consists of the subunits Ard1p and Nat1p (4Mullen J.R. Kayne P.S. Moerschell R.P. Tsunasawa S. Gribskov M. Colavito-Shepanski M. Grunstein M. Sherman F. Sternglanz R. EMBO J. 1989; 8: 2067-2075Crossref PubMed Scopus (246) Google Scholar). In addition, Nat5p was recently shown to be associated with Ard1p and Nat1p but does not seem to be important for NatA function (5Gautschi M. Just S. Mun A. Ross S. Rucknagel P. Dubaquie Y. Ehrenhofer-Murray A. Rospert S. Mol. Cell. Biol. 2003; 23: 7403-7414Crossref PubMed Scopus (172) Google Scholar). NatC consists of the subunits Mak3p, Mak10p, and Mak31p (6Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2286) Google Scholar). NatB consists of the subunits Nat3p (7Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google Scholar) and Mdm20p (8Polevoda B. Cardillo T.S. Doyle T.C. Bedi G.S. Sherman F. J. Biol. Chem. 2003; 278: 30686-30697Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Ard1p, Nat3p, and Mak3p are related by sequence homology and are the catalytic subunits of NatA, NatB, and NatC, respectively, and all exhibit acetyl-CoA binding sites (1Polevoda B. Sherman F. J. Mol. Biol. 2003; 325: 595-622Crossref PubMed Scopus (355) Google Scholar). NATs act on substrates with a specific but degenerated N-terminal amino acid sequence where certain amino acids in the N-terminal region are required for the activity of each NAT and where suboptimal amino acid residues can diminish the activity (9Polevoda B. Sherman F. J. Biol. Chem. 2000; 275: 36479-36482Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). In the case of NatB an N-terminal sequence of MD-, ME-, MN-, or MM- is required for acetylation. All proteins with an N-terminal sequence of MD- or ME- that have been characterized so far have been acetylated, while only a subset of the proteins with MN- or MM- at their N terminus are acetylated (10Polevoda B. Sherman F. Biochem. Biophys. Res. Commun. 2003; 308: 1-11Crossref PubMed Scopus (91) Google Scholar). Nat1p and Ard1p orthologs in mouse were shown recently to form a complex with acetyltransferase activity (11Sugiura N. Adams S.M. Corriveau R.A. J. Biol. Chem. 2003; 278: 40113-40120Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), and the presence of orthologs to both the catalytic subunits Nat3p, Ard1p, and Mak3p and the auxiliary subunits Mdm20p, Nat1p, and Mak10p in numerous higher eukaryotic model organisms and in humans indicates that the NATs may be found in all eukaryotes. Similarities between the N-terminal amino acid sequences of acetylated proteins in higher eukaryotes and in yeast indicate highly conserved molecular mechanisms of recognition and/or acetyl group addition over large evolutionary distances (10Polevoda B. Sherman F. Biochem. Biophys. Res. Commun. 2003; 308: 1-11Crossref PubMed Scopus (91) Google Scholar). Despite the fact that so many proteins are N-terminally acetylated few cases where the N-terminal group is of biological importance for protein function have been reported. The acetylation of the N terminus of the viral coat protein Gag catalyzed by NatC is essential for assembly or maintenance of the viral coat particle in yeast (12Tercero J.C. Wickner R.B. J. Biol. Chem. 1992; 267: 20277-20281Abstract Full Text PDF PubMed Google Scholar). The unacetylated form of fetal hemoglobin, HbF, has been shown to stabilize the hemoglobin tetramer compared with the acetylated form (13Manning L.R. Manning J.M. Biochemistry. 2001; 40: 1635-1639Crossref PubMed Scopus (29) Google Scholar). Tropomyosin isolated from striated muscles is dependent on N-terminal acetylation to polymerize and to bind to F-actin in the correct way (14Urbancikova M. Hitchcock-DeGregori S.E. J. Biol. Chem. 1994; 269: 24310-24315Abstract Full Text PDF PubMed Google Scholar), and acetylation of actin in Dictyostelium has been shown to affect the weak interactions between actin and myosin (15Abe A. Saeki K. Yasunaga T. Wakabayashi T. Biochem. Biophys. Res. Commun. 2000; 268: 14-19Crossref PubMed Scopus (44) Google Scholar). From a medical perspective it is also interesting to note that the human NAT1 ortholog NATH has been shown to be strongly overexpressed in papillary thyroid carcinomas (16Fluge O. Bruland O. Akslen L.A. Varhaug J.E. Lillehaug J.R. Oncogene. 2002; 21: 5056-5068Crossref PubMed Scopus (45) Google Scholar); however, the mechanistic implications of this for the disease are not known at present. S. cerevisiae strains lacking NatA, NatB, or NatC are viable but exhibit various phenotypes. The phenotypes are most severe in strains lacking NatB, and the effects are believed to be related to the lack of acetylation of the two NatB substrates actin and tropomyosin 1 (8Polevoda B. Cardillo T.S. Doyle T.C. Bedi G.S. Sherman F. J. Biol. Chem. 2003; 278: 30686-30697Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Both these protein need to be acetylated to interact and to form stable actin filaments (17Singer J.M. Shaw J.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7644-7649Crossref PubMed Scopus (73) Google Scholar). nat3Δ cells lacking functional NatB have been reported to exhibit many phenotypes including slow growth, sensitivity to various growth inhibitors when grown on agar plates (e.g. deficiency in utilization of nonfermentable carbon sources), reduced mating of MATa cells, inability to grow at 37 °C, abnormal mitochondrial and vacuolar inheritance (8Polevoda B. Cardillo T.S. Doyle T.C. Bedi G.S. Sherman F. J. Biol. Chem. 2003; 278: 30686-30697Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 17Singer J.M. Shaw J.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7644-7649Crossref PubMed Scopus (73) Google Scholar), and random budding polarity in diploid cells (18Ni L. Snyder M. Mol. Biol. Cell. 2001; 12: 2147-2170Crossref PubMed Scopus (238) Google Scholar). In this work we investigated the protein expression pattern of the nat3Δ strain during growth in high salt. We showed that the protein expression for this mutant in basal medium is very similar to the protein expression found in salt-adapted wild-type cells. Furthermore we identified Tfs1p as a novel NatB substrate. Tfs1p is known to be an inhibitor of the protease carboxypeptidase Y (CPY) (19Bruun A.W. Svendsen I. Sorensen S.O. Kielland-Brandt M.C. Winther J.R. Biochemistry. 1998; 37: 3351-3357Crossref PubMed Scopus (49) Google Scholar), and we showed that N-terminal acetylation is important for the inhibitory activity of Tfs1p. Furthermore we found experimental evidence supporting that Tfs1p negatively regulates signaling in the protein kinase A (PKA) pathway and that this inhibitory effect is acetylation-dependent. Strains, Media, and Growth Conditions—The S. cerevisiae strains used in this study are listed in Table I. Strains in the FY1679 background were kindly provided by Dr. Bogdan Polevoda (7Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google Scholar). The multicopy plasmid pKT1067 with the TFS1 gene and a ura marker was kindly provided by Dr. J. Winter with the kind permission of Dr. K. Tatchell. Double deletion mutants were obtained by mating of single mutants, sporulation of diploids, and dissection of tetrads followed by subsequent selection on plates containing 200 μg/ml kanamycin. Deletions were confirmed by PCR. Transformations were performed using the lithium acetate/polyethylene glycol method. Cells were grown at 30 °C in SD medium (0.14% yeast nitrogen base without amino acids (Difco), 2% (w/v) glucose, 0.5% ammonium sulfate, 1% succinic acid) supplemented with the appropriate amino acids. The medium was buffered to pH 5.8. Cells from stationary phase cultures were inoculated to OD610 = 0.07. The cell cultures were grown with continues shaking at 180 rpm. Cells grown for protein extraction were harvested by centrifugation at 3000 rpm for 4 min at 4 °C. All samples prepared were grown, harvested, and analyzed in triplicates. Microcultivation was performed in 350-μl cultures for 48 h in a Bioscreen analyzer C (Labsystems Oy, Helsinki, Finland) according to procedures in an earlier report (20Warringer J. Ericson E. Fernandez L. Nerman O. Blomberg A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15724-15729Crossref PubMed Scopus (183) Google Scholar). Duplicates of all samples were run on each plate. Two identical plates with cells from separate overnight cultures were run in parallel. The caffeine concentrations used were 0.5, 1, and 1.5 mg/ml. Strains from the FY background were used for two-dimensional PAGE analysis, preparation of protein extracts for MS/MS analysis, and for phenotype analysis in flask culture, while strains from the BY background were used for CPY activity measurements and phenotype analysis in microtiter culture.Table IS. cerevisiae strains used in this workIndicated as in this workGenotypeRef.Wild-type FY1679FY1679 Matα ura3-527Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google Scholarnat3Δ FY1679FY1679 Matα ura3-52 nat3Δ::kanMX47Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google ScholarWild typeBY4741 Matahis3Δ1, leu2Δ0, met15Δ0, ura3Δ0EUROSCARFnat3ΔBY4741 Matahis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, nat3Δ::kanMX4EUROSCARFmdm20ΔBY4741 Matahis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, mdm20Δ::kanMX4EUROSCARFtfs1ΔBY4741 Matahis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, tfs1Δ::kanMX4EUROSCARFprc1ΔBY4741 Matahis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, prc1Δ::kanMX4EUROSCARFprc1Δ tfs1ΔBY4741 Matahis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, prc1Δ::kanMX4, tfs1Δ::kanMX4This studynat3Δ tfs1ΔBY4741 Matahis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, nat3Δ::kanMX4, tfs1Δ::kanMX4This studyprc1Δ TFS1BY4741 Matahis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, prc1Δ::kanMX4, pKT1067This studynat3Δprc1Δ TFS1BY4742 Matα his3Δ1, leu2Δ0, lysΔ0, ura3Δ0, prc1Δ::kanMX4, nat3Δ::kanMX4, pKT1067This study Open table in a new tab Two-dimensional Electrophoresis—For the preparation of radiolabeled proteins 10 μCi of [35S]methionine (Amersham Biosciences catalog no. SJ-235) was mixed with 10 ml of cell culture for one-fourth of a generation time under continuous shaking at 30 °C. The isotope was added at A610 = 0.35. Protein extracts of radiolabeled proteins were prepared by glass bead disruption, and determination of incorporated [35S]methionine was performed as described previously (21Blomberg A. Methods Enzymol. 2002; 350: 559-584Crossref PubMed Scopus (20) Google Scholar). Protein concentrations were determined with trichloroacetic acid precipitation using a Lowry-based commercial kit (Sigma catalog no. P-5656). Protein extract with a total radioactivity of 2 × 106 dpm was loaded on each analytic gel, and 1 mg of protein was loaded on each preparative gel. The immobilized pH gradient strips used were 18 cm long and covered pH 3–10 (non-linear gradient) (Amersham Biosciences). Two-dimensional electrophoresis was performed as described previously (21Blomberg A. Methods Enzymol. 2002; 350: 559-584Crossref PubMed Scopus (20) Google Scholar) using a Multiphor II (Amersham Biosciences) for running the first dimension and an Ettan DALT II (Amersham Biosciences) for running the second dimension. Gels with radiolabeled proteins were dried and exposed to image plates for roughly 72 h. The plates were scanned using a phosphorimaging system (Bio-Rad Molecular Imager FX) with a resolution of 200 × 200 μm. The raw files were processed and put together in a match set in the two-dimensional gel analysis software PDQuest version 6.2.0 (Bio-Rad). The spot detection was checked manually, and matching of all spots was manually performed. Signal quantities in the individual spots on the gels were normalized to the total signal from all spots in each gel, and comparative quantification between corresponding spots on the different gels in the match set was performed. The proteins on the preparative gels were visualized with Coomassie Blue staining according to a published protocol (21Blomberg A. Methods Enzymol. 2002; 350: 559-584Crossref PubMed Scopus (20) Google Scholar). Protein Trypsinization, Mass Spectrometry, and Analysis of MS/MS Data—Gel pieces were cut out and in-gel trypsinized as described elsewhere (21Blomberg A. Methods Enzymol. 2002; 350: 559-584Crossref PubMed Scopus (20) Google Scholar). Peptides were eluted in 8 μl of elution buffer containing 2% acetonitrile and 0.05% formic acid. Peptide separation was performed on a Finnigan Surveyor chromatography work station (Finnigan Corp., San Jose, CA) using a 150 × 0.18-mm C18 HyPurity column (Thermo Hypersil) or a 150 × 0.18-mm C18 column produced in-house. Mobile phase 1 consisted of 95% water, 4.95% acetonitrile, and 0.05% formic acid, and mobile phase 2 consisted of 99.95% acetonitrile and 0.05% formic acid. Liquid chromatography was performed using a linear gradient starting at 0% and reaching 50% phase B after 50 min. The flow rate was 2 μl/min. The liquid chromatography device was directly coupled to a Finnigan LCQ DECA ion trap mass spectrometer (Thermofinnigan Corp., San Jose, CA). The mass spectrometer was run in data-dependent scan mode where the three most dominant ions in each MS scan were selected for MS/MS analysis. Dynamic exclusion was set to a maximum of three MS/MS scans in a row, and an exclusion time of 1 min was applied. Where indicated mass spectrometry was performed using MALDI-TOF as described elsewhere (22Molin M. Larsson T. Karlsson K.A. Blomberg A. Proteomics. 2003; 3: 752-763Crossref PubMed Scopus (8) Google Scholar). The SEQUEST search algorithm was used to correlate experimental mass data to theoretical mass data derived from the sequence in the yeast FASTA data base from the National Center for Biotechnology Information (NCBI). Protein identifications based on tandem mass spectra correlating to one tryptic peptide were considered valid for identification. For singly charged peptides, only spectra with a cross-correlation to a tryptic peptide of 1.5 or more were accepted. The corresponding value for multiple charged peptides was 2.0. Only peptides with a ΔCn score larger than 0.1 were accepted. In cases where the identity of the protein had already been established additional peptides with lower scores were taken into account after manual control of the MS/MS spectrum. MALDI-TOF spectra were interpreted using Mascot software. The MALDI-TOF analysis was performed at the SWEGENE proteomics center in Göteborg on a Micromass mass spectrometer. CPY Activity Measurement—Protein extracts for CPY activity measurement were prepared by glass bead disruption in MES buffer containing 50 mm MES, 1 mm EDTA, and 2.5% methanol with a pH of 6.5. Protein concentrations were determined with a Bradford assay with bovine serum albumin as standard. CPY activity was determined by measuring hydrolysis of N-(3-[2-furyl]acryloyl)-Phe-Phe (FA-Phe-Phe) (Sigma) over time in MES buffer. Protein samples were mixed with substrate in a total volume of 1 ml and a FA-Phe-Phe concentration of 0.3 mm. Absorbance at 337 nm was measured with a Beckman DU7400 spectrophotometer. Endogenous CPY activity was measured by mixing 150 μg of whole cell protein extract with substrate. To measure CPY activity in protein mixtures with a known CPY/Tfs1p ratio, commercially available CPY (Sigma catalog no. 21943) was mixed with protein extracts from prc1Δ strains lacking endogenous CPY. The purity of the CPY was confirmed on one-dimensional minigels using a standard protocol (data not shown). The protein mixture was incubated at room temperature for 10 min and then mixed with FA-Phe-Phe. The CPY concentration used was 24 nm. The amount of Tfs1p in the protein extracts was quantified by two-dimensional PAGE analysis. Substrate hydrolysis was measured at fixed CPY concentration and various Tfs1p concentrations. The following equation was used to calculate Ki(app). [I]01−(Vi−V0)=Ki(app)(Vi−V0)+E0(Eq. 1) Vi represents the CPY activity in the presence and V0 represents the CPY activity in the absence of inhibitor. If [I]0/(1 – (Vi/V0) is plotted against 1/(Vi/V0) the slope of the line equals Ki(app) (23Henderson P.J. Biochem. J. 1972; 127: 321-333Crossref PubMed Scopus (479) Google Scholar). Bright Field and Fluorescence Microscopy—Bright field microscopy was performed on cells harvested from flask cultures. All samples were diluted to OD610 = 0.1 before cells were observed. Approximately 200 cells were examined for each sample. Microscopy pictures were taken using a Leica DM R microscope. Actin staining was performed using phalloidin-TRITC (Sigma catalog P-1951). Staining was preceded by fixation in 3.7% formaldehyde for 40 min. Calculation of Growth Variables and Growth Ratios— Growth rate was calculated as described elsewhere (24Warringer J. Blomberg A. Yeast. 2003; 20: 53-67Crossref PubMed Scopus (204) Google Scholar). To standardize the growth behavior of each strain, a wild type was included as a reference in each run, and a logarithmic strain coefficient (LSC) was calculated according to Equation 2, LSCij=∑r=13[13[∑k=12log(refkjr)]−log(xijr)]2(Eq. 2) where refkj is the growth variable of the kth measurement of the reference strain in environment j, xkj is the growth variable of strain i in environment j, and r indicates the run. From the LSC values a logarithmic phenotypic index (LPI), which describes the sensitivity of the strain for a specific inhibitor, was calculated according to Equation 3. LPIi=LSCt,inhibitor−LSCi0(Eq. 3) nat3Δ Exhibited Biphasic Growth—To study the salt-imposed stress response in the nat3Δ strain, wild-type FY1679 and nat3Δ cells were grown and labeled in SD medium and SD medium supplemented with 1 m NaCl in flask cultures. In both media the nat3Δ strain grew slower than the wild type. However, an interesting feature of nat3Δ growth was that the growth rate in early exponential phase was faster then in late exponential phase independent of the salinity of the medium. The breakpoint between the two phases was recorded at around OD610 = 0.5 (Fig. 1A). The generation time of the nat3Δ in basal medium was 3.2 ± 0.1 h in the first exponential phase (wild type grow in basal medium at 1.7 ± 0.1 h) and 5.8 ± 0.05 h in the second exponential phase, and a similarly prolonged doubling time was also observed in the second exponential phase for the saline cultures. In a previous study nat3Δ was reported to be salt-sensitive when grown on agar plates (7Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google Scholar). We found that the generation time for the nat3Δ strain was indeed longer in saline medium compared with the wild type, 7.8 ± 0.9 h (first exponential phase) compared with 4.0 ± 0.1 h for the wild type. However, the ratio between growth rates in the presence and absence of salt was roughly 2 for both strains, indicating that the nat3Δ strain was not specifically slow growing in salt but that it had a general growth defect. In contrast, the stationary phase yield of nat3Δ grown in saline medium was ∼50% lower than for salt-free nat3Δ culture, while the corresponding diminished yield value for the salt-grown wild type was less than 10% (Fig. 1A). We conclude that the reported agar plate salt defect (7Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google Scholar, 8Polevoda B. Cardillo T.S. Doyle T.C. Bedi G.S. Sherman F. J. Biol. Chem. 2003; 278: 30686-30697Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) is a combined effect from a generally slower rate of growth for the nat3Δ strain but most significantly from a decreased yield in salt. In addition, bright field microscopy revealed that nat3Δ cells exhibited an increased number of multiple buds. The highest number of cells with two buds or more was observed for exponentially growing cultures where almost half of the nat3Δ cells had multiple buds (Fig. 1, B and C) independent of which of the two exponential phases were analyzed. On the contrary, less then 10% of wild-type cells had two buds, and no cells with three buds or more were observed. In addition, as reported earlier (17Singer J.M. Shaw J.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7644-7649Crossref PubMed Scopus (73) Google Scholar), the mutant displayed defective actin cable formation, which was observed in both the first and second exponential phases (data not shown). Mass Spectrometric Confirmation of Act1p and Rnr4p as NatB Substrates—Protein expression was analyzed by two-dimensional PAGE with protein extracts from wild type and nat3Δ exponentially growing in SD medium in the absence and presence of salt. The lack of N-terminal acetylation on substrate proteins in NAT mutant strains is characterized by a shift in the isoelectric point of the protein. The horizontal shift in two-dimensional gels can thus be used to identify NAT substrates. Initially two earlier identified NatB substrate candidates (7Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google Scholar), Act1p and Rnr4p, were isolated from two-dimensional gels and characterized from both wild-type and nat3Δ extracts. Their N-terminal acetylation status was confirmed using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Proteins of wild-type origin were found to be acetylated, while proteins in nat3Δ lacked the acetyl group in both Act1p (Fig. 2, A and B) and Rnr4p (Fig. 2G and data not shown). The wild-type Act1p N-terminal peptide ion was found at m/z 993.4 (charge state, +2), while the corresponding Act1p ion in the nat3Δ was found at m/z 972.4 (charge state, +2). The difference of 42 Da indicated an N-terminal acetylation difference for the two Act1p forms in its N-terminal peptide. The unacetylated status of the N terminus of the Act1p produced in the nat3Δ strains was confirmed in the MS/MS mode where all detected b-series ions, which include the N-terminal end of the peptide, were shifted by 42 Da and thus indicated an acetylation difference. In this fragmentation series it was evident that the acetyl modification was present at one of the first two amino acids; the b1 fragments in this series could not be detected. However, in the case of Rnr4p we could identify the whole series of fragments and even score the acetylated methionine at its modified mass of 190.1 Da (Fig. 2, G and H; acetylated and oxidized methionine) confirming that this NatB-dependent modification occurs at the N-terminal methionine. Tfs1 Is a Novel Salt-induced NatB Substrate—Besides the earlier identified NatB substrates Act1p and Rnr4p we found a salt-induced (>4-fold) protein to be a NatB substrate (Fig. 3). In the wild type this protein was positioned on the two-dimensional gel corresponding to a mass of ∼24 kDa and a pI value of 6.5. In the nat3Δ this protein apparently shifted its pI to a slightly more basic value (Fig. 3). The protein spots were cut out of the two-dimensional gels from both the wild-type and the nat3Δ strains, in-gel trypsinized, and identified as YLR178C/Tfs1p by ESI-MS/MS analysis (Table II). The theoretical mass and pI value of Tfs1p is 24 kDa and 6.5, respectively, further supporting the identification. The N-terminal peptide from both the wild-type and the nat3Δ forms of Tfs1p were identified and characterized by MS/MS analysis. Similar to Act1p and Rnr4p, the wild-type form of the N-terminal peptide of Tfs1p was found to be 42 Da heavier than the nat3Δ form. In addition, the b-series fragment ions exhibited a 42-Da shift between the wild-type sample and the nat3Δ sample, supporting its N-terminal acetylation status (Fig. 2, D, E, and F). The y-series ions, which include the C terminus, were of the same m/z values in both samples. The reason why the pI shift of Tfs1p in nat3Δ was not recorded earlier is probably that previous studies of protein expression in nat3Δ (7Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google Scholar) have not included samples grown in salt; Tfs1p is poorly expressed in wild-type cells grown in the absence of salt and could therefore easily be missed.Table IITfs1p peptides from wild type and nat3Δ identified using ESI-MS/MSPeptide sequenceMassAa[M + H]+ ion mass (Da) calculated from the proposed sequence.XcorrbThe raw correlation score.ΔCncThe difference in correlation score of the top two candidate peptides (55).IonsdThe number of peptide" @default.
• W2048885488 created "2016-06-24" @default.
• W2048885488 creator A5083199812 @default.
• W2048885488 creator A5090536075 @default.
• W2048885488 date "2004-09-01" @default.
• W2048885488 modified "2023-10-18" @default.
• W2048885488 title "The Stress-induced Tfs1p Requires NatB-mediated Acetylation to Inhibit Carboxypeptidase Y and to Regulate the Protein Kinase A Pathway" @default.
• W2048885488 cites W139428495 @default.
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