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J. Biol. Chem. 2004; 279(37): p. 38532-38543

The stress induced  Tfs1p  requires NatB mediated acetylation  to inhibit carboxypeptidase Y  and to regulate  the protein kinase A pathway

Robert Caesar and Anders Blomberg

ABSTRACT

The Saccharomyces cerevisiae N-terminal acetyltransferase NatB consists of the subunits Nat3p and Mdm20p. We found by 2D-PAGE analysis that nat∆ 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 phosphatidylethanoamine­binding (PEBP) family member Tfslp was identified as a NatB substrate. The N-terminal acetylation status of Tfslp, Actlp and Rnr4p in both wild type and nat∆ was confirmed by tandem mass spectrometry. Furthermore, it was found that unacetylated Tfslp expressed in nat∆ showed an approximately 100-fold decrease in CPY inhibition compared to the acetylated form, indicating that the N-terminal acetyl group is essential for CPY inhibition by Tfslp. ΡΕΒΡ proteins in other organisms have been reported to be involved in the regulation of cell signaling. We here report that a number of proteins, whose expression earlier has been shown to be dependent on the activity in the protein kinase A (PKA) signaling pathway, was found regulated in line with low PKA activity in the nat∆ strain. The involvement of Nat3p and Tfslp in PKA signaling was supported by caffeine growth inhibition studies. Firstly, growth inhibition by caffeine addition (resulting in enhanced cAMP levels) was suppressed in tfs∆. Secondly, this suppression by tfs∆ was abolished in the nat∆ background, indicating that Tfslp was not functional in the nat∆ strain possibly because of a lack of N-terminal acetylation. We conclude that the NatB dependent acetylation of Tfslp appears essential for its inhibitory activity on CPY as well its role in regulating the PKA pathway.

Key word: N-terminal acetylation, NatB, carboxypeptidase Y, 2D-PAGE; phenotypic analysis

Introduction

N-terminal acetylation is, together with N-terminal methionine cleavage, 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 (1). N-terminal acetylation occurs co-translationally when nascent peptides are between 20 and 50 amino acids long (2,3). The addition of acetyl groups is catalyzed by N-terminal acetyl transferases (NATs). In Saccharomyces cerevisiae there are three known NATs: NatA, NatB and NatC (1). NatA consists of the subunits Ard1p and Nat1p (4). In addition, Nat5p was recently shown to be associated with Ard1p and Nat1p but does not seem to be important for NatA function (5). NatC consists of the subunits Mak3p, Mak10p and Mak31p (6). NatB, finally, consists of the subunits Nat3p (7) and Mdm20p (8). 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 (1).

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 (9). In the case of NatB an N-terminal sequence of MD-, ME-, MN- or MM- is required for acetylation. All proteins with N-terminal sequence 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-terminal are acetylated (10).

Nat1p and Ard1p orthologs in mouse were recently shown to form a complex with acetyltransferase activity (11) 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 indicate 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 (10).

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 (12), the unacetylated form of fetal hemoglobin, HbF, has been shown to stabilize the hemoglobin tetramer compared to the acetylated form (13), tropomyosin isolated from striated muscles is dependent on N-terminal acetylation to polymerize and to bind to F-actin in a correct way (14), and acetylation of actin in Dictylostelium has been shown to affect the weak interactions between actin and myosin (15). 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 (16), however, the mechanistic implications of this for the disease are not at present know.

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 (8). Both these protein need to be acetylated to interact and to form stable actin filaments (17). nat3∆ cells lacking functional NatB have been reported to exhibit many phenotypes including slow growth, sensitivity against 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 at 37°C, abnormal mitochondrial and vacuolar inheritance (8,17) and random budding polarity in diploid cells (18).

In this work we investigate the protein expression pattern of the nat3∆ strain during growth in high salt. We show 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 identify Tfslp as a novel NatB substrate. Tfslp is known to be an inhibitor of the protease carboxypeptidase Y (CPY) (19) and we show that N-terminal acetylation is important for the inhibitory activity of Tfslp. Furthermore, we found experimental evidence for that Tfslp negatively regulates signaling in the protein kinase A (PKA) pathway, and that this inhibitory effect also was acetylation-dependent.

Experimental Protocol

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 (7). The multi-copy plasmid pKT1067 with the TFS1 gene and a ura marker was kindly provided by Dr. J Winter with the kind permission by Dr. K. Tatchell. Double deletion mutants were obtained by mating of single mutants, sporulation of diploids and disection of tetrads followed by subsequent selection on plates containing 2000 g/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 w/o amino acids (YNB; DIFCO), 2% (w/v) glucose, 0.5% ammonium sulphate, 1% succinic acid) supplemented with appropriate amino acids. The medium was buffered to pH 5.8. Growth was inoculated with cells from stationary phase cultures 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 minutes at 4°C. All samples prepared were grown, harvested and analyzed in triplicates. Microcultivation was performed in 350 μl cultures for 48h in a Bioscreen analyzer C (Labsystems Oy, Finland) according to procedures in an earlier report (20). Duplicates of all samples were run on each plate. Two identical plates with cells from separate over-night 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 2D-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.

Two-Dimensional Electrophoresis

For the preparation of radiolabeled proteins 10  Ci of [³⁵S]-methionine (Amersham Bioscience; cat# SJ-235) was mixed with 10 ml of cell culture for 1/4 of a generation time under continuous shaking at 30°C. The isotope was added at OD610 = 0.35. Protein extracts of radio-labeled proteins were prepared by glass bead disruption and determination of incorporated {³⁵S}-methionine was performed as previously described (21). Protein concentrations were determined with TCA precipitation using a Lowry based commercial kit (Sigma, P-5656).

Protein extract with a total radioactivity of 2 x l0 6 dpm was loaded on each analytic gel and 1 mg of protein was loaded on each preparative gel. The IPG strips used were 18 cm long and covered pH 3-10; non-linear gradient (Amersham Bioscience). Two-dimensional electrophoresis was performed as previously described (21) using a Multiphor II (Amersham Bioscience) for running the first dimension and an Ettan DALT II (Amersham Bioscience) for running the second dimension.

Gels with radio-labeled proteins were dried and exposed to image plates for roughly 72 hours. The plates were scanned in a phosphoimager (Bio-Rad Molecular Imager FX) with a resolution of 200 x 200 μm. The raw files were processed and put together in a matchset in the 2D software PDQuest 6.2.0 (Bio-Rad Inc.). 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 matchset was performed. The proteins on the preparative gels were visualized with coomassie blue staining according to a published protocol (21).

Protein Trypsination, Mass Spectrometry and Analysis of MS/MS data

Gel pieces were cut out and in-gel trypsinated as described elsewhere (21). Peptides were eluted in 8 μl eluation buffert containing 2% acetonitrile and 0.05% formic acid. Peptide separation was performed on a Finnigan Surveyor chromatography workstation (Finnigan Corp., San Jose, CA) using a 150 x 0.18 mm C₁8 HyPurity column (Thermo Hypersil) or a 150 x 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 minutes. Flow rate was 2 μl/minute. The LC device was directly coupled to a Finnigan LCQ 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 minute was applied. Where indicated mass spectrometry was performed using MALDI-TOF as described elsewhere (22).

The BEQUEST search algorithm was used to correlate experimental mass data to theoretical mass data derived from the sequence in yeast.fasta database from the National Center for Biotechnology Information (NCBI). Protein identifications based on tandem mass spectra correlating to at least 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 ∆Cn 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 centre in Göteberg on a Micromass mass spectrometer.

CPY activity measurement

Protein extracts for CPY activity measurement were prepared by glass bead disruption in MES buffert containing 50 mM MES, 1 mM EDTA, and 2.5% methanol and with a pH of 6.5. Protein concentrations were determined with a Bradford assay with BSA 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 buffert. 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 whole cell protein extract with substrate. To measure CPY activity in protein mixtures with a known CPY/Tfslp ratio, commercially available CPY (Sigma, 21943) was mixed with protein extracts from prcl∆ strains lacking endogenous CPY. The purity of the CPY was confirmed on 1D mini-gels using a standard protocol (data not shown). The protein mix was incubated at room temperature for 10 minutes and then mixed with FA-Phe-Phe. The CPY concentration used was 24 nM. The amount of Tfslp in the protein extracts were quantified by 2D-PAGE analysis. Substrate hydrolysis was measured at fixed CPY concentration and various Tfslp concentrations. The following equation was used to calculate Ki(app):

V; represents the CPY activity in presence and Vo the CPY activity in absence of inhibitor. If {I}o/(1 - (Vi/Vo) is plotted against 1/(Vi/Vo) the slope of the line equals Ki(app) (23).

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, P-1951). Staining was preceded by fixation in 3.7% formaldehyde for 40 minutes.

Calculation of growth variables and growth ratios

Growth rate was calculated as described elsewhere (24). To standardize the growth behavior of each strain compared to a reference strain, a wild type was included as a control in each run and a Logarithmic Coefficient, LSC, was calculated according to the equation:

where ref^r_kj is the growth variable of the k:th measurement of the reference strain in environment j, x^r_ij 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 the equation:

Results

nat3∆ exhibited biphasic growth

In order to study the salt imposed stress response in the nat3∆ strain, wild-type and nat3∆ cells were grown and labeled in SD medium and SD medium supplemented with 1M NaCI in flask cultures. In both media the nat3∆ strain grew slower than the wild type. However, an interesting feature of nat3∆ 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 (Figure lA). The generation time of the nat3∆ in basal medium was 3.2±0.1h in the first exponential phase (wild type grow in basal medium at 1.7±0.1h) and 5.8±0.05h 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 nat3∆ has been reported to be salt-sensitive when grown on agar plates (7). We found that the generation time for the nat3∆ strain was indeed longer in saline medium compared to the wild type, 7.80.9 h (first exponential phase) compared to 4.0±0.1h for the wild type. However, the ratio between growth rates in the presence and absence of salt was roughly 2 for both strains, indicating the nat3∆ strain not being specifically slow-growing in salt but having a general growth defect. In contrast, the stationary phase yield of nat3∆ grown in saline medium was approximately 50% lower than for salt-free nat3∆ culture, while the corresponding diminished yield value for the salt grown wild-type was less than 10% (Figure lA). We conclude that the reported agar plate salt defect (7,8) is a combined effect from a generally slower rate of growth for the nat3∆ strain but most significantly from a decreased yield in salt.

In addition, bright-field microscopy revealed that nat3∆ 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 nat3∆ cells had multiple buds (Fig. 1B and 1C), independent of which of the two exponential phases were analysed. 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, the mutant displayed defective actin cable formation, as earlier reported (17), which was observed in both the first and second exponential phase (data not shown).

Mass spectrometric confirmation of Actlp and Rnr4p as NatB substrates

Protein expression was analyzed on 2D-PAGE gels with protein extracts from wild-type and nat3∆ exponentially growing in SD medium in 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 protein's isoelectric point. The horizontal shift in 2D gels can thus be used to identify NAT substrates. Initially, two earlier identified NatB substrate candidates (7), Act1p and Rnr4p, were isolated from 2D gels and characterized from both wild-type and nat3∆ extracts and their N-terminal acetylation status was confirmed using electro-spray ionization tandem mass spectrometry (ESI-MS/MS). Proteins of wild-type origin were found to be acetylated, while proteins in nat3∆ lacked the acetyl group in both Act1p (Fig. 2A, B, C) and Rnr4p (Fig. G, H). 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 nat3∆ was found at m/z 972.4 (charge-state +2). The difference of 42Da indicated a 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 nat3∆ 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 42Da 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 bl 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 weight of 190.1Da (Fig. 2G, H; acetylated and oxidised methionine) confirming 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 2D gel corresponding to a mass of approximately 24 kDa and a pI value of 6.5. In the nat3∆ this protein apparently shifted its pI to a slightly more basic value (Fig. 3). The protein spots were cut out of the 2D gels from both wild type and the nat3∆ strain, in-gel trypsinated and identified as YLR178C/Tfslp by ESI-MS/MS analysis (Table II). The theoretical weight and pI value of Tfslp is 24 kDa and 6.5, respectively, further supporting the identification. The N-terminal peptide from both the wild-type and the nat3∆ form of Tfslp were identified and characterized in MS/MS analysis. Similar to Act1p and Rnr4p, the wild-type form of the N-terminal peptide of Tfslp was found to be 42Da heavier than the nat3∆ form. In addition, the b-series fragment ions exhibited a 42Da shift between the wild-type sample and the nat3∆ sample, supporting its N-terminal acetylation status (Figure 2D, E, 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 Tfslp in nat3∆ was not recorded earlier is probably that previous studies of protein expression in nat3∆ (7) have not included samples grown in salt; Tfslp is poorly expressed in wild-type cells grown in the absence of salt and could therefore easily be missed.

N-terminal acetylation of Tfslp appears essential for its inhibitory activity on CPY

The NatB substrate Tfslp is reported to be an inhibitor of the vacuolar protease carboxypeptidase Y (CPY) (19). It has previously been shown that Tfslp expressed in E. coli inhibits CPY with lower efficiency than Tfslp expressed in yeast (25). Since prokaryotes do not express NATs it has been claimed that the lack of N-terminal acetylation is the reason for the difference in inhibitory activity. In addition, our 2D analysis revealed that many proteins in the high molecular weight region of the gels, e.g. Hsc82p, Hsp82p, Eft1p, Met6p and Aco1p, were down-regulated in nat3∆ (Figure 4). This may indicate an elevated protease activity, potentially caused by increased CPY activity in nat3∆ compared to wild-type because of the non-functional unacetylated CPY inhibitor Tfslp in this strain.

To substantiate that the N-terminal acetylation of Tfslp influences the inhibition of CPY, even for natively expressed Tfslp (to circumvent any secondary effects from heterologous expression in another host like E. coli resulting in inclusion bodies) aCPY-inhibition test of endogenous CPY activity was first performed. Breakdown activity on the CPY substrate FA-Phe-Phe was initially measured in whole cell extracts. The strains included in the study were wild-type, nat3∆, tfs1, prc1∆ (PRC1 coding for CPY), mdm20∆ (the second subunit of the NatB system) and nat3∆tfs1 ∆. CPY inhibition was measured in strains grown in basal medium. The prcl∆ strain, lacking CPY, did not exhibit any CPY activity indicating that the assay is specific for this single protease (Fig. 5). Most importantly, the nat3∆ and the mdm20∆ strain, both lacking NatB, showed a 50% increased CPY activity (loss of CPY inhibition) compared to wild-type. A similar increase in CPY activity was recorded for the tfs1 ∆ strain. In addition, the double mutant nat3∆tfs1 ∆ exhibited a CPY activity similar to tfs1 and nat3∆, indicating an epistatic relationship and that the N-terminal acetylation of Tfslp is of vital importance for its inhibition of CPY.

To ensure that the difference in CPY activity between nat3∆ and wild-type is not caused by differences in expression or modification of the endogenous CPY in the mutant background, a prcl∆ strain and a prcl∆nat3∆ were transformed with amulti-copy plasmid expressing TFS1. Varying amounts of protein extracts were mixed with a fixed amount of commercially available CPY and the CPY activity/inhibition was determined as described above. The amount Tfslp in the protein extract was quantified by 2D-PAGE analysis. In Figure 6 the residual CPY activity of the two different protein mixtures are plotted against the Tfslp/CPY ratio. The _Ki(app) for the acetylated and the unacetylated form of Tfslp were estimated to 3x10^-9 and 2x10-7^-7 M, respectively. These results clearly show that Tfslp with an N-terminal acetyl group inhibited CPY approximately 100-fold more efficiently then the unacetylated form of Tfslp.

Global protein expression in nat3∆ indicates a connection to the general stress response via PKA

In total 582 spots were matched between 2D gels of whole cell protein extracts from wild-type and nat3∆ cells grown in basal and saline medium. The proteins were labelled at OD610 = 0.35, when nat3∆ cells were in the first phase of exponential growth (Fig. 1). 64 proteins were found down-regulated three-fold or more and 51 were up-regulated three-fold or more in nat3∆ compared to wild-type when grown in basal medium (only including spots where the regulation were statistically significant by log Student t-test criteria). Regulated spots were, if enough material was available for analysis, identified by mass spectrometry (Table III).

Surprisingly we found that many of the expression changes scored in the nat3∆ strain in basal medium mimicked changes observed in the wild type during stress (Fig. 7A-D). A hierarchical clustering of wild-type and nat3∆ grown in presence and absence of salt revealed that nat3∆ grown in basal medium clustered close to wild-type grown in salt and that wild-type grown in basal medium cluster far from the other samples (Fig. 7E). The most strongly altered regulation in nat3∆ compared to the wild type was Zps1p that was found to be up-regulated 38-fold in basal medium. Zps1p, a homolog to the Candida albicans protein Pra1p with similarity to Zn-metalloproteases (26), has earlier been reported to be up-regulated in alkaline medium (27) and to be located in the vacuole in S. cerevisiae (28). The DNA damage induced (29) and flocculent specific (30) protein Ddr48p was up-regulated 5.4 fold in nat3∆. Ddr48p has previously been reported to be induced when exposed to various stresses like ethylmethane, sulfonate, heat shock and osmotic stress (31,32). The glycolytic enzymes enolase I and glyceraldehyde 3-phosphate dehydroxygenase I (Eno1p and Tdh1p) were both up-regulated in nat3∆. Tdh1p has been reported as induced in response to osmotic stress (33) as well as changes in the intracellular redox balance (34). The unacetylated form of Tfslp was up-regulated 4-fold in nat3∆ compared to the acetylated form in wild-type (Fig. 3). It is noteworthy that also many of the down-regulated proteins during salt stress in the wild type, e.g. Adh1p, Gdh1p and Met6p, displayed repression in nat3∆ grown in the absence of salt.

Tfslp, identified as a NatB substrate in the present work, has previously been reported as a multi-copy suppressor of the temperature sensitive cdc25-1 mutant (35). Cdc25p is an activator of the protein kinase A (PKA) pathway. The expression of many of the salt stress induced proteins have earlier been shown to be partly or fully dependent on a the level of PKA activity (33). Protein expression in nat3∆ was compared with the expression of proteins previously shown to be regulated by the PKA pathway. Among five proteins reported to be upregulated and eight proteins reported to be down-regulated in a fully or partly PKA dependent manner, all but one exhibited the same type of regulation in nat3∆ during growth in basal medium (Fig. 8) indicating low PKA activity in this strain background.

However, the salt regulation of protein expression is complex and various pathways besides PKA, like the HOG MAP kinase pathway, the cell integrity pathway, and the calceneurin/calcium dependent pathway, are involved. An intricate interplay between these different stress activated pathways sets the final stress output (36). In particular, several of these pathways exhibit partial requirement for PKA or even an antagonistic role to PKA. The latter can be exemplified by the opposing roles played by the calceneurin pathway and PKA (37), and the PKA repression of the nuclear localization of the important transcription factors Msn2/4p and Sko1p during osmotic stress (36). Thus, we don't find it surprising that the salt regulation observed in the nat3∆ strain differs markedly from the wild type response. In fact, 8 of the 46 proteins stress induced in the wild type was down regulated in nat3∆ during stress (e.g. Fig. 7A, C, D) which exemplifies the antagonistic roles played by PKA to certain other stress pathways.

Phenotypic analysis of caffeine sensitivity supported that NatB and Tfslp are linked to the PKA pathway

The putative link between NatB and the PKA pathway from the expression data presented here is interesting given that some mammalian homologues to the NatB substrate Tfslp has been shown to affect signaling via interaction with certain kinases (see discussion). Even more importantly, overexpression of TFS1 has been shown to suppress the temperature sensitivity of a point mutation in CDC25 (Cdc25p is believed to act as a positive factor for Ras (35)). The link to the PKA pathway can be experimentally studied by the use of the growth inhibitor caffeine, since caffeine inhibits cAMP phosphodiesterase and thereby increases the intracellular concentration of cAMP. High concentrations of cAMP results in a high PKA activity. Thus, to further investigate the connection between NatB, Tfslp and the PKA pathway, we compared the phenotype of various single and double deletion mutants in the presence of growth inhibiting concentrations of caffeine.

The concentrations of caffeine applied resulted in growth rate retardation in the wild type in the range 30 - 300% (Fig. 9 and data not shown). The link between Tfslp and PKA was apparent in the strong suppression of the caffeine imposed growth defect by a TFS1 deletion (Fig. 9A). The suppressive effect was most apparent at the higher caffeine concentrations, which is clearly seen in the step-wise enlarged LPI values for tfs1 ∆ with increased concentration of the inhibitor (Fig. 9C). A positive LPI indicates resistance to that environment compared to the response in a reference strain (24). However, the suppressive effect was totally absent if the TFS1 gene was deleted in the nat3∆ background, supporting the notion that the Tfslp link to PKA is fully dependent on its N-terminal acetylation (Fig. 9C). The suppressive effect of tfs1 (possibly from lowering the intracellular cAMP level) on caffeine growth inhibition, was in line with the observed protein expression change that indicated lower cAMP level in the nat3∆ strain (with a unacetylated and non-functional Tfslp).

Is the tfs1 suppression of growth inhibition by caffeine related to its role as a CPY inhibitor? To address this question we deleted the TFS1 gene in the prc1∆ background. Most importantly, and similar to the case where TFS1 was deleted in the nat3∆ background, the double deletion did not show any improvement in growth compared to the single prc1∆ strain indicating an epistatic relation. In addition, the single deletion, prc1∆, displayed resistance to caffeine in the same range as for the tfs1 (Fig. 9C). These results indicate that the tfs1 suppressive effect is epistatic and "upstream" of CPY.

Discussion

Sequence requirements for NatB substrates

N-terminal acetylation is believed to occur on almost 50% of all yeast proteins (1). However, the loss of the NAT3 gene, encoding the catalytic subunit for NatB, is reported to influence the N-terminal acetylation on a limited number of yeast proteins; ten NatB substrates have previously been identified, including Act1p, Tpm1p, Rnr4p, four ribosomal proteins (Rps21ap, Rps21bp, Rps28ap, and Rps28bp) and three subunits of the 26 S proteasome (Pre1p, Rpt3p, and Rpn11p)(Table IV) (7,17,38-40). The sequence requirement for NatB substrates is proposed to be rather strict and so far all native MD- and ME- termini studied have been found acetylated (10). However, the number of proteins in the whole yeast proteome with these termini is almost 600, indicating that many more proteins are potentially modified by the NatB system in yeast.

By extending our analyses of the nat3∆ strain to salt stress conditions, we here report on the identification of yet another NatB substrate, Tfslp. Tfslp is the first native yeast protein with a MN- N-terminus that has proven to be a NatB substrate; this N-terminal sequence specificity for NatB was earlier proposed by studies on mutated variants of iso-1-cytochrome c (Table IV). NatA dependent N-terminal acetylation in yeast requires the removal of the initial methionine before completion of the nascent protein chain by the action of either of the two methionine aminopeptidases, Map1p or Map2p (9). Experimental evidence has been presented that removal of methionine is dependent on whether the penultamate residue has a radii of gyration that is less than 1.29Å. Contrary to NatA, NatB acetylates on the first methionine of nascent chains not subjected to methionine cleavage. Since asparagine, similiarly to aspartate and glutamate, has a radii of gyration greater than 1.29Å, proteins with an asparagine as the second residue will retain their N-terminal methionine and are thus potentially good NatB substrates.

Tfslp has the N-terminal amino acid sequence MNQAI-. Some mutant proteins with an N-terminal MN- remain unacetylated; i.e. iso-l-cytochrome c which has an N-terminal MNQFL- or MNEKL (41). To our knowledge Tfslp is the first protein (native or mutant form) with an MNQ- N-terminus found to be acetylated. This indicates that the amino acid sequence determining if a protein with an MNQ- N-terminal will be acetylated by NatB stretches beyond the third amino acid. This is not surprising given that the sequence requirements for acetylation in general has even been reported to extend beyond the fifth amino acid residue for MC- and MT- termini (7). Thus, data indicates that upstream inhibitory residues are present in some putative NatB substrates with MN- terminal sequences, providing less precise theoretical predictions about the acetylation status of these proteins.

Tfslp belongs to the PEBP family of inhibitors

Tfslp is a member of the widespread PEBP (phosphatidylethanolamine-binding protein) family. Crystal structure determination of both mammalian and prokaryotic members of the family has revealed that they display almost identical []-fold topology, despite rather low sequence similarity, suggesting conserved function throughout the family (42). PEBP proteins have been associated with different forms of protein inhibition activity. In rat and human cells the Tfslp sequence homolog RKIΡ (Raf-1 Kinase Inhibitor Protein; formally known as PEBP) has been shown to suppress Raf-1 kinase activity. By binding to either Raf-1 or its substrate MEK-1, RKIΡ prevents phosphorylation of ΜΕΚ-1 and thereby regulates the mitogen-activated protein (MAP) kinase pathway that activates the extracellular signal regulated kinases (ERK) (43,44). In addition to inhibiting Raf-1, RKIΡ was recently shown to inhibit transcription factor nuclear factor kappa B (NF-kB) activation by physically interacting with NF-kB-inducing kinases (45). The multiple kinase inhibitory functions of RKIP, together with genetic studies that indicates that PEBP proteins in plants function in signal transduction pathways (46-49), have led to the suggestion that PEBP proteins represent a widely conserved family of protein kinase regulators. In this study we also show that Tfslp acts as an inhibitor of the Protein Kinase A signaling pathway in yeast, in line with its reported role as a multicopy suppressor of the cdc25-1 mutation (35).

Tfslp has earlier been characterized as a high-affinity inhibitor of the vacuolar enzyme carboxypeptidase Y (CPY) (19,25), which is a serine-type protease involved in the C-terminal processing of peptides and proteins. This CPY inhibitory function we have confirmed in vitro, and, in addition, we point out numerous high molecular weight proteins that were down-regulated in vivo in nat3 indicating a generally increased protease activity. Furthermore, PEBP from mouse brain, to which Tfslp shows 31 %sequence identity, has been shown to be an inhibitor of several different serine-proteases including thrombin, neuropsin and chymotrypsin (50), supporting a more general role of PEBP members as protease inhibitors in diverse organisms. The fact that Tfslp acts both on the Protein Kinase A

signaling pathway and as a potent inhibitor of the vacuolar protease CPY, indicates Tfslp as the first member of the PEBP/RKIP family with manifested dual inhibitory roles.

Functional significance of the N-terminal acetylation of Tfslp

Since the N-terminal portion of Tfslp has been shown to be important for the inhibitory action on CPY (19) and the fact that an unacetylated form of Tfslp, expressed as inclusion bodies in E. coli, displayed impaired inhibitory activity on CPY (25), we performed a CPY activity/inhibition assay to study the impact of the N-terminal acetylation of endogenously expressed Tfslp. The assay clearly showed that the acetylated form of Tfslp inhibited CPY much more efficiently than the unacetylated form; _Ki(app) of Tfslp expressed in nat3 was increased 100-fold compared to Tfslp expressed in wild-type. Tfslp expressed in E. coli has been shown to have K; values for anilidase and peptidase activity of CPY increased by 700- and 60-fold, respectively, compared to Tfslp expressed in yeast (25). We thus believe that our data together with earlier data on heterogenously expressed Tfslp firmly establish the inhibitory activity of Tfslp to be dependent on the existence of the N-terminal acetyl group.

What can be the molecular mechanisms for this major effect on inhibitory activity of Tfslp from only this minor protein modification? First of all, it would be expected that the acetylated N-terminus would be exposed on the surface of the Tfs 1 protein. The structure of the yeast Tfslp has not been determined, however, the structural conservation between the bacterial and mammalian homologs indicates that the yeast form would be structurally rather similar. Importantly, the determined crystal structures of both bacterial forms expose their N-terminus (42). Currently no structure for the complex between a PEBP inhibitor and a carboxypeptidase has been determined. However, the structure of the human carboxypeptidase A2 in complex with the leech inhibitor has been determined by X-ray crystallography (51). Even if the leech carboxypeptidase inhibitor does not display any sequence homology to the yeast Tfslp, it is interesting to note that it is the terminus of the leech inhibitor (however, in this case the C-terminus) that extends into the active site of the carboxypeptidase and displaces the reactive water molecule. It can be hypothesized that Tfslp mechanistically acts in a similar way, but instead protrudes its acetylated N-terminus into the CPY active site. Consistent with this view is the experimental finding that the specific CPY reagents PMSF and PCMB reacted more rapidly against the catalytic site residues Ser146 and Cys341 of CPY when in complex with the in unacetylated Tfslp complex compared to when Tfslp was properly acetylated (25).

General functional significance of NatB dependent acetylation

The overall importance of the NatB modification system in yeast is revealed during growth since nat3 cells grow at approximately half the rate of wild-type cells. Even though NatA appears to have many more substrates than NatB, strains deleted in NatA subunits grow better in normal growth media than strains lacking a functional NatB ((7), R. Caesar et. al, unpublished results). In this study we show that Tfslp is N-terminally acetylated by NatB and demonstrate that the acetyl group is essential for the inhibition of CPY and for regulating the PKA pathway. Tfslp is the third NatB substrates that depends on its acetyl group to be fully functional. Considering that none of the numerous known NatA substrates has been reported to exhibit altered function in its unacetylated form, the frequent functional importance of NatB mediated acetylation is striking.

The functional importance of NatB mediated acetylation of actin and tropomyosin has been reported. When expressed in strains defective in NatB these proteins partially lose their activity. N-terminal acetylation of both proteins are important for F-actin-tropomyosin interaction, which is required to stabilize actin filaments (17). The loss of actin-tropomyosin interaction in nat3 is believed to cause dramatic changes in the cell physiology and the co-occurrence of this defect with anon-functional Tfslp, and possibly other proteins with altered function, makes the analysis of the nat3 phenotype complex. However, in an elegant series of experiments Polevoda et al. established the great importance of acetylation of actin and tropomyosin for the recorded nat3 phenotypes on a great variety of growth conditions (8). It was shown that many of these nat3 phenotypes, like NaCI or thermal (37°C) sensitivity, could be suppressed by expressing alleles of actin and tropomysin that were altered in their N-terminal sequences and that had earlier been shown to be dominant suppressors of mdm20∆ (17) and thus believed to be functional even without the corresponding N-terminal acetylation. Full to partial suppression of the phenotypes were found on most of the environments tested, highlighting the importance of Act1p and Tpm1p acetylation in establishing the nat3 phenotype. Similar dominant acetylation-independent variants of Tfslp are not presently available, making corresponding analysis of the phenotypic consequences of non-acetylation of Tfslp in isolation not feasible.

The acetylation of Tfslp appears essential for its functional link to the PKA pathway As been described above Tfslp orthologs exhibit kinase inhibitory activity. The observation that over-expression of Tfslp suppresses the phenotype of cdc25-1 (35) suggests

a connection to the control of cell signaling in S. cerevisiae as well. Cdc25p functions as an activator of Ras, which in turn activates the downstream portion of the PKA pathway (Fig. 10). By comparing protein expression in nat3 with expression of proteins reported to be regulated by the PKA pathway we found indications of a low PKA activity in nat3d Transcriptional regulation by the promotor element STRE (stress-responsive element; CCCCT) has been demonstrated to be highly dependent on the level of PKA activity (52). All of the proteins listed as up-regulated in nat3 in Table III, except Eno1p and Zps1p, have two or more STRE element in their pomotors strengthening a possible PKA mediated regulation. TFS1 has two STRE elements and has been reported as strongly up-regulated during steady-state growth in 1 M NaCI (Alipour et al., submitted manuscript) and during various stress conditions including oxidative stress, osmotic stress and heat-shock (53). In this work the up-regulation during osmotic stress is confirmed at the protein level and we also found that Tfslp is strongly up-regulated in nat3 both in basal medium and in salt supplemented medium. Interestingly, the TFS1 regulation during salt-stress and osmotic stress is completely abolished in a MSN2/4 deletion strain indicating aPKA-dependent regulation (53). Since the TFS1 promoter contains STRE elements this provides a set-up for a negative feedback loop of the general stress response; as described below more Tfslp leads to greater inhibition of Ira2p, which leads to increased PKA activity resulting in lower expression of TFS1 (Fig. 10).

Caffeine inhibition of cAMP phosphodiesterase leads to increased levels of cAMP that results in high PKA activity. We predict from our phenotypic growth data that inactivation of Tfslp, via gene deletion or loss of the N-terminal acetyl group, decreases the cAMP level and thus suppresses the caffeine growth inhibition. If the target for inhibition by Tfslp is upstream of the action of caffeine, a deletion of TFS1 should counteract the caffeine-induced phenotype in wild-type background (Fig. 10). Our data, showing that tfs1 ∆ grew better than wild-type during caffeine inhibition, supports this assumption. In line with our model it was during the latter phase of this work reported that Tfslp physically interacts strongly with Ira2p and that Tfslp in that way inhibited the GTPase promoting activity of Ira2p on Ras (54).

In addition, a deletion of TFS1 in nat3 background should not show the same suppressive effect on caffeine growth inhibition if the N-terminal acetylation of Tfslp is essential for its activity. Interestingly, we found that nat3 and nat3∆tfs1 ∆ are equally sensitive against caffeine indicating the caffeine suppressive effect to be acetylation dependent. In line with the presented physical interaction between Tfslp and Ira2p by Benedetti and co-workers (54) one can predict that N-terminal acetylation of Tfslp is important for this interaction. Since Tfslp is dependent on its acetylation status it might at

first seem strange that the nat3 strain is more caffeine sensitive than the wild type (Fig. 9). However, the earlier mentioned strong influence on the nat3 phenotypes from unacetylated and not fully functional actin and tropomysin could mask the suppressive effect of the non-functional Tfslp on caffeine growth inhibition. In fact, at higher caffeine concentrations the relative resistance of nat3 was increased similarly to the tfs1 ∆ strain; if the caffeine data is displayed as a relative response (setting the LPI at the lowest caffeine concentration to 1) the caffeine dose-response pattern is almost identical between nat3 and tfs1. We interpret this relative dose-response behavior as a positive caffeine suppressive effect from anon-function Tfslp also in the nat3 strain..

Interestingly, we also found the suppression of growth inhibition by caffeine by TFS1 deletion to be dependent on an intact CPY. This would place CPY mechanistically down-stream of Tfslp. Bruun et al. earlier predicted that the suppressive effect on the cdc25-1 mutation from TFS1 overexpression was not caused by complete inhibition of CPY (which in principle could be the result from TFS1 overexpression) since a prc1∆ did not suppress the cdc25-1 mutation (19). However, TFS1 suppression of cdc25-1 could still be dependent on CPY activity but be operational via some other mechanisms than CPY inhibition, and we hey propose a model where Ira2p is selectively degraded in the vacuole by CPY and where this delocalisation is dependent on Tfslp physically interacting with both Ira2p and CPY (Fig. 10). This interesting putative interconnection between the dual functionalities of the Tfs1 protein certainly deserves further in depth analysis.

Acknowledgements

Financial support from the Swedish national research council (VR) and the foundation of strategic research (SSF) is gratefully acknowledged.

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