©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Phosphorylated Ribosomal Protein S7 in Tetrahymena Is Homologous with Mammalian S4 and the Phosphorylated Residues Are Located in the C-terminal Region
STRUCTURAL CHARACTERIZATION OF PROTEINS SEPARATED BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS (*)

(Received for publication, November 17, 1994)

Lisbeth Palm (§) Jens Andersen Henrik Rahbek-Nielsen Torben S. Hansen (¶) Karsten Kristiansen Peter Højrup (**)

From the Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A single basic ribosomal protein, protein S7, can be multiply phosphorylated in the ciliated protozoan Tetrahymena. Induction of phosphorylation is highly regulated, and the phosphorylation proceeds in a strictly sequential manner. The first site to be phosphorylated is a serine residue and the second a threonine. In this paper we report the complete primary structure of Tetrahymena thermophila ribosomal protein S7 including identification of the phosphorylated serine and threonine residues. Most of the sequence information was obtained from peptides generated by in situ digestion of S7 in two-dimensional gels using an approach that combined traditional protein chemistry with mass spectrometry. T. thermophila ribosomal protein S7 has a molecular mass of 29,459 Da and contains 259 amino acid residues. Phosphorylation takes place on Ser and Thr in the C-terminal region of the protein. Alignment of T. thermophila ribosomal protein S7 with known ribosomal proteins yielded the surprising result that T. thermophila S7 is homologous, not with mammalian ribosomal protein S6, but with mammalian ribosomal protein S4. These findings clearly distinguish the pattern of phosphorylation of ribosomal proteins in Tetrahymena from all other eukaryotes analyzed to date.


INTRODUCTION

In all eukaryotic cells, one or a few of the ribosomal proteins are subject to phosphorylation. The actual number of phosphorylated ribosomal proteins varies from species to species, but a single basic ribosomal protein (r-protein) (^1)with an M(r) around 30,000 is normally found to be the most prominent phosphoprotein in eukaryotic ribosomes (Wool, 1979; Cuny et al., 1985; Bratholm, 1986). Phosphorylation of this ribosomal protein, which in higher eukaryotes is designated S6, is regulated in response to a variety of external stimuli. Up to five phosphate groups can be incorporated into mammalian S6 (Thomas et al., 1980). The phosphorylation proceeds in a sequentially ordered manner, and all phosphorylation sites are clustered in the C-terminal region of S6 (Martin-Pérez and Thomas, 1983; Krieg et al., 1988; Ferrari et al., 1991; Bandi et al., 1993). The general consensus is that phosphorylation is confined to serine residues although threonine phosphorylation has been reported (Martin-Pérez and Thomas, 1983). Increased S6 phosphorylation is correlated with growth and activation of protein synthesis, and a substantial number of observations are compatible with the notion that increased S6 phosphorylation is a prerequisite for activation of protein synthesis in response to growth factors (Thomas et al., 1982). Recently, it was shown that the mitogen-activated S6 kinase, p70 is essential for G1 progression (Lane et al., 1993).

In lower eukaryotes, the number of phosphate groups incorporated into the major basic ribosomal phosphoprotein varies from two to four (for reviews, see Cuny et al.(1985) and Bratholm(1986)). In yeast, molecular cloning has unambiguously demonstrated that the major basic ribosomal phosphoprotein, S10, is indeed a true S6 homolog (Leer et al., 1982). However, the functional significance of S10 phosphorylation in yeast is unclear (Johnson and Warner, 1987; Vanoni and Johnson, 1991). In the majority of cases, the conditions leading to increased phosphorylation in lower eukaryotes resemble those leading to increased S6 phosphorylation in mammals, i.e. phosphorylation is increased when the cell growth is stimulated (Cuny et al., 1985; Bratholm, 1986). This, and the fact that the location on two-dimensional gels of the major phosphoprotein in lower eukaryotes is comparable to that of S6, has led to the general assumption that the major basic ribosomal phosphoprotein in lower eukaryotes is homologous with mammalian S6.

In the ciliated protozoan, Tetrahymena phosphorylation of ribosomal proteins is confined to a single basic 40 S protein, originally named S6, now named S7, and previously believed to be the Tetrahymena S6 homolog (Kristiansen et al., 1978). However, the conditions that induce phosphorylation in Tetrahymena are virtually opposite to those leading to increased phosphorylation of S6 or its homolog in other species. Thus, no phosphorylation of ribosomal protein S7 is observed in growing Tetrahymena cells, and phosphorylation of S7 is induced when cells are starved in the presence of sodium or lithium ions or high concentrations of Tris (Cuny et al., 1985; Bratholm, 1986). The order of phosphorylation is strictly regulated. The first amino acid residue to become phosphorylated is a serine residue, and the second a threonine residue (Bratholm, 1986). This led to the suspicion that Tetrahymena S7 was not homologous with mammalian S6, and, recently, by using partial sequence information, we presented evidence that the phosphorylated Tetrahymena S7 most likely is homologous to mammalian S4 (Palm et al., 1993).

Thus, a detailed structural analysis of Tetrahymena S7 seemed warranted. In this report we present the complete primary structure of Tetrahymena S7 including identification of the phosphorylated serine and threonine residues. Purification of ribosomal proteins is known to be difficult, due to sparse solubility and aggregation (Ghrist et al., 1990). Therefore, an objective of the work presented in this report was also to explore the potential and limitations in sequencing of an unknown protein isolated directly from polyacrylamide gels by using an approach that integrated traditional protein chemistry with mass spectrometry.


MATERIALS AND METHODS

Chemicals and Reagents

Trypsin (EC 3.4.21.4) was a gift from Novo-Nordisk A/S (Bagsværd, Denmark). Staphylococcus aureus V8 protease (EC 3.4.21.19) was from ICN Biochemicals. Endoprotease Asp-N was from Boehringer Mannheim (Mannheim, Germany). Chymotrypsin (EC 3.4.21.1) was from Merck (Darmstadt, Germany). Cyanogen bromide and alpha-cyano-4-hydroxycinnamic acid was from Aldrich.

Dynosphere resin was from Dynochrom Sverige AB (Sweden). Shandon C(8), 5-mm particle size, was from Macherey-Nagel (Düren, Germany). Reagents for the protein sequencer were from Knauer Biochemie (Berlin, Germany). HPLC grade acetonitrile was from Rathburn Chemicals (Walkerburn, UK), and ultra-high-quality water was obtained by an Elgastat (Elga, High Wycombe, Bucks, Scotland). All other chemicals and buffers used were analytical grade quality.

Growth of Tetrahymena thermophila Cells and Extraction of Ribosomal Proteins

T. thermophila cells were grown, starved, and labeled with [P]orthophosphate as described previously (Kristiansen et al., 1978). Ribosomes were isolated, and r-proteins were extracted as described (Kristiansen et al. 1978).

Analytical and Preparative Two-dimensional Polyacrylamide Gel Electrophoresis (Two-dimensional PAGE)

For polyacrylamide gel electrophoresis, the Hoefer SE 220 Tube gel system and SE 250 Mighty Small II system (Hoefer) were used.

Two-dimensional PAGE was carried out as described previously using the pH 8.6/dodecyl sulfate system (Dreisig et al., 1984). The precipitated r-proteins were dissolved in sample buffer to a final concentration of 5 mg/ml, and 30-50 µg of r-protein was applied for analytical purposes. Preparative gels were run with less complex r-protein mixture, and the amount of Tetrahymena S7 applied per gel was approximately 2 µg. Electrophoresis was carried out at 40 V for 1 h, before the voltage was increased to 100 V and the electrophoresis was continued for 9 h.

Electrophoresis in the second dimension was carried out at 60 V for approximately 3 h until the front marker had reached the bottom of the gel. Ribosomal proteins were visualized by Coomassie Brilliant Blue staining. For preparative use, the spots containing Tetrahymena S7 were cut out and stored in Eppendorf tubes at -20 °C.

Analytical One-dimensional Polyacrylamide Gel Electrophoresis

Aliquots of the reversed-phase HPLC fractions were redissolved in SDS-sample buffer and heated to 100 °C for 1 min. Electrophoresis was performed in a 16% separation gel with a 5% stacking gel using the same SDS-PAGE system as described above. After electrophoresis, the proteins were visualized by staining with Coomassie Brilliant Blue.

In Situ Digestion in Gels

The gel plugs containing Tetrahymena S7 were washed thoroughly with distilled water before incubation in digestion buffer. Digestion in the gel matrix was performed essentially as described by Karwan and Kamp(1988) and Kawasaki et al.(1990). The excised gel plugs containing approximately 20-25 µg of protein were transferred to a polypropylene tube, crushed with a glass spatula, and covered with 400-500 µl of digestion buffer. After preincubation at 37 °C for 1 h, the enzyme was added and the digestion was allowed to proceed overnight at 37 °C with slow stirring.

Digestion with trypsin was performed in 100 mM NH(4)HCO(3), pH 8.0, with two additions of enzyme, each time using an approximately 1:10 ratio (w/w) of enzyme to the estimated amount of Tetrahymena S7. Digestion with S. aureus V8 protease was performed in 100 mM NH(4)Ac, pH 4.2, or 100 mM NH(4)HCO(3), pH 8.0, using a 1:5 ratio (w/w) of enzyme to the estimated amount of Tetrahymena S7.

After collection of the supernatant, the gel plugs were washed with buffer and spun down, and the supernatants were combined. Prior to separation on narrow-bore reversed-phase HPLC, the combined supernatant was concentrated by partial lyophilization.

Reversed-phase High Performance Liquid Chromatography (HPLC) Purification of Ribosomal Proteins

Separation of ribosomal proteins was performed using an HPLC system consisting of two LKB 2150 HPLC pumps (LKB-Pharmacia, Sweden) controlled by a DOS-compatible personal computer. The eluate was monitored continuously with an LKB 2141 variable UV detector.

The first separation step was performed on a Dynosphere column (100 times 4 mm, 10.3-µm particle size). Following extraction and acetone precipitation, the r-protein mixture was redissolved in 8 M urea just before application to the column. The separation was carried out by gradient elution (0.8 ml/min) of buffer B (90% acetic acid in water) in buffer A (20% acetic acid in water). The fractions were collected manually and lyophilized, but care was taken not to take the samples to complete dryness.

Further purification of the fraction containing Tetrahymena S7 was performed using a column (250 times 4 mm) packed with Nucleosil 300 C(18), 5-µm particle size. The sample was applied to the column in buffer A (0.1% trifluoroacetic acid in water) and separated by gradient elution (1 ml/min) with buffer B (90% acetonitrile in water). The samples were collected manually and lyophilized.

The third purification step of Tetrahymena S7 was carried out using a column (100 times 4 mm) packed with Shandon C(8), 5-µm particle size. The sample was applied in buffer A (0.4% trifluoroacetic acid in water) and separated by gradient elution (1 ml/min with buffer B) (90% acetonitrile in water).

Digestion of Column-purified Tetrahymena S7

0.5 nmol of Tetrahymena S7 was dissolved in 50 µl of 50 mM Tris-HCl, pH 7.5, and 0.4 µg of endoproteinase Asp-N dissolved in 10 mM Tris-HCl was added to the protein solution. Digestion was performed for 24 h at 37 °C and terminated by injection of the solution on a narrow-bore reversed-phase HPLC column.

4% S. aureus V8 protease (w/w) was added to 1.5 nmol of purified Tetrahymena S7 dissolved in 50 mM NaH(2)PO(4), pH 7.8, and incubated for 5 h at 37 °C. Digestion was terminated by injection of the solution on a narrow-bore reversed-phase HPLC column.

Cyanogen bromide (5 M stock in acetonitrile) was diluted 10 times in acetonitrile, and 3 µl of this solution was added to 0.3-0.5 nmol of Tetrahymena S7 dissolved in 25 ml of 50% trifluoroacetic acid. The cleavage was allowed to proceed at 25 °C for 24 h, and the resulting peptides were purified by narrow-bore reversed-phase HPLC.

The cyanogen bromide peptide CNBr-2 was dissolved in 25 µl of 50 mM NaH(2)PO(4), pH 7.8, and 0.8 µg of S. aureus V8 protease was added. The digestion was carried out for 45 min at 37 °C and terminated by injection of the solution on a narrow-bore reversed-phase HPLC column.

Narrow-bore Reversed-phase HPLC Purification of Peptides

Narrow-bore reversed-phase HPLC separation of peptides was carried out using an ABI 140B HPLC (Applied Biosystems) with a dual-syringe solvent delivering system, and the peptides were monitored with an ABI 759A variable UV detector at 214 nm. The separation was performed using a column (100 times 2 mm) packed with Nucleosil 300 C(18), 5-µm particle size. The sample was applied to the column in buffer A (0.1% trifluoroacetic acid in water) and separated by gradient elution (0.2 ml/min) with buffer B (90% acetonitrile in water).

Phosphoamino Acid Analysis

P-Labeled phosphopeptides were lyophilized and hydrolyzed in 6 N HCl for 2 h at 110 °C in vacuo (Bylund and Huang, 1976).

The hydrolysates were lyophilized, dissolved in acetic acid/formic acid/water (78:22:900, v/v), pH 1.65, and separated by electrophoresis on 20 times 20 cm precoated cellulose thin layer sheets (Merck) using the same buffer (Hunter and Sefton, 1980). Electrophoresis was for 90 min at 1000 V using cooling with circulating water at 4 °C. 1 µg each of phosphoserine and phosphothreonine was added to each sample. The markers were detected by ninhydrin staining, and radioactive phosphoamino acids were detected by autoradiography at -80 °C on a Fuji RX x-ray film with a Cronex intensifying screen.

Plasma Desorption Mass Spectrometry

Mass spectra of peptides were recorded using a Cf time-of-flight plasma desorption mass spectrometer (Bio-Ion 20, Applied Biosystems AB, Uppsala, Sweden). The peptides were dissolved in 3-4 µl of 0.1% trifluoroacetic acid and applied to a nitrocellulose target as described (Johnsson et al., 1986). The spectra were accumulated for 1 times 10^6 fission events using an acceleration voltage of 15 kV.

Electrospray Mass Spectrometry (ESMS)

Electrospray mass spectra of the intact ribosomal protein S7 were recorded on a Vestec electrospray mass spectrometer (Vestec Corp.). The samples were dissolved in 2% acetic acid, 50% methanol to a concentration of approximately 0.2 mg/ml and introduced by a syringe pump at a flow rate of 1 ml/min. The spectra were recorded with a scan rate of 10 s/scan using a mass window of m/z 500-1500. The molecular weight of the protein was calculated by weighted averaging as described by Mann et al.(1989).

Laser Desorption Mass Spectrometry (LDMS)

Laser desorption mass spectrometric analysis of peptides obtained by digestion with S. aureus V8 protease of the CNBr-2 peptide was performed using an in-house developed laser desorption mass spectrometer equipped with a nitrogen laser (337 nm). The samples were dissolved in 0.1% trifluoroacetic acid and applied to the target before being mixed (1:1) with matrix (alpha-cyano-4-hydroxycinnamic acid, 20 mg/ml 30% acetonitrile).

The laser desorption mass spectra of phosphorylated ribosomal protein S7 was acquired on a prototype laser desorption mass spectrometer from Applied Biosystems AB (Uppsala, Sweden). LDMS spectra were recorded using a nitrogen laser (337 nm) and a 0.7 m flight tube. The samples were purified on reversed-phase HPLC and dissolved in 0.1% trifluoroacetic acid. 0.5 ml of the sample (approximately 2 pmol/ml) was mixed (1:1) with matrix (alpha-cyano-4-hydroxycinnamic acid, 20 mg/ml 30% acetonitrile).

Edman Degradation

Sequence determination of the intact protein and the generated peptides was performed on a Knauer model 810 pulsed-liquid sequencer (Knauer GmbH, Berlin, Germany) connected to an on-line narrow-bore HPLC system (ABI 140B/759A, Applied Biosystems) using Polybrene-coated polyvinylidene difluoride membranes (Immobilon P, Millipore) as immobilizing support.


RESULTS

As related in the introduction, only a single ribosomal protein is subject to phosphorylation in vivo in the ciliated protozoan T. thermophila. This protein was identified by two-dimensional gel electrophoresis where its migration in the second dimension corresponds to a M(r) of 29,300, and the sodium-induced phosphorylation results in the appearance of two more anodically migrating derivatives representing the mono- and diphosphorylated forms of the protein (Fig. 1). Ribosomal proteins were extracted from ribosomes isolated from exponentially growing cells and submitted to a preliminary fractionation on a C(8) reversed-phase HPLC column. The fraction enriched for T. thermophila S7 was identified by two-dimensional PAGE. This fraction was then used for two-dimensional PAGE using the maximum amount of protein compatible with good resolution. Proteins were visualized by brief Coomassie Brilliant Blue staining, and the area containing S7 was cut out from the gel and stored at -20 °C. For efficient subsequent in situ enzymatic digestion, thorough fixation in methanol/acetic acid (to remove SDS not bound to proteins) followed by extensive washes in distilled water was found to be essential.


Figure 1: Two-dimensional PAGE of basic ribosomal proteins from Tetrahymena thermophila. Fifty µg of ribosomal protein were analyzed by two-dimensional PAGE using the pH 8.6/SDS system. Proteins were visualized by staining with Coomassie Brilliant Blue. The positions of Tetrahymena S7 and S3 are indicated. The inset shows the mono- and diphosphorylated forms (S7P(1) and S7P(2)) of Tetrahymena S7 present in starved cells.



Sequence Analysis of Gel-purified T. thermophila S7

Tetrahymena S7 was digested in situ with trypsin, S. aureus V8 protease, or chymotrypsin. Peptides were eluted by diffusion and separated by narrow-bore reversed-phase HPLC. If sufficient material was available, fractions containing more than one peptide were rechromatographed using a different gradient or column material. Otherwise, simple mixtures were sequenced by Edman degradation, and ambiguities were resolved by mass spectrometric analysis.

Most of the Tetrahymena S7 sequence could be solved based on the tryptic digest which yielded a total of 24 peptides, accounting for 208 amino acid residues. The S. aureus V8 protease digest yielded 8 peptides accounting for 70 residues. Combining the results of the two digests and alignment with the mammalian r-protein S4 allowed deduction of 85% of the Tetrahymena S7 sequence (Fig. 2).


Figure 2: The amino acid sequence of Tetrahymena S7. Alignment of the peptides obtained after cleavage with S. aureus V8 protease (S1 to S8 and S1 to S20), trypsin (T1 to T24), and endoproteinase Asp-N (A1 to A17). - - -, peptides obtained from in situ digested protein; -, peptides obtained from column purified protein; / / /, residues determined by Edman degradation. The identity of each peptide was confirmed by mass spectrometric analysis.



During Edman degradation analysis of phenylthiohydantoin products corresponding to positions 182 and 209 of the tryptic peptides T18 and T20 revealed peaks eluting just after phenylthiohydantoin-glycine at a position that corresponded to no known phenylthiohydantoin derivative. Mass spectrometric analysis showed that the masses of each of the two peptides were in agreement with the predicted sequences if the unidentified residues were acrylamide-modified cysteine residues. This type of modification has previously been reported for reduced proteins in acrylamide gels (Ploug et al., 1989; Hall et al., 1993). The presence of cysteine residues at these two positions was later confirmed by mass spectrometric analysis of a cyanogen bromide fragment isolated from column-purified protein (CNBr-3, Table 1).



Purification of Tetrahymena S7 by HPLC

To determine the remaining 15% of the Tetrahymena S7 sequence, the protein was purified by several rounds of reversed-phase HPLC. The ribosomal proteins were extracted from the isolated ribosomes, precipitated with acetone, and redissolved in 8 M urea. The crude extract was separated on a Dynosphere column using an acetic acid gradient (Welinder and Sørensen, 1991), and the fraction containing Tetrahymena S7 was identified by two-dimensional PAGE. The use of acetic acid as the eluent resulted in considerably improved reproducibility and higher recoveries. S7 was further purified by two rounds of reversed-phase HPLC. As only a few ribosomal proteins have M(r) values close to 29,300, it was decided to use electrospray mass spectrometry (ESMS) for protein identification.

The molecular mass of intact Tetrahymena S7 could be determined to 29,458 ± 6 Da, which is in excellent agreement with the mass determined by two-dimensional PAGE (29,300 Da).

Establishment of the Tetrahymena S7 Sequence

The purified r-protein S7 was digested with S. aureus V8 protease and endoproteinase Asp-N, respectively. The resulting peptides were separated by narrow-bore reversed-phase HPLC, and the individual fractions were analyzed by mass spectrometry and sequenced by Edman degradation. The two peptide maps confirmed and established most of the sequence. However, the N-terminal peptide was not recovered from the endoproteinase Asp-N digest and neither were parts of the C-terminal region. Combining the two sets of data obtained from gel- and column-purified S7 allowed deduction of the sequence of 252 amino acid residues (Fig. 2). Comparing the calculated mass of the 252 residues with the mass of the intact protein as determined by ESMS revealed that 883 Da, corresponding to approximately 8 residues, were still unaccounted for.

A final cleavage of the intact protein with cyanogen bromide was carried out, and the resulting peptides were separated by narrow-bore reversed-phase HPLC. Three peptides, covering the entire sequence, were identified by ESMS (Table 1). The mass of peptide CNBr-1 and CNBr-3 matched exactly the masses calculated from the already established sequence. The mass of peptide CNBr-2 exceeded the mass calculated on the basis of the remaining part of the already established sequence, showing that the yet unsequenced region(s) of S7 were present in this fragment. The CNBr-2 fragment was subjected to digestion with S. aureus V8 protease followed by separation by HPLC. Four peptides, covering all of CNBr-2, were isolated and analyzed by laser desorption mass spectrometry (Table 1). Sequence analysis of peptide CNBr2 S4 revealed that the first 8 residues were identical with the last residues of tryptic peptide T6 and extended the known sequence with Val-Arg-Arg (residues 77-79 in Fig. 2). The following amino acid residues corresponded to the N-terminal part of tryptic peptide T7.

The presence of a basic residue, Lys or Arg, in position 38 was inferred from the tryptic peptide T4. Based on this assumption, mass spectrometric analysis of S1 and CNBr2 S1 showed that position 37 can only be leucine or isoleucine combined with an arginine in position 38. Since the corresponding amino acid residue in yeast, rat, and man is leucine, we assume this to be the case also for T. thermophila (Fig. 3).


Figure 3: Alignment of Tetrahymena S7 with homologous ribosomal proteins. HumS4x and HumS4y, human ribosomal proteins 4x and 4y; RatS4, rat ribosomal protein S4; ScS7, Saccharomyces cerevisiae ribosomal protein S7; TthS7, T. thermophila ribosomal protein S7; MvS4, Methanococcus vannielii ribosomal protein S4; HmS4, Halobacterium marismortui ribosomal protein S4. Residues identical with the Tetrahymena S7 sequence are shown in bold. The serine and threonine residues phosphorylated in T. thermophila are indicated by arrows.



Determination of the Phosphorylated Sites

T. thermophila cells were starved in 10 mM Tris containing 100 µCi/ml [P]orthophosphate. After 1 h, sodium chloride was added to a final concentration of 50 mM, and the incubation continued for 90 min. This treatment yield ribosomes with S7 almost exclusively in the diphosphorylated form S7P(2) (Bratholm, 1986).

Ribosomal proteins were extracted, the P-labeled S7P(2) was purified by three successive rounds of reversed-phase HPLC as described above. The molecular mass of the intact protein was by LDMS determined to be 29,627 ± 9 Da. The LDMS spectrum was calibrated using a 14,467-Da contaminating protein the mass of which had previously been determined by ESMS. Based on the determined sequence of S7, the calculated mass of the diphosphorylated form is 29,620 Da which is in good agreement with the mass determined by LDMS. The purified S7P(2) was digested with S. aureus V8 protease, and the resulting peptides were fractionated by reversed-phase HPLC. Two peptides, P1 and P2, containing phosphorylated residues were identified by Cerenkov counting. Portions of P1 and P2 were hydrolyzed in 6 N HCl, and phosphoamino acids were identified by electrophoresis on TLC plates. Fig. 4shows that P1 contained only P-Ser, whereas P2 contained exclusively P-Thr. Mass spectrometric analysis and Edman degradation analysis showed both peptides to be located in the C-terminal region of the S7. P1 corresponds to residues 253-259, and P2 corresponds to residues 201-252. Detection of phosphorylated serine and threonine residues during Edman degradation is not possible since these modified amino acid residues do not give rise to stable phenylthiohydantoin derivatives (Roach and Wang, 1991). As peptide P1 only contains a single serine residue, the phosphorylated residue could unequivocally be assigned to Ser. Peptide P2 on the other hand contains 2 threonine residues (positions 211 and 248). None of these threonine residues are located in sequences that conform to known protein kinase consensus sequences. Normally, V8 protease would cleave after Glu; however, cleavage in this position has never been observed in diphosphorylated S7. The absence of cleavage at this position can be explained by phosphorylation at Thr, and, therefore, we consider it most likely that Thr is the phosphorylated residue. The lack of cleavage after Glu is probably caused by the proline residue in the P-2 position (Mikkelsen et al., 1987).


Figure 4: Identification of phosphoamino acids in P-labeled phosphopeptides isolated from diphosphorylated Tetrahymena S7. The isolated phosphopeptides P1 and P2 (Fig. 6) were hydrolyzed in 6 N HCl, and phosphoamino acids were identified by electrophoresis on TLC plates.




DISCUSSION

The work presented in this report led to the surprising conclusion that the only ribosomal protein which is subject to phosphorylation in vivo in the ciliated protozoan T. thermophila is homologous, not with mammalian ribosomal protein S6/yeast ribosomal protein S10, but rather with mammalian ribosomal protein S4/yeast ribosomal protein S7. Mammalian S4 is not phosphorylated in noninfected cells, but a low level of phosphorylation has been reported for vesicular stomatitis virus-infected cells (Marvaldi and Lucas-Lenard, 1977). To the best of our knowledge, phosphorylation of yeast S7 has never been reported.

The similarity between T. thermophila S7 and mammalian S4/yeast S7 extends throughout the entire protein. However, the C-terminal region of T. thermophila S7 is slightly truncated, and Ser is not conserved. In mammalian S4/yeast S7, an alanine residue replaces the serine residue of T. thermophila S7 (Fig. 3). Since phosphorylation of Ser is required in order to allow phosphorylation of Thr, the absence of a serine residue equivalent to Ser of T. thermophila S7 may in part explain the absence of phosphorylation of mammalian S4/yeast S7.

The fact that phosphorylation of ribosomal proteins in T. thermophila in vivo is confined to S7 posed the question as to whether T. thermophila was lacking a ribosomal protein homologous with mammalian S6/yeast S10. Recently, we have by N-terminal sequencing identified a protein, T. thermophila ribosomal protein S3, which in the sequenced first 42 amino acid residues exhibited a striking similarity with mammalian S6 indicating that T. thermophila ribosomal protein S3 is the S6 homolog (Palm et al., 1993). Although this protein has never been found in a phosphorylated state in vivo, it is one of the major substrates for phosphorylation in vitro by a ribosome-associated kinase from T. thermophila; the other major substrate is S7 (Palm et al., 1993). Thus, the lack of phosphorylation in vivo of T. thermophila S3 is not due to the absence of potential sites for phosphorylation.

In a study by Hallberg et al.(1981), it was found that a strong correlation existed between the phosphorylation of S7 and the sensitivity to cycloheximide. Furthermore, they found that ribosomes isolated from starved cells with phosphorylated S7 were significantly more resistant to thermal denaturation than ribosomes isolated from growing cells with nonphosphorylated S7.

Thus, it appears that phosphorylation of T. thermophila S7 may influence structural and functional properties of the 40 S ribosomal subunits. Considering that phosphorylation of mammalian S6 is believed also to influence the functional properties of mammalian 40 S ribosomal subunits, it is interesting to note that mammalian S6 and mammalian S4 topologically seem to be located close to each other in the 40 S ribosomal subunits. UV cross-linking experiments have suggested that mammalian S6 is involved in mRNA binding (Terao and Ogata, 1979), and in keeping with this immunoelectron microscopy has indicated that S6 is located in the cleft where mRNA binds (Bommer et al., 1980). We have shown that Tetrahymena S7 is located at the interface between the two ribosomal subunits (Kristiansen et al., 1978), and the same localization has been reported for mammalian S4 (Nygård and Nika, 1982; Uchiumi et al., 1986). Thus, both ribosomal proteins occupy a position in the 40 S ribosomal subunit that is likely to be important for mRNA binding.

Apart from the complete structural characterization of T. thermophila S7, a major objective of this study was to investigate the potential and limitations in sequencing an unknown protein isolated directly from a polyacrylamide gel. Conventional purification of many biologically interesting proteins is often cumbersome or even not feasible due to low abundance or solubility/aggregation problems (Choli et al., 1989). Information on the primary structure of such proteins can be obtained by cDNA cloning, but information on post-translational modifications relies to a very large extent on analysis of the isolated protein. A number of different enzymes was used in this study for in situ digestion. Most information was obtained from the tryptic digests. This is probably related to the preservation of tryptic activity in the presence of low concentrations of SDS (Kawasaki and Suzuki, 1990) combined with efficient elution of the generated peptides, most of which were of low mass due to the high content of basic amino acid residues in T. thermophila S7. Generally, HPLC separation of the eluted peptides yielded very complex chromatographic patterns, and reproducibility from one experiment to another was poor. This lack of reproducibility most likely was caused by variability in the amounts of SDS, salts, and unpolymerized acrylamide eluted with the peptides (Ward et al., 1990). Here, mass spectrometric analysis of individual peaks proved extremely valuable for cross-identification of peaks in different chromatograms.

In the work presented here, we were able to deduce 85% of the S7 sequence from peptides generated by in situ enzymatic digestion of S7 in two-dimensional gels. Furthermore, in preliminary experiments, the presence of phosphorylated Ser was demonstated by mass spectrometric analysis of a tryptic peptide generated by in situ digestion. Thus, extensive information on primary structure and post-translational modifications can indeed be obtained by direct analysis of proteins separated by two-dimensional PAGE, and, in many cases, a complete sequence determination should be possible.


FOOTNOTES

*
This work was supported by a grant from the Danish National Science Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Novo Nordisk A/S, Protein Chemistry, Hagedornsvej 1, DK-2820 Gentofte, Denmark.

Present address: Dept. of Clinical Chemistry, Odense University Hospital, DK-5000 Odense C, Denmark.

**
To whom correspondence and reprint requests should be addressed. Fax: 45-65-93-27-81.

(^1)
The abbreviations used are: r-protein, ribosomal protein; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; ESMS, electrospray mass spectrometry; LDMS, laser desorption mass spectrometry.


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