(Received for publication, November 17, 1994)
From the
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.
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) ()with an M
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.
Dynosphere
resin was from Dynochrom Sverige AB (Sweden). Shandon C,
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.
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.
Digestion with
trypsin was performed in 100 mM NHHCO
,
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
Ac, pH 4.2, or 100 mM NH
HCO
, 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.
The first separation step was
performed on a Dynosphere column (100 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 4 mm)
packed with Nucleosil 300 C
, 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 4 mm) packed with Shandon
C
, 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).
4% S.
aureus V8 protease (w/w) was added to 1.5 nmol of purified Tetrahymena S7 dissolved in 50 mM NaHPO
, 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 NaHPO
, 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.
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 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.
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
(-cyano-4-hydroxycinnamic acid, 20 mg/ml 30% acetonitrile).
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 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
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 and S7P
) of Tetrahymena S7
present in starved cells.
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).
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).
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.
Ribosomal proteins were extracted, the P-labeled S7P
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
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.
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.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
All ASBMB Journals | Molecular and Cellular Proteomics |
Journal of Lipid Research | Biochemistry and Molecular Biology Education |