(Received for publication, August 7, 1995)
From the
We found a novel protein in the postmitochondria supernatant fraction of rat liver, which is soluble in 5% perchloric acid and strongly inhibits protein synthesis in a rabbit reticulocyte lysate system. The protein extracted from the supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The protein was shown to consist of two identical subunits with a molecular mass of 14 kDa. By immunoscreening with the rabbit antisera against the protein, a cDNA encoding the protein was cloned and sequenced. The cDNA contained an open reading frame of 411 base pairs encoding a 136-amino acid protein with a molecular mass of 14,149 Da. The deduced amino acid sequence was completely identical with that constructed from all of the above peptides. Interestingly, the perchloric acid-soluble protein inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner from RNase A. The protein is likely to inhibit an initiation stage of cell-free protein synthesis. Among the rat tissues tested, the protein was located only in liver and kidney. These findings are the first report on a new inhibitor that may be involved in the regulation of protein synthesis in those tissues.
High mobility group (HMG) ()proteins are a family of
nonhistone components in chromatin(1) . There are four major
HMG proteins (HMG1, -2, -14, and -17) in all the eukaryotic cells
examined to date(2) . Although the function of HMG proteins has
not been identified unequivocally, HMG proteins have been implicated in
transcription and in DNA replication(2) .
We showed that the
HMG proteins play an important role in nutritional modulations of chick
liver RNA synthesis (3) and isolated the HMG 2a cDNA from a
gt11 expression library of chick liver using polyclonal
antibodies, which encodes a protein of 201 amino acids(4) . In
Northern blotting, 2.0- and 1.2-kb mRNAs for the protein were detected
in the liver of newly hatched chick, but they were shown to decrease
during posthatched development(5) .
In the course of the investigation on chicken and rat chromatin, we found in the livers of the animals a novel protein that was co-extracted with the H1 and HMG proteins by 5% perchloric acid. The perchloric acid-soluble protein (PSP) showed inhibition of protein synthesis in a rabbit reticulocyte lysate system. Recently, Levy-Favatier et al.(6) reported the isolation of a 10-kDa perchloric acid-soluble protein from rat liver and the cloning of a cDNA encoding the protein. They suggest that the half of the deduced polypeptide sequence presents 27% similarity with a region of the 83-90-kDa heat shock protein (hsp). However, they have never elucidated the function of the 10-kDa protein. It is of great interest to clarify whether our protein is identical with that reported by Levy-Favatier et al.(6) and to elucidate the physiological function of the PSP.
In this study, we describe the purification and characterization of the PSP from rat liver and the cloning and sequencing of a cDNA encoding the protein. Its function and distribution in rat tissues were also examined. Furthermore, a comparison of our protein with that detected by Levy-Favatier et al.(6) is described.
Kidney and liver sections (4 µm thick) were placed on glass slides and immunohistochemically stained using the avidin-biotin-peroxidase complex method(11) . The slides were deparaffinized and soaked in 0.3% hydrogen peroxide in absolute methanol for 30 min at room temperature. After hydration and rinsing in PBS (10 mM phosphate buffer (pH 7.2) containing 0.85% NaCl), the sections were treated with a normal goat serum at 1:60 dilution for 20 min at room temperature to reduce nonspecific staining and then incubated at 4 °C overnight with 3 µg/ml of polyclonal antibodies against PSP in PBS containing 0.1% bovine serum albumin in a moist chamber. The sections were rinsed in PBS and incubated for 50 min at room temperature in a 1:200 dilution of biotinylated goat anti-rabbit IgG (Vectastain ABC kit, Vector Laboratories, Inc.). Following a rinse in PBS, the sections were incubated for 60 min at room temperature in the avidin-biotin complex, rinsed in PBS, stained for 5 min with 50 mM Tris-HCl (pH 7.6), containing 0.1% 3,3`-diaminobenzidine tetrahydrochloride, 0.02% hydrogen peroxide, and 0.65 mg/ml of sodium azide. After washing with PBS, the section were counter-stained for 10 min with 1% methylgreen, dehydrated, and mounted.
Controls were prepared in the above mentioned manner, except that nonimmunized rabbit IgG was used instead of the first antibody.
Figure 1:
Subcellular distribution of the PSP,
which was co-extracted with HMG and H1 (A) and
SDS-polyacrylamide gel electrophoresis of the purified PSP (B). A, the 5% PCA extracts from homogenate, crude
nuclear fraction (CN), mitochondria-lysosome fraction (ML), and postmitochondria supernatant fraction (PMS)
were precipitated with 25% trichloroacetic acid. Ten µg of protein
in each fraction were electrophoresed on a 15% SDS-polyacrylamide gel.
The proteins on the gel was stained with Coomassie Brilliant Blue
R-250. Molecular mass standards (in kDa) are shown on the left. B, four µg of the purified PSP were
electrophoresed on an SDS-polyacrylamide gel with or without
-mercaptoethanol. Molecular mass standards (in kDa) are shown on
the right.
Figure 2: ESI mass spectra of the N-terminal peptide of the PSP (A) and its deblocked peptide (B). The N-terminal peptide of the PSP was isolated as described in the text. The deblocked peptide was obtained from the N-terminal peptide by digestion with the acyl amino acid-releasing enzyme.
Furthermore the PSP was digested with lysyl endopeptidase, and the products were separated by reversed-phase HPLC (Fig. 3). Finally nine peptides were isolated. Among them, peptide I was insensitive to automated Edman degaradation, indicating that peptide I is the N-terminal peptide. Therefore peptide I was treated with the acyl amino acid-releasing enzyme, and the deblocked peptide was sequenced by automated amino acid sequencer. The other eight peptides were directly sequenced. The amino acid sequences determined of the nine peptides were summarized in Table 1. The above-mentioned C-terminal peptide was included in peptide IX. However, the peptides, except for peptide I and IX, could not be ordered on the amino acid sequence of PSP.
Figure 3: HPLC of the peptides obtained from the PSP by digestion with lysyl aminopeptidase. The PSP was digested, and the products were separated by HPLC as described under ``Experimental Procedures.'' Since peptides VII and IX were eluted together in the peak at a retention time of 54 min, the peptides were collected, further rechromatographed by elution with a slower linear gradient of acetonitrile in 0.05% trifluoroacetic acid, and separated from each other. The separated peptides were collected and evaporated by a centrifugal evaporator. The dried materials were used as the isolated peptides. The elution of the peptides was followed by monitoring the absorbances at 206 nm (solid line) and 280 nm (dashed line).
Figure 4: Nucleotide sequence of the cDNA insert of clone pPSP-1 and the deduced amino acid sequence of the PSP. The numbering of the nucleotide sequence is presented on the right, whereas the amino acid residue are given below the corresponding codons.
Figure 5: Comparison of the predicted amino acid sequence of the PSP and that of ORF 1 from A. vinelandii. Identical amino acids are boxed. Hyphens show the points at which gaps were introduced to maximize alignment. Sequence comparison was done using the program NBRF.
The pPSP-1 insert, encoding the PSP, was P-labeled by
random- primed synthesis and used as a probe in a Southern blot of rat
liver DNA digested with different restriction enzymes. The Southern
blot analysis demonstrated a single hybridization band for each
restriction digest (Fig. 6). These results suggest that in the
rat genome there is one copy of the PSP gene.
Figure 6:
Southern blot analysis of the PSP gene.
Total rat liver DNA (10 µg/lane) was digested with BamHI (lane 1), EcoRI (lane 2), and PstI (lane 3) and processed as described under ``Experimental
Procedures.'' The positions of HindIII fragments of
phage DNA are indicated at the left (23.1, 9.4, 6.5, 4.3, 2.3,
2.0, 0.5 kb in descending order).
Figure 7: Immunoblot analysis of the PSP from various rat tissues. Twenty µg of proteins in the homogenates from various tissues were electrophoresed on a 15% SDS-polyacrylamide gel. Immunoblot analysis was carried out as described under ``Experimental Procedures.''
Figure 8:
Northern blot analysis of the PSP mRNA
from various rat tissues. Total rat liver RNA (20 µg/lane) was
loaded on the gel and hybridized to a
[P]dCTP-labeled EcoRI fragment pPSP-1
as described under ``Experimental Procedures.'' The positions
of 28 and 18 S are indicated at the left. Also PSP mRNA of 0.9 kb is
indicated by the arrowhead.
Rat liver (Fig. 9A) and kidney (Fig. 9B), which were stained with the antisera, were observed by a light microscope. In the liver, positive reaction products were observed in both the nuclei and cytoplasm of most hepatocyte cells. Some of these cells along the interlobular connective tissue were more intensely stained than cells in other sites. The interlobular connective tissue, arteries, veins and bile ducts were less stained. In the kidney, immunopositive deposits were seen in both the nuclei and cytoplasm in some epithelial cells of the upper renal tubules of the cortex. The glomeruli were not immunostained. No reaction products were observed in any control sections prepared using nonimmunized rabbit IgG as the first antibody (data not shown).
Figure 9: Immunohistochemical localization of the PSP in rat liver and kidney. Immunohistochemical staining of rat liver and kidney were performed as described under ``Experimental Procedures.'' A, light micrograph of liver section stained with the antisera against the PSP was positive in both nuclei and cytoplasm of most hepatocyte cells. Some of these cells along the interlobular connective tissue are more intensely stained than other hepatocytes. Note less intense staining in the interlobular connective tissue, arteries, veins, and bile ducts. B, light micrograph of a kidney section shows intense deposits of immunoreactive products in some epithelial cells of upper renal tubules in the cortex, but not in the glomerulus. Original magnification is 400-fold.
Figure 10:
Inhibition of cell-free protein synthesis
by the PSP and RNase A. Protein synthesis by a rabbit reticulocyte
lysate mixture was measured in the presence of different concentrations
of the PSP () and RNase A (
) as described under
``Experimental Procedures.'' Each point represents the mean
value of [
H]leucine incorporation into protein
determined in five separate experiments and expressed as a percentage
of the incorporation (45,000 cpm) in the absence of the
PSP.
Figure 11:
Kinetics of inhibition of protein
synthesis. Reaction mixtures (50 µl) were incubated at 30 °C
without or with the indicated amount of the PSP. At various intervals,
5-µl aliquots were removed and assayed for
[C]leucine incorporation as described under
``Experimental Procedures.''
Figure 12:
Effect of the PSP on the disaggregation
of the polyribosomes. Reaction mixtures (50 µl) were incubated at
30 °C for 15 min without or with the PSP (10M). After incubation, 100 µl of a cold buffer
solution (20 mM Tris-HCl (pH 7.6), 75 mM KCl, 2
mM magnesium acetate) was added to each sample, and 50-µl
aliquots were applied on linear 10-50% sucrose gradient in the
same buffer. The gradient were centrifuged at 4 °C at 40,000 rpm
for 70 min with a Hitachi RPS-50-2 rotor, and the absorbance
profile was monitored in an ISCO density gradient monitor at 254 nm.
The figure in the panel expresses the 4-fold magnification of the
actual absorbance measured. A, the profile obtained without
the PSP; B, the profile obtained with the
PSP.
Figure 13:
RNase
activity and activity staining of the PSP and RNase A on
SDS-polyacrylamide gel. A, SDS-polyacrylamide gel
electrophoresis and RNase staining are carried out as described under
``Experimental Procedures.'' Lane 1, RNase A (1 ng
of protein); lane 2, PSP (10 ng); lane 3, PSP (1
µg). B, RNase activity was determined as described under
``Experimental Procedures.'' , RNase A;
, the
PSP.
We have purified to homogeneity a novel protein that was co-extracted with H1 and HMG proteins by perchloric acid. By polyacrylamide gel electrophoresis, its molecular mass was found to be 30 kDa under the nonreducing condition and 14 kDa under the reducing condition. This native protein thus consists of two identical subunits with a molecular mass of 14 kDa.
Recently, Levy-Favatier et al.(6) found a 10-kDa perchloric acid-soluble protein in rat liver and cloned a cDNA that consists of an open reading frame of 297 base pairs and encodes a 99-amino acid protein with a molecular mass of 10,355 Da. The first half of the deduced polypeptide sequence presents 27% similarity with a region of the 83-90-kDa hsp, which is highly conserved from Drosophila to human. The hsp has been shown to belong to the class of molecular chaperones that plays a role in the folding of proteins(16) . On the basis of this fact, they proposed that the perchloric acid-soluble 10-kDa protein could play a role as a molecular chaperone. The nucleotide sequence of the cDNA reported by Levy-Favatier et al.(6) was shown to be fairly consistent with that of the cDNA encoding our protein, although some differences were observed. Eventually, the amino acid sequence of the protein is completely identical with sequence 38-136 of our protein. We carefully examined the nucleotide sequence reported by Levy-Favatier et al.(6) and noticed that if G is inserted at nucleotide at 76, their nucleotide sequence could be fully identical with our nucleotide sequence, indicating that their nucleotide sequence codes for amino acid sequence 1-37 of our protein. We repeatedly carried out the sequencing of our cDNA and confirmed that the present nucleotide sequence of our cDNA is correct (Fig. 4). On the other hand, the nucleotide 442 of the cDNA reported by Levy-Favatier et al.(6) is G, the first base of the codon for a Val residue, whereas that of our cDNA is C and therefore codes for a Leu residue. The C-terminal amino acid residues of our protein was determined to be a Leu residue by amino acid sequencing (Table 1). The nucleotide sequence encoding the C-terminal region of our protein was repeatedly sequenced, and the nucleotide 442 was determined to be C (Fig. 4). These findings clearly demonstrate that our protein is identical with that reported by Levy-Favatier et al.(6) and that our protein is a 136-amino acid protein with a molecular mass of 14,149 Da, the N terminus of which is acetylated.
Interestingly, the PSP showed a high similarity (70%) with a hypothetical protein ORF1 from A. vinelandii (Fig. 5). Although the physiological function of the PSP is unknown, the conservation of the PSP during evolution indicates an important role in the cells.
An additional interest is
the repression of protein synthesis by the PSP in a rabbit reticulocyte
cell-free lysate system. The PSP inhibited the incorporation of
[C]leucine into proteins at low concentrations
(IC
, 8 nM). However, the PSP did not show any
RNase activity in the condition tested (Fig. 13). This finding
shows that the PSP inhibits protein synthesis in the rabbit
reticulocyte cell-free lysate system in a different manner from RNase
A. For the first several-minute period, the protein synthesis in the
lysate was not inhibited in the presence of the PSP, but with prolonged
incubations it was shown to be inhibited in a dose-response mode by the
PSP (Fig. 11). Furthermore, the disaggregation of the
polyribosomes in the lysate was stimulated, accompanied with the
inhibition of the protein synthesis (Fig. 12). These findings
show that the PSP inhibits the initiation stage of the protein
synthesis in the lysate. A similar phenomenon was observed during the
protein synthesis in a heme-deficient lysate(17, 18) .
The inhibition of the protein synthesis in the heme-deficient lysate is
due to the activation of a translational inhibitor (HRI) which acts at
an initial stage of the protein
synthesis(17, 18, 19, 20, 21) .
A macromolecular inhibitor similar to HRI has been shown to be in
Ehrlich ascites tumor cells (22) and rat liver(23) .
Interestingly, Delaunay et al.(23) reported that a
rat liver inhibitor phosphorylates the 38-kDa subunits of eukaryotic
initiation factor-2 and that the inhibitor may be a protein kinase,
although the entity of the inhibitor has never been characterized. The
PSP exhibited the same behavior as the rat liver inhibitor with respect
to the kinetics of the inhibition of protein synthesis in a cell-free
system and its effect on polyribosomes disaggregation. In order to
examine whether the PSP possesses any protein kinase activity, we
incubated the lysate in the presence of the PSP with
[
-
P]ATP but failed to detect any protein
kinase activity (data not shown). This fact indicates that the PSP
might regulate the protein synthesis in a different fashion from the
rat liver inhibitor found by Delaunay et al.(23) .
However, further detailed investigations on a protein kinase activity
in the PSP should be done. At present, the investigations as to the
mechanism by which the PSP inhibits the process of the above-mentioned
cell-free protein synthesis are actively in progress.
The PSP was shown to be only abundant in kidney and liver by immunoblotting, Northern blotting, and immunohistochemistry (Fig. 7Fig. 8Fig. 9). The protein is especially localized in some epithelial cells of the upper renal tubules in the kidney cortex and in the cells along the interlobular connective tissue of liver (Fig. 9). We have recently purified a PSP-like protein from rat heart. The antisera against the PSP-like protein was shown to cross-react with that from rat muscle, and the PSP-like protein inhibited cell-free protein synthesis at a concentration of 2 nM as the PSP did (data not shown). These findings are the first report on a new inhibition mode of cell-free protein synthesis by the PSP and PSP-like protein and indicate that these proteins may play an important role in the regulation of protein synthesis in rat tissues.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D49363[GenBank].