©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The rad21 Gene Product of Schizosaccharomyces pombe Is a Nuclear, Cell Cycle-regulated Phosphoprotein (*)

(Received for publication, September 8, 1994; and in revised form, January 13, 1995)

Rainer P. Birkenbihl (§) Suresh Subramani (¶)

From the Department of Biology, University of California at San Diego, La Jolla, California 92093-0322

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The rad21 gene of Schizosaccharomyces pombe is involved in the repair of double-strand breaks in DNA and is essential for mitotic growth (Birkenbihl, R. P., and Subramani, S.(1992) Nucleic Acids Res. 20, 6605-6611). We show that the [Medline] Rad21 protein migrates with an aberrantly slow mobility, has a thrombin cleavage site, and is multiply phosphorylated mainly at serine residues. The expression of the rad21 mRNA and the Rad21 protein is cell cycle-regulated, with the peak of mRNA and protein expression occurring near the G(1) to S transition. Following translation of the protein, hypophosphorylated forms of the protein appear. However, the most phosphorylated form of Rad21 appears only later in the cell cycle (in S to G(2)). Analysis of the radiosensitive mutant rad21-45 revealed that the mutant protein is permanently hypophosphorylated. The Rad21 protein is nuclear during the cell cycle. The nuclear localization signal was identified in the C-terminal third of the protein. Upon repression of the Rad21 protein expressed from the repressible nmt1 promoter, the unphosphorylated and hypophosphorylated forms of Rad21 disappeared first. When the concentration of the most highly phosphorylated form of Rad21 sank under a critical level, the cells underwent aberrant mitoses. They exhibited loss of proper nuclear organization and abnormal septation.


INTRODUCTION

In recent years, many of the radiation-sensitive (rad) mutants of the fission yeast Schizosaccharomyces pombe(2) have been cloned and sequenced, and their functions have been partially characterized (for reviews, see (3) and (4) ). Besides genes that have been identified as DNA repair genes, mainly by sequence homologies, e.g.rad15(5) , rad13(6) and rad16(7) , there are genes whose products monitor the integrity of the DNA. The Rad1, Rad3, Rad9, and Rad17 (8, 9, 10) proteins are involved in a checkpoint that senses the integrity of the DNA before entry into mitosis. In cells inflicted with DNA damage, they are necessary for cell cycle arrest in G(2), which provides time for DNA repair. Alternatively, if DNA replication is incomplete, the action of these proteins mediates cell cycle arrest rather than premature entry into mitosis.

The rad21 gene, whose gene product is the subject of this paper, is involved in DNA double-strand break repair(1) . The gene is represented by only one mutant allele, rad21-45. This mutant grows normally unless it is irradiated with UV or ionizing radiation. The resulting DNA lesions, mainly DNA double-strand breaks, cannot be repaired efficiently. Unlike the cells with mutations in the rad1, rad3, rad9, and rad17 genes, the rad21-45 cells arrest in G(2) after irradiation, but proceed after a cell cycle delay into mitosis without having repaired the DNA lesions. These observations indicate that in the rad21 mutant, the checkpoints recognizing DNA damage and mediating cell cycle arrest are still intact(1) .

Because only one mutant allele of the rad21 gene exists, it was important to find out whether the gene product is only important after overt DNA damage or if the mutant shows a partial phenotype not truly representative of a more profound function in growing cells. Deletion of the genomic gene resulted in spores that germinated well, but the cells died after two or three cell divisions. These results showed that the rad21 gene is essential for mitotic growth (1) .

To learn more about the rad21 gene and the Rad21 protein, which has no significant sequence homology to any other protein sequence in the data bases (nonredundant PDB + SwissProt + SPupdate + PIR + GenPept + GPupdate; August 30, 1994), we investigated the protein biochemically and with cell biological methods. In numerous cases, it has been shown that the activity and even function of proteins are modulated by their phosphorylation status. Using biochemical methods, we investigated the protein's expression pattern and its post-translational modification by phosphorylation during the cell cycle. The subcellular localization of the Rad21 protein and the nuclear localization signal were determined. A strain in which the genomic rad21 gene was placed under the control of the repressible nmt1 promoter (11) was used to deplete cells of the Rad21 protein and to correlate the observed phenotype with the loss of the Rad21 protein.


MATERIALS AND METHODS

Yeast Strains and Culture Conditions

All S. pombe strains were derived from wild-type 972 h and are described below. Cells were grown in YPD medium (yeast/peptone/dextrose) or in Edinburgh minimal medium at 30 °C. Multicopy plasmids pART1 (12) and pFL20leu (the Saccharomyces cerevisiae LEU2 gene was inserted into the EcoRV site of pFL20, destroying the URA3 gene as a result) were used for introducing mutated rad21 genes into strains. Transformation of S. pombe strains was performed by electroporation(13) .

Construction of a Rad21-repressible Strain, SPR61

Genomic DNA upstream of the rad21 gene (2.1-kilobase BamHI-ClaI fragment of p21-5)(1) , the S. pombe ura4 gene(14) , and the nmt1 promoter (11) were cloned into the HindIII site 8 bp (^1)upstream of the rad21 ORF. The 6.2-kilobase BamHI-PstI fragment was then used to transform SPR16 (ura4-D6 leu1-32 h). Correct integrants, replacing the immediate 5`-upstream region of the rad21 ORF, were found by their inability to grow on medium containing thiamine and subsequent Southern blot analysis.

Antibody to the Rad21 Protein

The rad21 ORF (1.9-kilobase HindIII-BglII fragment, blunt-ended) was cloned in-frame into the XbaI-HindIII sites (blunt-ended) of vector pGEX-KG (15) downstream of the glutathione S-transferase coding sequence. The construct was transformed into Escherichia coli DH5alpha. The culture was grown to A = 1, induced with 0.4 mM isopropyl-1-thio-beta-D-galactopyranoside, and harvested after 4 h. The cells were lysed in lysis buffer (phosphate-buffered saline with Triton X-100 containing 2 mM EDTA, 0.1% beta-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine) by sonication. The insoluble pellet was washed with water and 50 mM Tris, pH 7.5, 250 mM NaCl and solubilized with 1% Sarkosyl, 2 mM dithiothreitol. After precipitation with 25% (w/v) ammonium sulfate, the fusion protein was digested with thrombin under the conditions described(15) . The 68-kDa peptide was isolated from an 8% SDS-polyacrylamide gel and used to immunize rabbits.

Southern and Northern Blots

Preparation of DNA and RNA from yeast cells was carried out as described by Birkenbihl and Subramani (1) . Southern blotting and Northern blotting were performed according Sambrook et al.(16) . In both cases, the alkaline transfer protocol was used. Hybridization conditions were the same for Southern and Northern blots and are described by Chomczynski and Qasba(17) .

In Vivo Cell Labeling, Cell Lysates, and Immunoprecipitation

In vivo cell labeling, preparation of cell lysates, and immunoprecipitation were carried out as described earlier (18) using 20 µl of anti-Rad21 serum for incubation with preswollen protein A-Sepharose.

Protein Gels and Western Blots

Proteins were separated by SDS-PAGE as described by Laemmli(19) . For Western blots, the proteins were transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature with 5% nonfat milk in PBST. After rinsing, the membranes were incubated for 1 h with Rad21 antiserum diluted 1:1000 in PBST and then were washed three times for 5 min with PBST. The immune complexes were detected by incubation with horseradish peroxidase-conjugated protein A at 1:3000 dilution in PBST for 30 min. After extensive washing in PBST, blots were developed with either the enhanced chemiluminescence system (ECL, Amersham Corp.) or horseradish peroxidase color development (Bio-Rad) according to the instructions of the supplier.

In Vitro Translation

The coding sequences of interest were cloned into the T7 expression vector pT7-7 (20) and transcribed with T7 RNA polymerase. For translation, the untreated rabbit reticulocyte lysate (Promega) was used following the instruction of the supplier with [S]methionine as radioactive substrate.

Protein Expression in E. coli

For protein expression in E. coli, the T7 expression system was used. Different parts of the rad21 ORF were cloned into vector pT7-7 (20) and transformed into E. coli strain BL21(DE3)(21) . After induction with 0.4 mM isopropyl-1-thio-beta-D-galactopyranoside for 4 h, 20 µl of cells were boiled in Laemmli buffer(19) , and the Rad21 peptides were analyzed by Western blotting.

Phosphoamino Acid Analysis

In vivoP-labeled Rad21 protein was immunoprecipitated and separated by SDS-PAGE. After localization by autoradiography, the most phosphorylated Rad21 band was excised and subjected to phosphoamino acid analysis(22) . The protein was eluted with ammonium bicarbonate, trichloroacetic acid-precipitated, and hydrolyzed in 6 N HCl for 1 h at 110 °C. After lyophilization, the sample was resuspended in pH 1.9 buffer (25 ml of 88% formic acid, 78 ml of glacial acetic acid, 897 ml of H(2)O) containing 100 µg/ml each phosphoserine, phosphothreonine, and phosphotyrosine. The sample was subjected to two-dimensional thin-layer electrophoresis (first dimension: 20 min at 1.5 kV in pH 1.9 buffer; second dimension: 16 min at 1.3 kV in pH 3.5 buffer (50 ml of glacial acidic acid, 5 ml of pyridine, 945 ml of H(2)O)). Labeled individual phosphoamino acids were identified by alignment of the phosphoamino acids markers (stained with ninhydrin reagent) with the signals obtained by autoradiography.

Phosphatase Treatment of Rad21

Potato acid phosphatase (Boehringer Mannheim) in ammonium sulfate precipitates was dissolved in potato acid phosphatase buffer (40 mM Pipes, pH 6.0, 1 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin) and dialyzed against potato acid phosphatase buffer. Immunopurified Rad21 protein from S. pombe cells, also dialyzed against potato acid phosphatase buffer, was digested with 1 unit of potato acid phosphatase for 30 min at 30 °C.

Thrombin Digestion of Rad21

Phosphorylated and dephosphorylated Rad21 proteins were dialyzed against cleavage buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM CaCl(2), 0.1% beta-mercaptoethanol) and treated with 1 unit of thrombin (Sigma) for 20 min at 30 °C.

Microscopy

After fixation with 3.7% formaldehyde, cells were stained for fluorescence microscopy with 4`,6`-diamidino-2-phenylindole (DAPI) (Sigma), calcofluor (fluorescence No. 28, Sigma), and fluorescein isothiocyanate-conjugated phalloidin (Sigma). For indirect immunofluorescence, we followed the protocol of Hagan and Hyams(23) . We used affinity-purified anti-Rad21 antibody (1:200, 707/6) as the primary antibody.


RESULTS

The Rad21 Protein Migrates Aberrantly on Denaturing SDS-Polyacrylamide Gels and Contains a Thrombin Cleavage Site

Western blot analysis showed that the Rad21 protein of S. pombe had an aberrant mobility when analyzed by SDS-PAGE under reducing conditions (Fig. 1, lane 4, without thrombin). While the calculated molecular mass of the protein is 68 kDa, the Rad21 protein isolated from S. pombe cells resulted in a 92-100-kDa ladder consisting of at least five bands (Fig. 1, lane 4, without thrombin). When the complete ORF was expressed in E. coli (Fig. 1, lane 3, without thrombin) or in vitro in rabbit reticulocyte lysates, the products showed the same mobility of a 92-kDa protein. Using deletion constructs, we could show that the discrepancy in the mobility was lowered with decreasing size of the truncated peptides (see examples in Fig. 2B).


Figure 1: The Rad21 protein migrates aberrantly on SDS-PAGE and contains a thrombin cleavage site. Complete lysates from E. coli cell expressing the rad21-GST fusion protein (lane 1) and the complete rad21 ORF (lane 3) and from growing S. pombe wild-type cells (lane 4) were separated by SDS-PAGE using reducing conditions and subjected to Western blot analysis using the antibody to Rad21. The lysates were mock-treated(-) or digested (+) with thrombin (Throm.). Lane 2 shows the purified 68-kDa Rad21 peptide (truncated at the N terminus) that was used for immunization of rabbits. In crude cell extracts of S. pombe cells, the highest phosphorylated form of Rad21 is protected against thrombin cleavage. This is not the case when Rad21 purified from E. coli is used.




Figure 2: Mapping of the thrombin site in Rad21. Three constructs coding for the whole Rad21 protein (rad21; lane 1), the N-terminal 329 amino acids (rad21Pst; lane 2), and the C-terminal part of Rad21 starting with amino acid 265 (rad21Nae; lane 3) were expressed in vitro in rabbit reticulocyte lysates using [S]methionine. Half of each sample was treated (+) with thrombin (Throm.), whereas the other half was not(-). The samples were separated by SDS-PAGE and transferred to nitrocellulose. A, autoradiograph of the nitrocellulose filter; B, map constructed from the cleavage data. Italicizednumbers give the calculated (calc.) molecular masses and Romannumbers the observed (obs.) molecular masses on SDS-PAGE. N (NaeI) and P (PstI) represent the restriction sites used for construction of the truncated genes. The thrombin cleavage site is after Thr-215.



To create antibodies against the Rad21 protein, we expressed the complete rad21 ORF as a glutathione S-transferase fusion protein(15) . When the fusion protein, which also migrated aberrantly at 120 kDa (Fig. 1, lane 1, without thrombin) instead of at the calculated 95 kDa, was treated with thrombin to release the glutathione S-transferase polypeptide, we found that Rad21 contained an internal thrombin site dividing Rad21 into a 68-kDa (C-terminal) and a 28-kDa (N-terminal) peptide (Fig. 1, lane 1, with thrombin). The C-terminal peptide corresponding to the 68 kDa-band was used to make antibodies against the Rad21 protein.

To map the internal thrombin site in Rad21, two deletion constructs were made, one encoding the N-terminal 329 amino acids of the Rad21 protein (Rad21Pst) and the other encoding the C-terminal part (amino acids 265-628) of the Rad21 protein (Rad21Nae). Both, together with the full-length construct, were expressed in rabbit reticulocyte lysates using [S]methionine. The products were cleaved with thrombin and analyzed by Western blotting (data not shown) and autoradiography of the Western filter (Fig. 2A). Only the full-length (Fig. 2A, lane 1) and N-terminal (lane 2) peptides were cleaved. They resulted in a common 28-kDa N-terminal fragment, only detectable by autoradiography (Fig. 2A, lanes 1 and 2, with thrombin) and not by Western blotting. Besides this 28-kDa fragment, the full-length construct resulted in a 68-kDa fragment recognized by the antibody. For the Rad21Pst protein, a 12-kDa C-terminal fragment should also be detectable by the Rad21 immune serum. However, this fragment was not resolved by 15% SDS-PAGE.

To determine the exact cleavage site, we sequenced, by Edman degradation, the first five amino acids of the N-terminal end of the 68-kDa fragment generated by thrombin cleavage of the full-length Rad21 protein. This resulted in the sequence SVHSD, indicating Ser-216 as the N-terminal amino acid of this fragment.

The Rad21 Protein Is Phosphorylated at Multiple Sites

In S. pombe, the Rad21 protein is post-translationally modified. On Western blots, the modifications result in multiple mobility shifts of the 92-kDa band up to 100 kDa.

To investigate if the rad21 gene product is phosphorylated, exponentially growing wild-type cells and cells transformed with the overexpressing plasmid p21adh, carrying the complete rad21 ORF downstream of the S. pombe adh promoter(12) , were labeled with P, and the Rad21 peptides were immunoprecipitated. After SDS-PAGE, the proteins were transferred to nitrocellulose and analyzed by Western blotting using the antibody to Rad21 (Fig. 3A, lanes 2 and 4). On an autoradiograph taken from the same filter (Fig. 3A, lanes 3 and 5), the strongest bands lined up perfectly with the Rad21 ladder on the Western blot. While the lower molecular mass bands of the Rad21 ladder were clearly labeled in the cells overexpressing the Rad21 protein, in the lysate from wild-type cells, the highest modified band contained the most label. It is possible that in cells overexpressing Rad21, the responsible phosphorylation pathway is saturated. This could mean that the highest modified form is the mature form, while the lower forms are only intermediates.


Figure 3: The Rad21 protein is multiply phosphorylated. A, identification of Rad21 phosphorylation. Growing S. pombe wild-type cells (lanes 2 and 3) and cells overproducing Rad21 (lanes 4 and 5) were labeled with [P]orthophosphate. The Rad21 protein was immunoprecipitated, separated by SDS-PAGE, and transferred to nitrocellulose. The Rad21 protein was visualized by Western blotting (lanes 2 and 4) and by autoradiography (lanes 3 and 5). Lane 1 contains purified p68. B, potato acid phosphatase digest of the Rad21 protein. The Rad21 protein from growing S. pombe cells (lanes 3-7) and truncated Rad21Pst (lanes 8-12) were immunoprecipitated with anti-Rad21 antibodies and then digested with potato acid phosphatase (PAP) for 0, 4, 8, 12, and 20 min, respectively. By this treatment, the phosphorylated forms were converted to the unphosphorylated protein. The full-length phosphorylated substrate (lane 3) was also treated with thrombin (Thromb.; lane 2) prior to potato acid phosphatase digest (lane 1). Note that thrombin treatment results in a single band and that subsequent digest of this 68-kDa band with potato acid phosphatase does not result in any additional increase in mobility. C, phosphoamino acid analysis of Rad21. P-Labeled Rad21 protein from growing wild-type cells was immunoprecipitated and subjected to SDS-PAGE. The protein contained in the highest modified band was hydrolyzed with HCl, and the products were separated by two-dimensional electrophoresis. The dots on the autoradiograph of the electrophoresis plate are as follows: phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y).



To examine if the mobility shifts are only the result of multiple or differential phosphorylation, we treated immunopurified Rad21 protein and a C-terminal truncated peptide (Rad21Pst), which also showed the phosphorylation pattern, with potato acid phosphatase. By this treatment (Fig. 3B), all the higher molecular mass bands of each ladder were converted to the 92-kDa band or to a 46-kDa band, respectively, indicating that the mobility shifts to higher molecular masses were caused by phosphorylation.

To determine the amino acids that were phosphorylated, the most highly modified, P-labeled protein band was excised from a polyacrylamide gel and processed for phosphoamino acid analysis (Fig. 3C). Scanning the electrophoresis plate with a PhosphorImager revealed that about 90% of the radioactivity was contained in phosphoserine and about 10% in phosphothreonine. No phosphotyrosine was detected.

A rough mapping of the phosphorylation sites was achieved by digestion of the protein with thrombin. The larger fragment, containing the C-terminal 413 amino acids, produced a single 68-kDa band on a protein gel. This peptide was not phosphorylated, as indicated by the fact that subsequent treatment with potato acid phosphatase did not change its mobility (Fig. 3B, lane 1). Furthermore, after thrombin cleavage of the P-labeled full-length protein, the resulting 68-kDa band was unlabeled (data not shown).

Analysis of deletion constructs expressed in S. pombe supported this finding. While Rad21Pst, which contains the N-terminal half of the Rad21 protein, showed at least four additional bands in the expected pattern (Fig. 3B, lane 8), Rad21Nae, containing more than the C-terminal half, was unmodified (Fig. 2A, lane 3).

Rad21 Is Expressed and Phosphorylated Periodically in the Cell Cycle

We examined the expression of the rad21 RNA and the Rad21 protein and the phosphorylation of Rad21 during the cell cycle. For this, cdc25-22 cells, released synchronously after cell cycle arrest at the restrictive temperature, were collected every half hour, and their lysates were subjected to Western (Fig. 4C) and Northern (Fig. 4B) blot analyses. Cell cycle synchrony and position were monitored by cell number and septation index (Fig. 4A). Transcription (Fig. 4B) and translation (Fig. 4C) indicated by the level of mRNA and unphosphorylated protein were clearly periodic in the cell cycle. They peaked just before the peak of septation. These data indicate that rad21 mRNA and Rad21 protein expression are highest in the G(1)/S phase. The most phosphorylated form peaked later in G(2), when transcription and new synthesis of protein were lowest (Fig. 4C). The phosphorylated form was stable during mitosis. At the time of new synthesis in the next cycle, the level of the most phosphorylated form of Rad21 dropped to its lowest point.


Figure 4: Expression level and phosphorylation state of the Rad21 protein during the cell cycle. A culture of cdc25-22 cells was synchronized by shifting to the restrictive temperature (36 °C) for 2.5 h and shifting back to the permissive temperature (25 °C). A, cell synchrony monitored by septation index and cell number. B, Northern blot. At 0.5-h intervals, equal numbers of cells were lysed and analyzed by Northern blotting for rad21 mRNA. (The same amounts of total RNA were used. The graph is normalized to leu1 mRNA in the samples.) C, Western blot of amount and phosphorylation state of the protein. Equal amounts of protein were loaded in each lane, and this was confirmed by Ponceau S staining of the gel prior to Western blot analysis. The mRNA and protein are expressed in arbitrary units.



To confirm this finding, lysates from arrested cdc mutants (3 h at restrictive temperature) were analyzed for their rad21 mRNA and Rad21 protein (Fig. 5, A and B). The cdc10-129 and cdc17-K42 mutants, arresting in the G(1) and S phases, respectively, showed the highest levels of mRNA. While cdc13-117, which arrests in G(2), showed an intermediate level of mRNA, cdc16-116 (defective in septum formation) exhibited the lowest level of rad21 mRNA (Fig. 5A).


Figure 5: Levels of rad21 mRNA and different phosphorylation states of the Rad21 protein in arrested cdc mutants. Different cdc mutants (c10, cdc10-129; c13, cdc13-117; c16, cdc16-116; c17, cdc17-K42) were arrested by holding them for 3 h at the restrictive temperature (36 °C). Equal numbers of cells were lysed and analyzed by Northern (A) and Western (B) blotting. On the Northern blot, equal amounts of total RNA were used, which were additionally standarized to leu1 mRNA and expressed as a percentage of mRNA in growing wild-type cells (WT) at 36 °C. The amounts of Rad21 protein species were determined by scanning the x-ray film of the enhanced chemiluminescence reaction. The lowermost and uppermostbands correspond to the unphosphorylated and hyperphosphorylated forms of the Rad21 protein, respectively.



At the protein level, as analyzed by Western blotting (Fig. 5B), the hypophosphorylated forms of the Rad21 protein were overrepresented in the lysate from arrested cdc10 cells compared with that from growing wild-type cells. This was probably the result of a kinase activity that phosphorylates Rad21 partially at this stage of the cell cycle. The most phosphorylated band was underrepresented, probably because a different kinase activity appearing later in the cell cycle was missing in these arrested cells. In lysates from arrested cdc17 cells, the most phosphorylated band was much enhanced, and the hypophosphorylated bands were weaker. The kinase(s) responsible for the hyperphosphorylation of the Rad21 protein must have been active in these arrested cdc17 cells.

In rad21-45 Mutant Cells, the Rad21 Protein Is Hypophosphorylated

The radiosensitive rad21-45 mutant of S. pombe is not able to efficiently repair DNA double-strand breaks(1) . To address whether the mutant protein is altered in its phosphorylation pattern, we compared total cell extracts of growing wild-type and rad21-45 cells by Western blot analysis (Fig. 6). Both strains contained the same amount of Rad21 protein. However, while the most phosphorylated form was the most abundant in wild-type cells, it was almost nonexistent in the mutant cells. Instead, the hypophosphorylated forms were overrepresented in the mutant. In the rad21-45 cdc10 and rad21-45 cdc17 double mutants placed at the restrictive temperature for the cdc mutations, the Rad21 protein remained hypophosphorylated (Fig. 6). This experiment shows that the mutant protein cannot be modified efficiently to the hyperphosphorylated form, as it is in wild-type cells.


Figure 6: The protein made by the rad21-45 mutant is hypophosphorylated. As described in the legend to Fig. 5, the phosphorylation status of the Rad21 protein in different strains was analyzed by Western blotting. The strains used were wild-type (WT), rad21-45 (r21), cdc10-129 rad21-45 (c10 r21) and cdc17-K42 rad21-45 (c17 r21) and were incubated at 25, 30, or 36 °C.



We also examined S. pombe wild-type and rad21-45 cells for changes in their pattern of Rad21 phosphorylation after treatment with 90 kilorads of -radiation. No changes in Rad21 phosphorylation were apparent, relative to that seen in unirradiated cells, after 2, 4, or 6 h following the irradiation.

The rad21 Gene Product Is Present in the Nucleus throughout the Cell Cycle and Carries the NLS in the C-terminal Part of the Protein

Based on our earlier studies (1) showing that the rad21-45 mutant is deficient in DNA double-strand break repair, the Rad21 protein was expected to be a nuclear protein. We examined exponentially growing S. pombe wild-type cells by indirect immunofluorescence microscopy. Fig. 7shows cells from different phases of the cell cycle labeled with the Rad21 antibody. In all of them, a clear nuclear staining was present. No labeling was observed with preimmune serum.


Figure 7: The Rad21 protein is localized in the nucleus during the whole cell cycle. Shown are wild-type cells during interphase, mitosis, and cytokinesis labeled with the antibody to Rad21 (a-21), with DAPI (to label DNA), and with fluorescein isothiocyanate conjugated to phalloidin (Phal.; labels F-actin). Scalebar = 10 µm.



Strain SPR61, in which the genomic rad21 gene is under the control of the S. pombe nmt1 promoter, which can be repressed by the addition of thiamine, was used to map the Rad21 NLS. SPR61 was transformed with three different constructs expressing the whole rad21 ORF, or parts of it, from the S. pombe adh promoter. The adh promoter usually causes a high constitutive expression of the gene under its control(12) . Thiamine was added to the exponentially growing cells to stop expression of the genomic rad21 gene. After 6 h, cells were examined by indirect immunofluorescence for the subcellular localization of the proteins expressed from the plasmids (Fig. 8A).


Figure 8: A, mapping of the Rad21 nuclear localization signal. In SPR61 and SPR61 transformed with p21adh, p21adhPst, and p21adhHinc, endogenous expression of Rad21 was stopped by adding thiamine to the cultures. 5 h later, cells were checked by indirect immunofluorescence (a-rad21) for subcellular localization of the peptides expressed from the adh promoter. Cells were simultaneously stained with DAPI to localize the DNA. B, characterization of full-length and truncated Rad21 polypeptides. Under the Rad21 protein (box), the full-length and truncated peptides that were expressed from the adh promoter in SPR61 are shown. They were tested for their subcellular localization (local.; nucl, nuclear; cyt, cytoplasmic; nd, not determined) and for their ability to rescue viability (viab.; +, viable; -, not viable) after endogenous expression from the nmt1 promoter had been stopped. The NLS is shown by its amino acid sequence (basic residues are boldface and underlined).



All constructs except p21adhPst, which covered the N-terminal half of Rad21, generated proteins that were imported into the nucleus. p21adhPst produced a protein (Rad21Pst) that was cytoplasmic (Fig. 8A), indicating that it did not contain an active NLS. Other truncated forms of Rad21 tested for nuclear localization are shown in Fig. 8B. The shortest peptide (Rad21Hinc), which was still imported into the nucleus, was derived from construct p21adhHinc. It consisted of the C-terminal 252 amino acids and contained only one NLS consensus sequence. Between amino acids 438 and 455, two clusters of basic amino acids are separated by 10 amino acids (Fig. 8B). This fits perfectly the bipartite NLS(24, 25) . There are no other clusters with three basic amino acids within five amino acids that could serve as a single cluster NLS. Like Rad21 protein expressed in wild-type cells, the truncated forms targeted to the nucleus colocalized with the DNA (Fig. 8A, DAPI staining).

Repression of Rad21 Expression Leads to Degradation of the Rad21 Protein and Cell Death

Because the rad21 gene disruption was lethal(1) , we examined cells after the expression of Rad21 had been shut off. For this, we placed the genomic rad21 gene under the control of the repressible nmt1 promoter. In the presence of >0.5 µM thiamine, transcription from this promoter is blocked(11) . The nmt1 promoter, together with the S. pombe ura4 gene, was used to replace 1.5 kilobases of DNA immediately upstream of the rad21 ORF. Two stable Ura strains were isolated (SPR61 and SPR62) that grew only in the absence of thiamine. Southern blot analysis showed that in these strains, the construct had been integrated into the rad21 locus at the correct position while replacing the rad21 promoter (Fig. 9).


Figure 9: A, construction of SPR61. a, map of the genomic rad21 region in wild-type cells; b, linear construct used to replace the rad21 5`-untranslated region with the nmt1 promoter (nmt1p); c, the same region after exchange. B, BamHI; C, ClaI; G, BglII; H, HindIII; P, PstI. Restriction sites lost are indicated in parentheses. B, Southern blot analysis of the following strains: wild-type (lane 1), parental SPR16 (lane 2), SPR61 (lane 3), and SPR62 (lane 4). Genomic DNAs were digested with BglII and hybridized with the HindIII-BglII fragment containing the rad21 ORF. kb, kilobases.



When growing SPR61 cells were transferred to medium containing 10 µM thiamine (+T medium), it took about five generations before the cells stopped dividing (Fig. 10A) and died (Fig. 10B) due to depletion of the rad21 gene product. When YPD medium was used, the culture stopped growing earlier and showed an accelerated growth rate immediately after transfer into the rich medium. When SPR61 was transformed with plasmid p21Fleu, which carries the complete rad21 region, the cells grew fine in +T and YPD media (Fig. 10A).


Figure 10: A, growth curves of SPR61 under conditions in which Rad21 expression is on or off. SPR61 cells growing on -T plates were diluted to 1 times 10^5 cells/ml in -T (circle), +T(bullet), and YPD () media, and the titer was determined at the indicated time points. Included in the graph is also SPR61 transformed with p21Fleu (which contains the rad21 gene behind its own promoter) in +T medium (). B, survival of SPR61 after exposure to thiamine. After 1 h of growth of SPR61 in Edinburgh minimal medium (EMM)-T, EMM+T, and YPD media, cells were streaked onto a EMM-T plate. Only cells from the -T culture survived. C, depletion of Rad21 in SPR61 cells by repression of the nmt1 promoter. Growing SPR61 cells were diluted into -T, +T, and YPD media. At different time points, the same numbers of cells were lysed, and the amounts and the phosphorylation states of Rad21 were analyzed by Western blotting. Expression is compared with that in growing wild-type cells (WT) in -T medium at the same density.



During the experiment, we monitored the degradation of the Rad21 protein pool. SPR61 cells were grown in -T medium to a titer of 1 times 10^7 cells/ml. Cells were diluted to a titer of 1 times 10^6 cells/ml in prewarmed -T medium as well as in +T and YPD media to stop Rad21 expression and were incubated for further growth. At various time points, cells were removed for Western blot analysis (Fig. 10C). On the same Western blot, we also compared expression of Rad21 in wild-type cells growing in -T medium. Estimating the total amount of Rad21 protein isolated from the same number of wild-type and SPR61 cells, Rad21 was overexpressed approximately 10-fold when it was under the control of the nmt1 promoter. Interestingly, while the lower bands of the Rad21 ladder were overrepresented in the strain with the nmt1 promoter, the highest phosphorylated band had almost the same intensity in lysates from wild-type and SPR61 cells. Within 30 min after the rad21 transcription had been stopped, a decrease in the concentrations of the hypophosphorylated forms of Rad21 was observed clearly, while the highest phosphorylated form seemed stable beyond the 1-h time point, re-establishing the band pattern of growing wild-type cells. After synthesis of Rad21 stopped, presumably the less modified forms got degraded faster, while the highest phosphorylated form was more stable, or the lower bands got converted to the highest modified form before degradation. After 6 h of repression, the Rad21 protein was almost undetectable (Fig. 10C).

This result was confirmed by immunofluorescence. The nuclear staining of SPR61 cells with the Rad21 antibody decreased constantly after the addition of thiamine to growing cells. After 5 h, no nuclear staining could be observed anymore (Fig. 8A, top left panel).

The survival of cells exposed to thiamine for various times was tested by plating them onto plates lacking thiamine (-T plates). The viability of the cells dropped to zero after 1 h in thiamine (Fig. 10B). Since it takes about 10 h to reactivate the nmt1 promoter(26) , this was long enough to deplete the Rad21 pool, and as a consequence, cells were damaged irreversibly.

Fig. 11shows the final phenotype of cells after 15 h in YPD medium. The nuclear organization was disturbed severely, often resulting in cells with dispersed staining of nuclear material (Fig. 11C, DAPI staining). At this and later time points (24 h), aberrant septation also occurred (Fig. 11, D and F). It is not clear yet whether cell death is a direct result of the Rad21 depletion or whether it is caused by a secondary effect.


Figure 11: Morphological changes in SPR61 after the expression of Rad21 was repressed. A and B, exponentially growing SPR61 cells in -T medium were shifted to YPD medium. After 15 h (C and D) and 24 h (E and F), cells were analyzed by fluorescence microscopy using DAPI (stains DNA) and calcofluor (stains septa). Scale bar = 10 µm.




DISCUSSION

The Rad21 Protein Has an Aberrant Apparent Molecular Mass

The rad21 gene product from S. pombe was identified by Western blotting. An antibody raised against a peptide expressed in E. coli recognized a protein with the mobility of a 92 kDa-protein and additional peptides with reduced mobilities of up to 100 kDa. Despite the fact that the calculated molecular mass of Rad21 is 68 kDa, there is no doubt that the peptides detected by the Rad21 antibody correspond to the authentic rad21 gene product. Expression of the 1884-bp ORF cloned into different vectors and expressed in three different systems (E. coli, pT7-7; rabbit reticulocyte lysates, pT7-7 linearized 35 bp downstream of the ORF; and S. pombe, pART1) always resulted in the 92-kDa band. When cloned in S. pombe downstream of the overexpressing adh promoter, the intensity of the bands corresponding to Rad21 peptides increased up to 50-fold, and after repression of the expression of Rad21 under the control of the nmt1 promoter, the signal disappeared. In addition, truncated versions of the rad21 ORF in pART1 generated proteins migrating with higher mobilities of the immunodetected band after SDS-PAGE.

When analyzed by Western blotting ( Fig. 1and Fig. 2), an aberrantly low mobility was also observed for most of the truncated forms of Rad21 and even for the Rad21-GST fusion protein. For other proteins(27, 28) , this phenomenon of aberrant mobility has been explained by the high polarity of the proteins. We believe that this is also true for Rad21 since 156 of its 628 amino acids are charged at pH 7.0, and its polarity index is 51(1) .

Post-translational Phosphorylation of Rad21

The Rad21 protein is post-translationally modified as clearly indicated by the appearance of an additional four to five bands of decreased mobility when the protein is analyzed by Western blotting (Fig. 3A). Labeling with [P]orthophosphate and limited treatment with potato acid phosphatase (Fig. 3B), which gradually converted all bands to the 92-kDa band (or 46-kDa band in a truncated version of Rad21), revealed that multiple phosphorylation is the only reason for these band shifts.

The phosphorylation sites in Rad21 reside in the N-terminal 215 amino acids of the protein. After treatment with the protease thrombin, which recognizes a unique cleavage site in Rad21, the 413-amino acid C-terminal fragment (68 kDa) appeared unphosphorylated as judged by its appearance as a clean single band on Western blots that could not be converted to lower molecular mass species by subsequent treatment with potato acid phosphatase (Fig. 3B, lane 1). The N-terminal fragment (28 kDa) could not be detected by Western blotting since the antibody was made against the 68-kDa C-terminal fragment. Also, after cleavage of P-labeled Rad21, the resulting 68-kDa band was unlabeled. Supporting this result was the observation that phosphorylation occurred only in Rad21Pst, which contains the N-terminal 374 amino acids, while Rad21Nae, containing the C-terminal 364 amino acids, appeared unmodified (Fig. 2A, lanes 2 and 3, without thrombin).

Phosphoamino acid analysis revealed that Rad21 is mainly phosphorylated at serine and much less at threonine residues. Since there are 28 serines and 11 threonines in the N-terminal fragment generated by thrombin cleavage, we have not yet attempted to map the exact phosphorylation sites.

Phosphorylation of the Rad21 Protein in the Cell Cycle

The Rad21 protein is phosphorylated at two distinct stages of the cell cycle. The first kinase acts immediately after translation of the Rad21 protein in the G(1)/S phase and phosphorylates the Rad21 protein (see bands up to the third or fourth band on Western blots in Fig. 5B). The last step, leading to the most phosphorylated form of Rad21, does not occur at this time, as seen in lysates from arrested cdc10 cells (Fig. 5B). A second kinase is probably responsible for phosphorylating Rad21 after the cdc10 execution point, but before the cdc17 execution point. Western blots of arrested cdc17 lysates reveal mainly the most phosphorylated Rad21 species. The degradation pathway for Rad21 is probably not active during the generation of this highly phosphorylated form of Rad21. This leads to an accumulation of the most phosphorylated band (Fig. 5B).

Despite the facts that the rad21 gene is expressed at its highest level in G(1) and the gene contains two MlwI cell cycle box-like sequences (29) (bp -223 to -211 in sequence(1) ) 40 bp upstream of the potential TATA box, its expression is not dependent on the presence of p85 because transcription of the rad21 gene continues under cdc10-restricted conditions as well as in arrested cdc17 cells.

The Protein Made by the rad21-45 Mutant Is Permanently Hypophosphorylated

When we analyzed the protein made by the rad21 mutant, we got a first hint of the significance and biological importance of Rad21 phosphorylation. Rad21-45 protein appeared always hypophosphorylated, no matter if isolated from exponentially growing cells or from cdc mutants arrested at distinct points in the cell cycle (Fig. 6). Even in arrested cdc17 cells, where the most phosphorylated form of the wild-type protein accumulates, the protein made by the rad21-45 mutant remained hypophosphorylated.

The rad21-45 mutation causes a substitution of Ile-67 by Thr-67(1) . This by itself does not remove a potential phosphorylation site, but it might alter a kinase recognition site. It is also possible that the wrong amino acid causes a conformational change in the protein. This idea is supported by the fact that computer analysis of the amino acid sequence of the mutant revealed a higher probability for a turn at the mutated position compared with the wild-type sequence. This could result in the inaccessibility of phosphorylation sites due to abnormal folding of the mutant protein. It is not clear yet if the putative conformational change, the hypophosphorylation, or a combination of both is the reason for the radiosensitivity of the rad21-45 mutant. This will have to be resolved by identification and site-directed mutagenesis of the phosphorylation sites.

Rad21 Is a Nuclear Protein

Previously, we described the putative involvement of Rad21 in DNA double-strand break repair(1) . From this, it was expected to be a nuclear protein. We proved this by using indirect immunofluorescence to examine cells in which Rad21 was expressed from three different promoters. In wild-type cells, indirect immunofluorescence for the Rad21 protein resulted in a clear nuclear signal. This signal increased in intensity when Rad21 was expressed from the nmt1 or adh promoter, which increased the expression level to about 10- or 50-fold, respectively, as estimated by Western blotting. When expression was repressed with thiamine in SPR61 cells, in which the genomic rad21 gene is under the control of the nmt1 promoter, the nuclear signal disappeared after 6 h.

By expressing truncated forms of Rad21 in SPR61, the NLS was identified in the C-terminal third of the protein (Fig. 8, A and B). While N-terminal Rad21Pst was excluded from nuclear import and stayed in the cytoplasm, Rad21Hinc, the shortest peptide tested, containing the C-terminal 252 amino acids, was still imported. Two kinds of NLS motifs have been described that contain one or two clusters of basic amino acids. In the latter, the clusters are usually divided by a 10- or 11-amino acid spacer. There is only one region in the smallest imported peptide that fulfills the consensus requirements for both motifs (Fig. 8B). An N-terminal basic cluster (RKRK) is followed by a 10-amino acid spacer and a second cluster in which three of four amino acids are basic (KHqR). Since histidine does not appear within any downstream basic clusters of nuclear proteins (listed in (24) and (25) ), the N-terminal cluster may act alone as a single cluster NLS.

Subcellular Location of Rad21 Phosphorylation

Phosphorylation of Rad21 happens in the cytosol and perhaps in the nucleus and does not seem to have any influence on the nuclear import of Rad21. Rad21 was definitely phosphorylated in the cytosol as shown by Rad21Pst, which was excluded from nuclear import (Fig. 8A) and was phosphorylated (Fig. 3B). But, phosphorylation is not required for nuclear import because unphosphorylated Rad21Nae and Rad21Hinc were imported into the nucleus (Fig. 8B).

Although truncated forms of Rad21 can be phosphorylated in the cytosol, phosphorylation of Rad21 probably occurs in the nucleus. Upon overexpression, most of the nuclear Rad21 protein is hypo- or unphosphorylated (Fig. 10C). As suggested from the Rad21 depletion experiment, these forms get phosphorylated when new synthesis of Rad21 is repressed (Fig. 10C, 0.5 h). It seems more probable that this phosphorylation takes place in the nucleus. The alternative model (in which hypophosphorylated Rad21 is exported into the cytoplasm and, after full phosphorylation, is reimported into the nucleus) seems less likely.

Functional Role of the Rad21 Protein

Although the precise function of the Rad21 protein in the mitotic growth of S. pombe cannot be answered yet, it is likely that its fully phosphorylated form is the most important one. The hypophosphorylated forms of the rad21-45 mutant are not sufficient to support DNA double-strand break repair. Furthermore, in the Rad21 depletion experiment, the timing of loss of cell viability correlates quite well with the disappearance of the hyperphosphorylated forms of Rad21. Interestingly, in unirradiated cells analyzed 15 h after shutoff of Rad21 expression from the nmt1 promoter, the S. pombe chromosomes remained intact as analyzed by pulsed-field gel electrophoresis. Thus, although Rad21 is required for repair of DNA double-strand breaks, its absence does not appear to promote the accumulation of double-strand breaks in the DNA of unirradiated cells.

The final phenotype, which develops a few generations after Rad21 expression is switched off, was without doubt triggered by dilution and depletion of Rad21 in the daughter cells. We cannot exclude the possibility that the disperse staining of the nuclear material and abnormal septation are secondary consequences resulting from the aberrant mitoses since these phenotypes were observed about 5 h after visible depletion of the Rad21 protein. It might be easier to address the question of the primary phenotype by using a temperature-sensitive mutant, which unfortunately is not available at present.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM31253 (to S. S.). 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.

§
Supported in part by a fellowship from the Deutsche Forschungsgemeinschaft. Present address: Inst. für Genetik, Universität Köln, Zülpischer Str. 47, 50674 Köln, Germany.

To whom correspondence should be addressed: Dept. of Biology, Rm. 3226, Bonner Hall, University of California at San Diego, La Jolla, CA 92093-0322. Tel.: 619-534-2327; Fax: 619-534-0053.

(^1)
The abbreviations used are: bp, base pair(s); ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; DAPI, 4`,6`-diamidino-2-phenylindole; NLS, nuclear localization signal.


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