(Received for publication, September 8, 1994; and in revised form, January 13, 1995)
From the
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 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
). 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.
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, 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 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.
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.
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).
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 and
S phases, respectively, showed the highest levels of mRNA. While cdc13-117, which arrests in G
, 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.
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.
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).
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 10
cells/ml in
-T (
), +T(
), 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
10
cells/ml. Cells were diluted to a titer of 1
10
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.
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) .
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.
Despite the
facts that the rad21 gene is expressed at its highest level in
G 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 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.
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.
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.
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.