(Received for publication, November 23, 1994)
From the
We have performed a screen for genes that affect chromosome stability when overexpressed in the budding yeast Saccharomyces cerevisiae. Two of the genes recovered in the screen, CST17 and CST20, share a number of phenotypic properties, suggesting their involvement in the same cellular process. DNA sequence analysis of these genes revealed that they encode components of the Ran/RCC1 molecular switch system: CST17 is Ran itself (Ras-like nuclear protein) and CST20 is a novel yeast protein with a high degree of similarity to mammalian RanBP1, which is known to interact with Ran-GTP in vitro. We demonstrate that the CST20 protein can interact with Ran-GTP in vitro under similar conditions, indicating that it is the functional yeast homolog of mammalian RanBP1. The results of immunoprecipitation experiments show that the two yeast proteins form a complex in vivo. Deletion of the gene encoding RanBP1 revealed that it is essential for viability, as are Ran and RCC1. Similar phenotypic consequences of overproduction of either Ran or RanBP1 indicate that the latter protein is a functional component of the Ran/RCC1 molecular switch system, which is implicated in the control of a number of nuclear functions. Our finding that overproduction of two components of this system results in mitotic chromosome nondisjunction and sensitivity to an anti-microtubule drug benomyl suggest their involvement in mitosis as well. Thus RanBP1 is a functional component of a highly conserved molecular system that affects diverse cellular processes. The availability of this gene in S. cerevisiae provides a genetic system for the analysis of RanBP1 function in vivo.
Components of the cell that regulate nuclear organization are likely to play an essential role in proper execution of chro-mosome replication and segregation. One of the components important for the control of nuclear structure is the Ran/RCC1 molecular switch system. Ran is a conserved GTP-binding protein that is homologous to members of the Ras superfamily(1, 2, 3, 4, 5, 6) . A conserved nuclear protein RCC1 catalyzes the exchange of guanine nucleotides on Ran(2, 7) . The low intrinsic GTPase activity of Ran is activated upon interaction with a GTPase-activating protein RanGAP1, leading to hydrolysis of Ran-bound GTP to GDP(8) . By analogy with Ras, it can be postulated that Ran is activated when GTP is exchanged for bound GDP by RCC1 and inactivated when GTP is hydrolyzed by Ran upon activation by RanGAP1. A number of proteins of unknown function that can interact with Ran-GTP in vitro have been reported(9, 10) .
The
components of the Ran/RCC1 molecular switch system are implicated in a
variety of biological functions, including regulation of gene
expression and progression into the S-phase(11) , RNA
processing and export from the
nucleus(6, 12, 13, 14) , protein
import into the nucleus (15, 16) , chromatin
decondensation after completion of mitosis(4) , nuclear
envelope growth (17) and integrity, ()DNA
replication(17) , and chromosome
stability(3, 11) . Much of these data are compatible
with the hypothesis that the activity of the Ran/RCC1 pathway is
necessary for bringing nuclear organization into an active, interphase
state upon exit from mitosis(4, 17) .
Many events critical for genomic stability are dependent on proper stoichiometric ratios of the components involved(18, 19, 20, 21) . We have exploited this requirement to search for genes important for chromosome stability (CST genes) in Saccharomyces cerevisiae. Here we describe the identification and analysis of two genes, CST17 and CST20, which encode components of the Ran/RCC1 molecular switch system.
The diploid yeast strain YPH275 contains a supernumerary
chromosome fragment (CF) ()that allows detection of
chromosome loss by colony color-sectoring
assay(22, 23) . The yeast cDNA library under the
control of a galactose-inducible promoter (24) was transformed
into YPH275, and clones showing both plasmid-dependent and
galactose-dependent increases in CF loss were selected.
To obtain a
genomic clone of CST20, polymerase chain reaction
amplification was performed using genomic DNA and primers flanking the
gene (640 bp 5` and 110 bp 3`). The fragment was cloned into
pBluescript to make pIL28 or into pRS316 to make pIL30-1. The CST20 deletion construct (pIL29) was made by replacing a
515-bp BglII fragment of CST20 sequence in pIL28 with
a 3.5-kb Kan-LEU2 fragment. To obtain
epitope-tagged CST20, it was fused with the HA epitope of pETXHA and
cloned into pRS316-GAL1(24) to make pAM10.
Protein
analysis techniques were as described(25) . Wild-type spi1/fyt1 cDNA was expressed in Escherichia coli from
pHisFyt (a gift of X. He) as an N-terminal His-6 fusion. The protein
was purified on nickel-nitrilotriacetic acid resin (Qiagen) and labeled
with [-
P]GTP(9) . Immunoblots were
probed either with the 12CA5 anti-HA monoclonal antibody or with a
rabbit antibody against the Schizosaccharomyces pombe spi1/fyt1 protein (a gift of X. He). Probing the blots with
Ran-[
P]GTP was as described(9) . For
immunoprecipitation, the anti-HA 12CA5 or control ascites fluid was
added at a dilution of 1:100 to 200 µl of the extract containing
0.6 mg/ml protein.
Quantitation of the rates of chromosome loss, nondisjunction, and mitotic recombination were performed as described(18, 23) .
Plasmid pCST17 contains a cDNA insert of 0.8 kb. A sequence of the insert revealed its identity to a previously described gene GSP1/CNR1, which is the more abundantly expressed of the two genes encoding Ran in budding yeast(5, 6) .
The
sequence of the 0.8-kb insert of CST20 is identical to a
stretch of genomic DNA ()on chromosome IV. It contains an
open reading frame potentially encoding a protein of 211 amino acids,
with a molecular mass of 24.15 kDa. The deduced protein sequence is 50%
identical and 65% similar to the mouse RanBP1 (9) over most of
its length (Fig. 1). Such a high degree of conservation suggests
that the yeast CST20 is a structural and perhaps functional
homolog of mammalian RanBP1. Two possible leucine zipper regions (asterisks in Fig. 1) and a potential RNA-binding motif
(residues 81-90) have been identified in the mouse RanBP1
sequence(9) . Leucine residues of the potential zipper regions
are largely conserved between the two species. However, the region
corresponding to the RNA-binding consensus in the mouse protein is not
conserved in the yeast sequence, suggesting that this protein does not
bind RNA in this species.
Figure 1: Comparison of predicted amino acid sequences of mouse RanBP1 and yeast CST20. Asterisks underneath the sequence indicate partially conserved leucines in the potential zipper regions.
The gene encoding the RCC1 homolog (SRM1) and the more abundantly expressed of the two genes encoding Ran (GSP1/CNR1) are also essential in budding yeast(5, 6, 11) . The fact that RanBP1 is as necessary for cell viability as the other components of the Ran/RCC1 pathway points to its important role in the function of this molecular system.
To distinguish between the endogenous RanBP1 and the protein
expressed from the plasmid, an HA epitope was added at the N terminus
of CST20. This fusion was placed under the control of the GAL1 promoter, making pAM10. The resulting fusion protein is
biologically active, since growth of the cells on galactose produces
the same phenotypic effects (described below) as overexpression of the
unaltered gene. Protein extracts were prepared from cells containing
pAM10, grown on glucose or on galactose. Three identical sets of
samples were separated in parallel on a polyacrylamide gel; two of them
were transferred onto nitrocellulose and probed with either the anti-HA
monoclonal antibody (Fig. 2A) or
Ran-[P]GTP (Fig. 2B). The panel
of the gel containing the third set was stained for total protein (Fig. 2C). Induction of the expression of HA-CST20 resulted in the appearance of an HA-reactive protein of 30 kDa (Fig. 2A, lane2). This is in
reasonable agreement with the predicted value of 26 kDa and with the
fact that the 23.6-kDa mouse RanBP1 migrates with a mobility of 27
kDa(9) . When a duplicate blot was probed with
Ran-[
P]GTP, a single protein of 28 kDa was
detected in all samples (Fig. 2B). When expression of HA-CST20 is induced, an additional 30-kDa Ran-binding protein
appears (Fig. 2B, lane2). Its
mobility is identical to that of HA-CST20 (Fig. 2A, lane2). This establishes the identity of CST20 as
the RanBP1 of S. cerevisiae.
Figure 2:
Interaction of Ran (CST17) and RanBP1
(CST20) in vitro. A, immunoblot of yeast protein
extracts probed with a monoclonal antibody to the HA-epitope tag. B, an identical blot probed with
Ran-[P]GTP. C, a control part of the
gel showing samples identical to A and B, stained
with Coomassie Blue. Lane1, GAL1-HA-CST20 on glucose (promoter repressed); lane2, GAL1-HA-CST20 on galactose (promoter induced); lane
3, control cells (containing the parental vector) on glucose; lane 4, control cells on galactose. M, molecular mass
markers (kDa). The arrow points to
HA-CST20.
Protein interaction in
vitro indicates their likely functional association in the cell.
To test for the in vivo interaction between Ran (CST17) and
RanBP1 (CST20), we used a rabbit polyclonal antiserum to Ran
(spi1/fyt1) from S. pombe. ()Its specificity to S. cerevisiae antigens was determined by immunoblotting of
protein extracts from cells bearing CST17 (Ran) under the
control of GAL1 promoter (Fig. 3A). Under
non-inducing conditions, the antiserum reacted with a protein of 25 kDa (lane2), which has nearly identical mobility to
spi1/fyt1 (lane3), consistent with the predicted
molecular masses of the Ran proteins in the two species (24.8 and 24.5
kDa, respectively). Upon induction of CST17, the intensity of
staining of the 25-kDa band increased significantly (lane1). Thus the anti-spi1/fyt1 antiserum recognizes the
product of CST17 in S. cerevisiae.
Figure 3: Interaction of Ran (CST17) and RanBP1 (CST20) in vivo. A, immunoblot probed with anti-Ran (S. pombe spi1/fyt1) antiserum, showing the specificity of the antiserum for the S. cerevisiae Ran (CST17). Lanes 1 and 2, S. cerevisiae containing GAL1-CST17 grown on galactose (1) or glucose (2); lane 3, S. pombe. B, immunoblot of S. cerevisiae proteins, probed with the anti-Ran antiserum. Proteins of GAL1-HA-CST20 cells grown on glucose (lane1) or galactose (lanes2 and 3) were immunoprecipitated with anti-HA antibody (lanes1 and 2) or a control antibody YOL1/34 (lane3). Positions and molecular mass (in kDa) of the marker proteins are shown at the right. The arrow points to Ran.
To assess in vivo association of Ran and RanBP1, protein extracts from cells containing the GAL1-HA-CST20 fusion were immunoprecipitated using anti-HA antibodies and subjected to immunoblotting with anti-Ran antiserum (Fig. 3B). When expression was induced, prominent staining of CST17 was visible (lane2). This protein could be detected neither in immunoprecipitates with a control antibody (lane3) nor in immunoprecipitates of cells grown on glucose (lane1), even though they still contained endogenous CST20. Thus, Ran (CST17) and RanBP1 (CST20) form a complex in yeast cells in vivo.
Chromosome instability can manifest itself as chromosome loss (1:0 segregation) or nondisjunction (2:0 segregation). The first type of missegregation is characteristic of defects in DNA metabolism. Chromosome nondisjunction is primarily induced by defects in mitotic chromosome segregation (e.g.(19) and (21) ). We determined the rate of loss and nondisjunction of the CF in cells overexpressing CST17 or CST20 (Table 1). Overexpression of each of the genes causes a very strong increase in both 1:0 and 2:0 events, implicating possible defects in both DNA metabolism and mitosis.
To further characterize the chromosome transmission defect we measured the rate of mitotic recombination (Table 2), which is directly related to the presence of DNA lesions(26) . Overexpression of either CST17 or CST20 induces a strong increase in the frequency of mitotic recombination, indicating that DNA damage is occurring in the cells. Additionally, these data provide evidence that these CST genes affect the stability of a native yeast chromosome, thus showing that the mechanism is not specific for the CF.
Hydroxyurea (HU) is an inhibitor of DNA synthesis. Cells with a reduced efficiency of DNA replication, repair, or S-phase checkpoint can be expected to be sensitive to HU. We tested diploid cells overexpressing CST17 or CST20 for the ability to grow in the presence of 100 mM HU. Wild-type cells and cells containing the parental vector can grow under these conditions. Cells containing either pCST17 or pCST20 grow normally on glucose plus HU but are unable to form colonies on galactose plus HU (data not shown).
Since CST17- and CST20-overexpressing cells show a chromosome nondisjunction pattern suggestive of mitotic defects, we tested them for sensitivity to a microtubule-depolymerizing drug, benomyl. Yeast mutants with defects in the function of the mitotic spindle have an increased sensitivity to this drug (e.g.(19) and (21) ). Neither CST17- nor CST20-overexpressing diploid cells form colonies in the presence of 10 µg/ml benomyl at 23 °C, whereas the control cells grow well under these conditions (data not shown). This is consistent with a defect in the mitotic spindle function.
The elevated rates of mitotic recombination and sensitivity to HU, induced by overexpression of either CST17 (Ran) or CST20 (RanBP1), provide an indication that some aspect of DNA metabolism is affected by both of these genes. At the same time, the increased rates of mitotic chromosome nondisjunction and sensitivity to benomyl suggest that both genes also induce a defect in mitotic chromosome segregation. Taken together with the data on their association in vivo and the fact that both genes are essential for viability, this strongly indicates that RanBP1 is a functional component of the Ran/RCC1 system.
Although chromosome instability was detected in RCC1
mutants(3, 11) , involvement of this system in mitotic
processes has not been characterized. Fission yeast pim1 (RCC1) mutants display no gross defects of mitotic spindle
structure(3) . The only possible connection between the
Ran/RCC1 system and the mitotic spindle function may be the finding
that overexpression of RCC1 in budding yeast can suppress some
-tubulin mutations(27) . It is possible that, since the
mitotic spindle in S. cerevisiae is entirely intranuclear,
abnormal nuclear organization presumably induced by CST17 overexpression may interfere with spindle function. Alternatively,
abnormal chromatin organization could prevent the formation of
functional mitotic chromosomes.
The available biochemical data
indicate that RanBP1 potentiates the action of RanGAP1 on Ran, ()thus playing the role of a negative regulator of the Ran.
In this case, overproduction of RanBP1 might lead to accumulation of
Ran in its inactive GDP-bound state, thus mimicking the effect of RCC1
inactivation. This would be in good agreement with the data that RCC1
is necessary for a number of nuclear functions in mammalian and yeast
cells and in Xenopus egg extracts.
In conclusion, the data presented here indicate that RanBP1 is an essential, functional component of the Ran/RCC1 molecular switch system and demonstrate the involvement of this system in chromosome segregation. The high degree of sequence identity of RanBP1 and other characterized components of this pathway between yeast and mammals indicates the evolutionary conservation of the functions of this system in eukaryotes. The Ran/RCC1 molecular switch system controls many diverse essential functions, and it is not yet clear whether RanBP1 participates in all or a subset of them. However, identification of RanBP1 in yeast will allow the genetic dissection of its role and will aid in the future resolution of these issues.