Regulation of the localization and stability of Cdc6 in living yeast cells

BBRC Biochemical and Biophysical Research Communications 306 (2003) 851–859 www.elsevier.com/locate/ybbrc

Regulation of the localization and stability of Cdc6 in living yeast cells Kathy Q. Luo,a,b,* Suzanne Elsasser,a Donald C. Chang,b and Judith L. Campbella b

a Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China

Received 26 May 2003

Abstract The Cdc6 protein is an essential regulator for initiation of DNA replication. Following the G1/S transition, Cdc6 is degraded through a ubiquitin-mediated proteolysis pathway. In this study, we tagged Cdc6 with green fluorescent protein (GFP) and used site-specific mutations to study the regulation of Cdc6 localization and degradation in living yeast cells. Our major findings are: (1) Cdc6-GFP distributes predominantly in the nucleus in all cell cycle stages, with a small increase in cytoplasmic localization in G2/M cells. (2) This nuclear localization is critical for Cdc6 degradation. When the N-terminal nuclear localization signal (NLS) was mutated, Cdc6-GFP no longer accumulated in the nucleus, and the mutant cdc6 was stabilized compared to wild type. (3) The putative CDK phosphorylation sites are not required for Cdc6 nuclear localization, but are important for protein stability. These observations suggest that the stability of Cdc6 protein is regulated by two factors: nuclear localization and phosphorylation by CDK1. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Cdc6; GFP; Yeast; G1/S transition; Nuclear localization and phosphorylation

Cdc6 is one of the essential proteins that regulate the initiation of DNA replication in eukaryotic cells. In Saccharomyces cerevisiae, Cdc6 is expressed in G1 phase and degraded quickly after G1/S transition [1]. Cdc6 can bind to ORC and together with Cdt1 promotes the loading of Mcm2-7 proteins to the origins of DNA replication, which forms pre-replicative complexes (preRCs) during G1 phase [2–7]. Because of the functional importance of Cdc6, we are interested in studying the regulation of its subcellular localization and degradation during the progress of the cell cycle. At present, it is not clear whether Cdc6 is localized exclusively in the nucleus of yeast cell or not. An earlier indirect immunofluorescent study had indicated that a Cdc6-myc fusion protein, when overproduced from the GAL promoter, was accumulated predominantly in the nucleus during G1 phase [8]. Results from another immunostaining study using antibody against Cdc6, however, suggested that the majority of Cdc6 protein was distributed in the cytoplasm [9]. One possible ex* Corresponding author. Fax: +852-2358-7323. E-mail address: [email protected] (K.Q. Luo).

planation is that Cdc6 localization may vary during the cell cycle. It has been demonstrated that the human Cdc6 homolog, HsCdc6, localizes to the nucleus during the G1 phase and translocates to the cytoplasm at the start of S phase [10–13]. The subcellular localization of HsCdc6 was thought to be regulated by multiple mechanisms including phosphorylation, the signal for nuclear import (NLS) at the N-terminus, and the signal for nuclear export (NES) at the C-terminus [11,12,14,15]. It was proposed that HsCdc6 is synthesized and imported into the nucleus in an NLS-dependent manner before DNA replication. After entry into S phase, phosphorylation of HsCdc6, probably by cyclinA/CDK2, leads to its export from nucleus to the cytoplasm via NES [15]. The mechanisms controlling the protein localization of Cdc6 are not yet understood in yeast cells. In this study, we want to examine the effects of NLS and phosphorylation on translocation of Cdc6 in various stages of the cell cycle by measuring the distribution between wild-type Cdc6 and its mutants in living yeast cells using a GFP fusion protein technique. A second important question about Cdc6 is what regulates its protein level during the cell cycle. In

0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01082-9

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S. cerevisiae, the CDC6 mRNA is expressed mainly during late M and G1 phases [1,16–19]. The protein levels mirror the mRNA pattern because Cdc6 is targeted for rapid degradation during late S and G2/M phases, though degradation is slower in G2/M [1,20,21]. It has been proposed that degradation of Cdc6 could help to prevent rebuilding of preRCs in the S and G2/M phases, thus ensuring that the chromosome goes through only one round of DNA replication per cell cycle [22]. It has been shown that Cdc6 is very unstable in G2 phase, and its N-terminus (1–48 amino acid) is important for regulating its degradation. When the Nterminus was deleted, the half-life of the protein was greatly lengthened [23,24]. It is known that the N-terminus of Cdc6 contains three-consensus CDK1 phosphorylation sites [25]. And they were shown to be important for Cdc6 degradation. The N-terminus of Cdc6, however, also contains a nuclear localization signal (NLS), which can control the translocation of an NLS-LacZ fusion protein into the nucleus [9]. Furthermore, the N-terminus also contains the binding sites for Cdc28/Clb kinase [26] and Cdc4, which is thought to be a component of the Skp1-Cullin-F-box (SCF)-Cdc4 ubiquitin ligase [23,24]. Thus, the stabilization of Cdc6 resulting from its N-terminal deletion may be due to a number of different factors, including the removal of the three phosphorylation sites at the N-terminus, the removal of the binding domain for Cdc4, or the failure of nuclear localization caused by deletion of the NLS. In order to sort out these issues under in vivo conditions, we employed Cdc6-GFP to examine the translocation and degradation of Cdc6 in living cells harboring the following mutations: (1) mutations of the three putative N-terminal phosphorylation sites, (2) mutations of all six putative phosphorylation sites, and (3) mutations of the NLS site. We found evidence that both nuclear localization and phosphorylation by CDK1 can affect the stabilization of Cdc6 in yeast cells. Materials and methods Plasmid constructions. Plasmid pRS424-CDC6-GFP was used to complement a temperature-sensitive allele of CDC6, cdc6-1. It was generated as follows: (a) The XhoI and EcoRI fragment of CDC6 gene was excised from plasmid yCP50-CDC6 and cloned into a Bluescript vector. (b) The stop codon of the CDC6 gene in pBS-CDC6 was replaced with a NdeI site by PCR. (c) The NdeI–SacI fragment of GFP(S65T) was excised from pRSET-GFP provided by Dr. Roger Tsien [27] and cloned at the 30 end of CDC6 in the pBS-CDC6 vector. (d) The XhoI/SacI fragment of CDC6-GFP was excised from plasmid pBS-CDC6-GFP and cloned into vector pRS424. pRS424 is a yeast 2 l (multicopy) plasmid containing a TRP1 marker. To clone the CDC6-GFP fusion gene behind a regulatable GAL1,10 promoter, the C-terminal-half of CDC6 and the full-length GFP gene was inserted into plasmid pSE43 at the BglII and blunt-ended SpeI sites. Plasmid pSE43 has the GAL1,10 promoter linked with the CDC6 gene including its 30 -untranslated region (30 UTR) in a Bluescript vector [24]. The resulting construct is named pBS-GAL-CDC6-GFP. The

SacI–HindIII fragment of GAL-CDC6-GFP was then cloned into a multi-copy plasmid, pRS425, containing a LEU2 marker. The procedures used to generate three plasmids carrying cdc6 mutant genes, pSE156, pSE200, and pSE159x, have been described in [24]. In pSE156, the NLS sequence of CDC6 was changed from K29 RKK32 to four alanines and the mutant called cdc6-DNLS. In pSE200, the three N-terminal phosphorylation sites of CDC6 were changed from T7 T23 S43 to three alanines and the mutant called cdc6-3ALA. In pSE159x, the three C-terminal sites of CDC6 were mutated from T134 S354 S372 to three alanines. To tag cdc6-DNLS with GFP, the SacI/PstI fragment of pSE156 containing GAL promoter and N-terminus of cdc6-DNLS was ligated with SacI/PstI digested pRS425-GAL-CDC6-GFP to generate a pRS425-GAL-cdc6-DNLS-GFP construct. To label cdc6-3ALA with GFP, the region containing GAL promoter and cdc6-3ALA was excised from plasmid pSE200 and fused with SacI/PstI-digested pRS425-GALCDC6-GFP to generate a pRS425-GAL-cdc6-3ALA-GFP construct. To prepare and tag cdc6-6ALA with GFP, the fragment containing alanine mutations on the three C-terminal phosphorylation sites of CDC6 was removed from plasmid pSE159x and ligated with PstI/BclI digested pRS425-GAL-cdc6-3ALA-GFP to generate pRS425-GAL-cdc6-6ALAGFP. Complementation of cdc6 mutants by CDC6-GFP. Plasmid DNAs of pRS425-GAL-CDC6-GFP, pRS425-GAL-cdc6-DNLS-GFP, pRS425GAL-cdc6-3ALA-GFP, and pRS425-GAL-GFP were transformed into strain K4055 (MET-CDC6), and transformants were selected on synthetic minimal (SD) medium plates lacking methionine and leucine at 30 °C. Single colonies were re-streaked on leucine-deficient SD plates containing 2 mM methionine and 2% galactose. Cells were grown on plates at 30 °C for 2 days. Plasmid DNA of pRS424-CDC6-GFP or pRS424-CDC6 was transformed into strain RJD 610 containing a temperature sensitive allele of cdc6-1. This strain can grow on YPD plate at 23 °C, but not at 37 °C, and it cannot grow on a tryptophan deficient SD plate. After transformation, single colonies were re-streaked onto tryptophan deficient SD plates and incubated at 23 or 37 °C for 2 days. Induction of CDC6-GFP in W303 cells. Strain W303 carrying CDC6-GFP and its mutant constructs was grown in leucine minus SD medium plus 2% dextrose overnight at 30 °C, then diluted into fresh medium plus 2% raffinose, and grown to A600 of 0.8–1.2. Galactose was added into the cell culture to a final concentration of 2% to induce CDC6-GFP expression at 30 °C for 3–4 h. SDS–PAGE and Western blot analysis. Yeast cells were lysed in 2% SDS containing 0.1 N NaOH and boiled for 7 min. Several drops of 1 N HCl were used to adjust the pH of the cell lysates to neutral. The centrifuged cell extracts were subjected to SDS–PAGE (10% acrylamide gel) and transferred to nitrocellulose membrane. Protein blot was probed with anti-GFP antibody (Clontech, CA) at 1:3000 dilution and detected by ECL (Amersham, Arlington Heights, IL). Flow cytometry and FACS analysis. Logarithmically grown yeast cells expressing CDC6-GFP and its mutant constructs were analyzed by flow cytometry using a FACScan flow cytometer (Coulter Elite) with Argon 488-nm laser at 15 mW. Living yeast cells were briefly sonicated and sorted by the FACS. GFP intensity of each cell was measured on a four-decade log scale. Fluorescence of propidium iodide stained nuclei was measured on a 1024 channel with a linear scale. Microscopy techniques. W303 cells containing CDC6-GFP and its mutant constructs were induced as described above. A permeable DNA dye, Hoechst 33342, was added into the culture medium at a final concentration of 5 lg/ml to stain DNA at 30 °C for 30 min. Three microliters of cell culture was directly placed onto a microscope slide and the cover glass was sealed with nail polish. For fixation, cells were fixed with 4% paraformaldehyde plus 0.3% glutaraldehyde at room temperature for 1 h. Cells were then stained with DAPI at a final concentration of 2.5 lg/ml at room temperature for 30 min and mounted with 50% Mowiol on a poly-lysine treated slide. Images were taken on a Olympus fluorescence microscope equipped with a 100 objective lens, a 100 W Hg epifluorescence illuminator, DAPI, and GFP filter sets. Digital images were obtained using a cooled charged-

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coupled device camera that was controlled by ‘‘MetaMorph’’ software (Universal Imaging, West Chester, PA).

Results Fusion protein of Cdc6-GFP retains the essential function of the endogenous Cdc6 To determine whether Cdc6-GFP fusion protein retains the function of endogenous Cdc6, we performed two complementation experiments. First, we tested whether the Cdc6-GFP could perform the biological function of Cdc6 when expression of endogenous CDC6 was repressed. We expressed the CDC6-GFP with a GAL promoter in MET-CDC6 cells that have only one copy of the CDC6 gene under the regulation of a repressible MET3 promoter [1]. In the presence of methionine, expression of the endogenous CDC6 was repressed and cells were not viable. When the GAL promoter was induced on a culture plate containing 2% galactose and 2 mM methionine, the expression of CDC6-GFP rescued the MET-CDC6 cells (Fig. 1a). On the other hand, overexpression of GFP alone under the GAL promoter could not support the growth of METCDC6 cells. This result indicates that the Cdc6-GFP fusion protein can retain the function of endogenous Cdc6 in S. cerevisiae. We further tested whether expression of CDC6-GFP from the endogenous promoter of CDC6 can rescue a temperature sensitive (ts) mutant of CDC6. Due to a genetic defect in CDC6 gene, cdc6-1 cells can grow at the lower temperature of 23 °C, but not at a higher temperature of 37 °C. When the mutant cells of cdc6-1 were transformed with either CDC6-GFP or CDC6 alone, both of the transformants grew well at 37 °C on SD culture plates lacking tryptophan, while no cell growth was seen from the cdc6-1 control cells under the same conditions (data not shown). These results again verify that the fusion protein of Cdc6-GFP retains the essential function of the endogenous Cdc6. In this study, we have also generated Cdc6-GFP chimeras in which either the nuclear localization signal (NLS) or CDK1 dependent phosphorylation sites in the N-terminus were mutated. As can be seen in the following sections, these mutations were found to affect the stability of Cdc6, but not its function. For example, expression of either cdc6-DNLS-GFP or cdc6-3ALAGFP was shown to rescue the MET-CDC6 cells under the methionine repressing condition (Fig. 1a). Cdc6 is normally localized in the nuclei of living yeast cells To study the sub-cellular localization of Cdc6, the CDC6-GFP fusion gene was expressed using a GAL

Fig. 1. (a) Complementation of a CDC6 repression strain with Cdc6GFP and its mutants. Strain K4055 (MET-CDC6) was transformed with recombinant plasmids expressing GAL-wild-type CDC6-GFP (wtCdc6), GAL-cdc6-DNLS-GFP (cdc6-DNLS), GAL-cdc6-3ALA-GFP (cdc6-3ALA), and GAL-GFP (GFP), respectively. Transformants were streaked onto medium containing 2 mM methionine for repressing the endogenous CDC6 expression and 2% galactose for inducing GAL1,10 promoter. Plates were incubated at 30 °C for 2 days and then photographed on a light box with a transmitting light source. It was evident that the GFP alone cannot support growth, while all cdc6-GFP mutants can. (b) Localization of wild-type Cdc6-GFP in living yeast cells. W303 cells containing CDC6-GFP fusion construct were induced with 2% galactose at 30 °C for 3 h. Nuclei of living cells were visualized by staining with Hoechst 33342. The distribution of Cdc6-GFP fusion protein in living cells was imaged using a fluorescence microscope. Panels A–C represent the images of Hoechst staining from three unbudded and small budded cells. Panels D–F are GFP images from corresponding cells. The outlines in D–F show the cell shape.

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promoter in W303 cells. After CDC6-GFP was induced with 2% galactose at 30 °C for 3 h, a permeable DNA dye, Hoechst 33342, was used to stain the nuclei. When these cells were examined under a fluorescence microscope, the Cdc6-GFP protein was found to be co-localized with the Hoechst stained nuclei in living yeast cells (Fig. 1b). The Cdc6-GFP was found mainly from G1 and S phase cells, while most G2/M phase cells did not display a high level of Cdc6-GFP (Figs. 1b and 2). This finding suggests that the destruction of the Cdc6GFP in living cells is cell-cycle-dependent with timing similar to that of the endogenous protein [1]. In this living cell imaging study, Cdc6-GFP was always found concentrated in the cell nuclei. We have conducted a detailed analysis of the distribution of Cdc6-GFP by digitally analyzing its fluorescent intensity using a computer program ‘‘MetaMorph’’ as described in the legend to Table 1. We found that in the G1 phase, 83% of the Cdc6-GFP signal was concentrated in the nucleus; while in the S phase, the nuclear fraction of Cdc6-GFP accounted for 84% (Table 1). Under the GAL promoter, a small percentage of yeast cells in G2/ M phase was found to contain Cdc6-GFP; their GFP signals were also predominantly localized in the nuclei (Table 1). Analysis of G2/M cells, however, shows a slight but significant increase in the cytoplasmic population of GFP-Cdc6 at that point in the cell cycle, consistent perhaps with slower mode 3 degradation of Cdc6 [21]. Using a fluorescence microscope, we have further

Fig. 2. Distribution of wild-type Cdc6 and DNLS mutant in yeast W303 cells. Cells were visualized by DIC (differential interference contrast) optics (panels A and D). Fluorescence images of the same cells using a DAPI filter set (panels B and E) or a GFP filter set (panels C and F) were also shown here. Panels A–C: yeast cells expressing Cdc6-GFP. Panels D–F: cells expressing cdc6-DNLS-GFP.

Table 1 Percentage of Cdc6-GFP signal detected in the nucleus

Wild-type DNLS

G1

S

G2/M

83%  7% (n ¼ 10) 28%  4% (n ¼ 5)

84%  9% (n ¼ 10) 19%  6% (n ¼ 13)

71%  24% (n ¼ 7) 13%  5% (n ¼ 19)

Note. The fluorescent intensity of Cdc6-GFP was measured from the digital images using the MetaMorph software. Briefly, the area of each cell was marked based on phase image of the cell. The area of nucleus was determined based upon DNA staining by Hoechst. After subtracting the background fluorescence, we integrated the fluorescent signals in the cytoplasm + nucleus to calculate the relative amount of Cdc6-GFP proteins in (nucleus)/(nucleus + cytoplasm). n, number of cells analyzed.

examined, though not quantitated, the cellular localization of Cdc6-GFP in more than 200 cells. In all cases, we observed that Cdc6-GFP was distributed predominantly in the nucleus. Nuclear translocation of Cdc6 is dependent on a NLS in the N-terminus This dominant nuclear localization of Cdc6-GFP suggests that the protein may be quickly transported into the nucleus after it is synthesized in the cytoplasm. Previous biochemical studies suggested that Cdc6 contains a conserved nuclear localization signal (NLS) close to its N-terminus [9]. To investigate whether nuclear localization of Cdc6 is dependent on the presence of NLS in an in vivo system, we changed the amino acids within the NLS from K29 R30 K31 K32 to four alanines. The resulting cdc6 mutant was then fused with GFP and the fusion gene was expressed using a GAL promoter in W303 cells. The distribution of this GFP-labeled mutant protein, designated as cdc6-DNLS-GFP, is shown in Figs. 2 and 3. Unlike the wild-type Cdc6-GFP, which was observed predominantly in the nucleus (Fig. 2, panel C), the cdc6-DNLS-GFP was distributed in both nucleus and cytoplasm (Fig. 2, panel F and Fig. 3, panel D). Using a digital analysis, we have measured the relative intensity of Cdc6 in the nucleus. We found that the fluorescent signal of cdc6-DNLS-GFP was mainly concentrated in the cytoplasm, regardless of the stage of the cell cycle (Table 1). As with wild-type Cdc6-GFP, we observe a small but significant increase in cytoplasmic cdc6-DNLS-GFP in the G2/M phase of the cell cycle. This may suggest increased stability of Cdc6 in the cytoplasm. It is possible, but less likely, that there is reduced import of Cdc6 to the nucleus, since nuclear import is already reduced by deletion of the NLS. Nuclear localization is important for Cdc6 degradation During the examination of Cdc6-GFP and cdc6DNLS-GFP fluorescence intensity, we noticed that there

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Table 2 Percentage of Cdc6-GFP-positive cells at different cell cycle stages

Wild-type (n ¼ 27) DNLS (n ¼ 37)

G1 (%)

S (%)

G2/M (%)

37 14

37 35

26 51

Note. Three hours expression.

Fig. 3. Morphology and distribution of Cdc6-GFP in yeast cells expressing the 6ALA and DNLS mutants. Panels A and C, DIC images of GFP-positive cells; panels B and D, fluorescence images of cdc66ALA-GFP and cdc6-DNLS-GFP expressing cells.

are many more GFP-positive cells in the DNLS mutantcontaining cell population than in cells containing the wild-type Cdc6. This could be explained if mislocalization of Cdc6 leads to an increase in stability and hence an increase in intracellular levels. The number of GFPpositive cells expressing either wild-type Cdc6 or cdc6DNLS was determined by cell counting. In a random population, only 7.8% (33/425) of the cells transformed with the wild-type CDC6-GFP have the GFP signal, while 29% (107/370) of the cdc6-DNLS-GFP cells are GFP-positive. The 3.6-fold increase in the number of cells containing the cdc6-DNLS-GFP suggested that cdc6-DNLS is more stable than the wild-type Cdc6. By analyzing the cell cycle distribution of the GFP-positive cells, we found that unlike cells expressing the wild-type Cdc6-GFP, in which the GFP-positive cells were concentrated in the G1 and S phases, a higher percentage (51%) of cells expressing cdc6-DNLS-GFP was found in the G2/M phase (Table 2). These results imply that nuclear localization is important for Cdc6 degradation in the G2/M phase. Phosphorylation of Cdc6 in both N- and C-termini is important in regulating its protein stability The Cdc6 protein contains six putative CDK1 phosphorylation sites [25] and three of them are located at the N-terminus. Phosphorylation of Cdc6 or Cdc18 has been shown to be required for its destruction in both budding and fission yeast [21,24,28]. To assess whether phosphorylation of Cdc6 is important for its degradation in living cells of S. cerevisiae, we compared the

frequency of GFP-positive cells from wild-type and the mutant stains. The first mutant, 3ALA, has three Nterminal phosphorylation sites mutated from T7 T23 S43 to three alanines. In the second mutant, 6ALA, all six sites (T7 T23 S43 T134 S354 S372 ) were mutated to alanines. Both cdc6-3ALA-GFP (Fig. 1A) and cdc6-6ALA-GFP (data not shown) could support cell growth of METCDC6 cells. When cells were induced with 2% galactose for 4 h, about 7.8% (33/425) of cells with Cdc6-GFP were GFP-positive. In cells carrying cdc6-3ALA-GFP, the relative population of GFP-positive cells increased to 18% (61/345). The population of GFP-positive cells increased even further to 36% (139/388), when all six phosphorylation sites were mutated. This result suggests that the phosphorylation sites in the C-terminal region of Cdc6 are important in regulating its protein stability. Furthermore, we observed that the mutant proteins were localized mainly in the nucleus, similar to that of the wide-type Cdc6-GFP (Fig. 3, panel B). Nuclear localization signal plays a more important role than phosphorylation in controlling the stability of Cdc6 One possible explanation for the increased stability of the DNLS mutant of the Cdc6 protein is that the kinase responsible for the six CDK1 phosphorylation sites may be mainly located in the nucleus. In that case, the cdc6DNLS would have less chance to be phosphorylated and thus be degraded more slowly than the wild-type Cdc6. If this hypothesis is true, one might expect Cdc6 to be more stable in the 6ALA mutant than in the DNLS mutant. With the availability of the GFP tagged Cdc6, we can test this hypothesis by using a Fluorescent Activated Cell Sorter (FACS) analysis to examine the stability of the different cdc6 mutant proteins in yeast cells. Approximately 10,000 W303 cells expressing wild-type and mutant forms of CDC6-GFP were examined by FACS analysis using a 488-nm laser to excite the GFP from live cells. The cell number is plotted as a function of cellular fluorescence intensity in Fig. 4A. W303 cells containing pRS425-GAL1,10 were used as a negative control to set the basal level of auto-fluorescence in yeast cells. The basal fluorescent intensity from the control cells was less than 2 units. A small fraction of the wild-type Cdc6-GFP cells had GFP intensity varying from 2 to 20 units. A slightly larger fraction of the cdc66ALA-GFP mutant cells had GFP signal ranging between 2 and 30 units. The most significant increase of GFP intensity was in the cdc6-DNLS-GFP cells, which

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had a GFP intensity ranging from 2 to 500 units. This means that cells attain more than 10-fold higher level of GFP signal in the cdc6-DNLS-GFP expressing cells in comparison to the cdc6-6ALA-GFP expressing cells. This finding suggests that the NLS mutant is more stable than the 6ALA mutant. Consistent with our FACS data, Western blotting analysis also showed that the NLS mutant has a higher Cdc6-GFP protein level than the 6ALA mutant (Fig. 4B). To further examine this point, we conducted a promoter turn-off experiment to determine the rate of disappearance of GFP positive cells in cultures of the cdc6-DNLS-GFP and cdc6-6ALA-GFP mutants. In this experiment, cdc15, a temperature-sensitive mutant that can be arrested in anaphase at 37 °C, was used as the host cell. Thus, the host cells can be synchronized by a temperature shift. Exponentially growing cdc15 cells containing cdc6 mutant constructs were arrested at 37 °C for 4 h. During this time, the cdc6-DNLS-GFP and cdc6-6ALA-GFP expression was induced by addition of 2% galactose. Cells were then released from the cdc15 block by shifting the culture from 37 to 23 °C, and the expression of cdc6 mutants was repressed by addition of 2% glucose. The number of GFP-positive cells was determined at different time points by cell

counting (Fig. 5). When the expression of cdc6-DNLSGFP was repressed, the percentage of GFP-positive cells decreased from 53% to 3.2% in 7 h. In contrast, the number of cdc6-6ALA-GFP-positive cells decreased more rapidly, and almost no GFP-positive cells were observed after 2 h of repression (Fig. 5). These results again suggest that the cdc6-DNLS-GFP protein is more stable than the cdc6-6ALA-GFP protein. Taken together, these results suggest that nuclear localization of Cdc6 may have a greater effect on protein stability than the regulation by phosphorylation, and thus, the regulatory effect of DNLS on Cdc6 stability is not due solely to its preventing Cdc6 from being phosphorylated. High level of Cdc6 tends to arrest cells at G2/M phase It is known that the endogenous Cdc6 is normally degraded in G2 and early M phases. We wondered whether there might be a checkpoint mechanism to monitor the degradation of Cdc6. Previously, Bueno and Russell [17] had observed a cell cycle delay when Cdc6 was overexpressed from the GAL promoter. Their observations, however, were not confirmed in later studies [1,8]. Thus, we have conducted two experiments to investigate whether blocking the degradation of Cdc6

Fig. 4. (A) FACS analysis of wild-type and mutant form of CDC6-GFPs. W303 cells expressing pRS425-GAL1,10 promoter (control), wild-type CDC6-GFP (wtCdc6), cdc6-DNLS-GFP (cdc6DNLS), and cdc6-6ALA-GFP (cdc6-6ALA) were examined by FACS analysis. The intensity of green fluorescence was measured at 4 log scales from 0 to 1000 units. The area below the large peak with GFP intensity less than 2 units represents the cell population exhibiting only background auto-fluorescence. (B) Western blot analysis of wild-type Cdc6-GFP and its mutants. Fifty micrograms (1) and 100 lg (2) of cell lysates from W303 cells expressing wild-type Cdc6-GFP (wtCdc6), cdc6-DNLS-GFP (cdc6DNLS), cdc6-6ALA-GFP (cdc66ALA), cdc6-3ALA-GFP (cdc6-3ALA), and vector pRS425-GAL1,10 promoter (control) were analyzed by Western blot using anti-GFP antibody. The specific Cdc6-GFP interactive band with a molecular weight at around 87 kDa is shown in the upper panel.

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Fig. 5. Time course of cdc6-GFP degradation in different mutants. cdc15 cells containing recombinant plasmid GAL-cdc6-DNLS-GFP (}) or GAL-cdc6-6ALA-GFP (d) were arrested at 37 °C for 4 h. During this time, expression of the mutant proteins was induced with 2% galactose. Cells were then shifted back to 23 °C to lift this M phase block and the expression of cdc6 mutants was repressed by addition of 2% glucose. The number of GFP-positive cells was determined by cell counting at the indicated times after glucose addition.

may cause cells to arrest at a specific stage of the cell cycle. In the first experiment, we analyzed the morphology of yeast cells that expressed various cdc6 mutant proteins. In cells that overexpressed cdc6-6ALAGFP for 4 h, the majority of the GFP-positive cells were either large budded cells or cells with elongated buds (Fig. 3, panel A). In cells overexpressing cdc6-DNLSGFP, most of the GFP-positive cells also have the morphology of large, elongated bud cells (Fig. 3, panel C). These results suggest that by blocking degradation of Cdc6, cells may be arrested (or at least delayed), at the G2/M phase. To further examine this point, we conducted a second experiment to measure the DNA content from the GFPpositive cells by FACS analysis. GFP positive cells expressing either cdc6-DNLS-GFP or cdc6-6ALA-GFP were separated from GFP-negative cells using FACS. As much as 3.5 million GFP-positive cells were recovered after the sorting experiment, and 93.3% of the collected GFP-positive cells were confirmed to be GFP positive under a fluorescence microscope. Then, we determined the DNA content of the propidium iodide-stained GFPpositive cells by flow cytometry and compared it with control cells that did not contain the cdc6 mutant proteins. The results are shown in Fig. 6. We found that the great majority of cdc6-DNLS-GFP and cdc6-6ALAGFP expressing cells have 2C DNA content, while the control asynchronous cells show a random population with DNA content varying from 1C to 2C (Fig. 6). These results suggest that by preventing Cdc6 degradation, the cdc6 mutant cells were arrested at G2/M phase. Indeed, when we used FACS analysis to measure the

857

Fig. 6. Determination of DNA content of control and GFP-positive cells by FACS analysis. W303 cells expressing pRS425-GAL1,10 promoter (control), and GFP positive cells expressing GAL-cdc6-DNLSGFP (DNLS), or GAL-cdc6-6ALA-GFP (6ALA) obtained by cell sorting were fixed and stained with propidium iodide. Nuclear DNA content from each sample was then determined by FACS analysis. Cells from a ts mutant, cdc15, which were arrested in M phase were analyzed similarly for the purpose of comparison.

DNA content of cdc15 cells that had been arrested at 37 °C in anaphase with 2C DNA content, their population profile of DNA content was almost identical to that of DNLS and 6ALA cells (Fig. 6). These results suggest that high level of Cdc6 tended to arrest cells in G2/M phase. To further determine whether the cells containing stabilized Cdc6 were accumulated before or after nuclear division, we analyzed the number of nuclei in the propidium iodide-stained GFP-positive cells from the cdc6-DNLS-GFP and cdc6-6ALA-GFP mutants. The results are summarized in Table 3. We found that 59.7% of the cdc6-DNLS cells have one nucleus and the rest have two nuclei (Table 3). Similarly, 61.1% of the cdc6-6ALA cells have one nucleus and the other 38.9% have two nuclei. Taken together, the results of Fig. 6 and Table 3 imply that the presence of high levels of Cdc6 protein could affect the processes of both nuclear division and cytokinesis. Table 3 Percentage of GFP-positive cells arrested before and after nuclear division

cdc6-DNLS cdc6-6ALA

One nucleus

Two nuclei

59.7% (71/119) 61.1% (44/72)

40.3% (48/119) 38.9% (28/72)

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Discussion Results of this study clearly indicate that the wildtype Cdc6 predominantly accumulates in the nucleus, and, this nuclear localization remains unchanged during the progression of the cell cycle. The cellular localization of yeast Cdc6 protein has been studied previously in fixed yeast cells using immunostaining. When Myc-tagged CDC6 was expressed from a GAL promoter, the Myc-Cdc6 was accumulated to a relatively high level in the nuclei of post-anaphase and G1 phase cells, but was more evenly distributed between nucleus and cytoplasm in cells at other stages of the cell cycle [8]. Based on these results, it was proposed that the cytoplasmic Cdc6 was derived from Cdc6 released from the nucleus following the dismantling of the preRC after the initiation of DNA replication [8]. Results of our study suggest that virtually all-mature wild-type Cdc6-GFP protein accumulated in the nucleus. If there was release of Cdc6 from nucleus to cytoplasm following the initiation of DNA replication, this fraction of the Cdc6 protein must be degraded quickly. It is possible that some of the cytoplasmic fraction of Cdc6 observed in earlier studies could be freshly synthesized protein; these proteins might not have reached the mature stage to be transported into the nucleus. Since the GFP molecule needs to go through a maturation process for its fluorophores to form, some of the newly synthesized Cdc6-GFP protein may not be detectable in our fluorescence measurement. Nevertheless, we do see a small but significant increase in cytoplasmic Cdc6 during G2/M phase, which might indicate that there is export of Cdc6 from the nucleus but that it is more slowly degraded at this point in the cell cycle. In fact, Diffley [21] has shown that mode 3 (G2/M) degradation of Cdc6 is slower than mode 2 (G1/S). As regards the relationship between nuclear localization of Cdc6 and its phosphorylation by CDK1, we found no evidence that phosphorylation plays a significant role in nuclear localization. Instead, our results showed that the nuclear translocation of the protein is mainly dependent on a consensus NLS sequence found in its N-terminus. When the NLS was mutated, the Cdc6 protein could no longer be concentrated in the nucleus. Hence, the NLS must play a critical role in mediating the nuclear transport of the Cdc6 protein. These findings are also consistent with those observed in human cells, in which nuclear localization of HsCdc6 is dependent on the NLS rather than phosphorylation. It has been shown that mutations in those NLS sites resulted in changing the localization from nuclear to cytoplasmic, while mutating four putative CDK phosphorylation sites from serines to alanines had no effect [14,15]. In agreement with observation made by others, we also found that mutation in the NLS sequence of Cdc6 can stabilize the protein. This result suggests that the

initiation of Cdc6 degradation mainly occurred in the nucleus. Our observation that the cdc6-DNLS mutant is more stable than the cdc6-6ALA mutant has ruled out the possibility that the DNLS effect is due to prevention of phosphorylation of Cdc6 by a nuclear kinase. A more plausible interpretation of our results is that the ubiquitin-targeting apparatus may interact with Cdc6 inside the nucleus. This notion is consistent with the finding that Cdc4, a component of the ubiquitin ligase E3 for Cdc6 [24], is a nuclear protein [29]. In the absence of Cdc4 function, the Cdc6 protein was found to become more stabilized [8,23] and cannot be ubiquitinated [24,30]. Thus, when the NLS is mutated, most Cdc6 proteins are localized in the cytoplasm and thus cannot interact with the E3 complex. Cdc6 has six putative CDK1 phosphorylation sites and three of them are located in the N-terminus. In our studies, we found that mutation of the N-terminal sites in Cdc6 can only partially stabilize the protein. Mutation of all sites, including the ones located in the center and the C-terminal end of Cdc6, can greatly increase the stability of the Cdc6 protein. This conclusion is consistent with a recent report demonstrating that Cdc6 has two Cdc4-interaction domains; one is at the N-terminus and the other at the C-terminus. Moreover, phosphorylation sites, particularly those in the C-terminal domain, are critical for Cdc6 degradation in M phase [21]. Acknowledgments We thank Dr. Rochelle A. Diamond for her assistance in conducting FACS analysis. We also thank Dr. Pin Lu for his help in the imaging studies. We are grateful to Dr. Susanna Boroant (California Institute of Technology) for critical reading of the manuscript. This work was supported by USPHS Grant 25508 awarded to JLC and RGC Grants awarded to D.C.C. (HKUST 6109/01M and 6104/02M).

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