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© The Rockefeller University Press,
0021-9525/2000//1115 $5.00
The Journal of Cell Biology, Volume 148, Number 6,
, 2000 1115-1122
Brief Report |
Nucleocytoplasmic Shuttling of the Cdc42p Exchange Factor Cdc24p
ra2{at}mrc-lmb.cam.ac.uk
Cdc24p, the GDP/GTP exchange factor for the regulator of actin cytoskeleton Cdc42p, localizes to sites of polarized growth. Here we show that Cdc24p shuttles in and out of the yeast nucleus during vegetative growth. Far1p is necessary and sufficient for nuclear accumulation of Cdc24p, suggesting that its nuclear import occurs via an association with Far1p. Nuclear export is triggered either by entry into the cell cycle or by mating pheromone. As Far1p is degraded upon entry into the cell cycle, cell cycle–dependent export of Cdc24p occurs in the absence of Far1p, whereas during mating similar export kinetics indicate that a Cdc24p–Far1p complex is exported. Our results suggest that the nucleus serves as a store of preformed Cdc24p–Far1p complex which is required for chemotropism.
Key Words: nucleocytoplasmic shuttling • GDP/GTP exchange factor • Far1p • Cdc24p • polarized growth
© 2000 The Rockefeller University Press
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Introduction |
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 TopAbstract Introduction Materials and MethodsResults and DiscussionReferences |
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and is localized to the shmoo tip (Nern and Arkowitz 1998, Nern and Arkowitz 1999). Cdc24p is a member of a ubiquitous class of small G protein regulators that are critical for cell growth and polarity in virtually all eukaryotes. Recently, Cdc24p has been shown to localize to the nucleus of budding cells (Toenjes et al. 1999). Here we show that Far1p is necessary and sufficient for Cdc24p nuclear localization and that Cdc24p is exported from the nucleus upon either entry into the cell cycle or in response to pheromone. Our results suggest that Cdc24p nuclear export in budding cells is triggered by degradation of Far1p, whereas in response to pheromone a complex of Cdc24p–Far1p is exported. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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N FAR1 (lacking codons 1–393 which encode the first 131 amino acids) were amplified by PCR and cloned into pRSR424 with the GAL1/10 promoter and a modified multiple cloning site.
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3 h before microscopy for nuclear staining of live cells. 
-Factor was added to 24 µM in growth media. Expression levels were compared by SDS-PAGE of glass bead lysed cell extracts (Nern and Arkowitz 1999), immunoblotting, followed by ECL visualization. Anti-GFP rabbit polyclonal serum (used at 1:5,000), anti-Far1p rabbit polyclonal serum (used at 1:1,000), and anti-HA mAb (12CA5) tissue culture supernatant (used at 1:40) were used for immunodetection. |
Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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2/3 of the Cdc24GFP was in the nucleus. Nuclear accumulation of Cdc24GFP was observed in haploid cdc24 (Fig. 1 A) and CDC24 but not diploid cells despite the similar localization of Cdc24p to growth sites in both cell types (Fig. 1 B). These results suggest that Cdc24p nuclear localization is dependent on a haploid specific protein whose level varies during the cell cycle.
Far1p accumulates in the nucleus of haploid, but not diploid, cells in the G1 cell cycle phase (Henchoz et al. 1997). Because Far1p interacts both in vivo and in vitro with Cdc24p (Butty et al. 1998; Nern and Arkowitz 1999) we examined whether Far1p was necessary for Cdc24p nuclear localization. Cdc24p was not observed in the nuclei of far1 cells and furthermore the Cdc24p mutant Cdc24-m1p, which is defective in Far1p binding, was not localized to the nucleus in either cdc24 or CDC24 cells (Fig. 1 B and data not shown). In both cases Cdc24p growth site localization and expression levels were similar to wild-type strains (Fig. 1 B and data not shown). In addition, cells with the far1 mutation, far1-H7, which is defective in Cdc24p binding, had a reduction in the percentage of cells with nuclear Cdc24p. Together, these results demonstrate that Far1p and its ability to bind Cdc24p are necessary for Cdc24p nuclear localization, either by promoting nuclear retention or by cytoplasmic association and subsequent nuclear import.
As Far1p is not expressed in diploids, we expressed a stabilized mutant of Far1p, Far1-22p (Henchoz et al. 1997), to determine if it was sufficient to direct Cdc24p to diploid cell nuclei. Expression of Far1-22p from a GAL promoter for 3 h resulted in Cdc24GFP nuclear localization in 65% of diploid cells (Fig. 1 B). There was a significant decrease in cells with growth site localized Cdc24GFP, suggesting that nuclear accumulation of Far1p depletes cytoplasmic Cdc24GFP. In contrast, expression of Far1-22p did not shift Cdc24-m1GFP to the nucleus (data not shown). These results demonstrate that Far1p is necessary in haploids and sufficient in diploids for Cdc24p nuclear localization.
Overexpression of Far1-22p is toxic, resulting in G1 cell cycle arrest, which has been suggested to be due to Cln/Cdc28p inhibition by Far1p (Henchoz et al. 1997). However, the localization of Cdc24GFP in cells expressing Far1-22p raised the possibility that Far1-22p toxicity might instead be the result of nuclear sequestration of this exchange factor that would remove it from sites of polarized growth thereby physically separating it from the Rho G protein Cdc42p which it normally activates. To test this hypothesis we first determined if mutations in Far1p which block Cdc24p binding (Butty et al. 1998; Nern and Arkowitz 1999) alleviated the toxicity of Far1-22p overexpression. Strikingly the combination of two different Far1p mutations, either Far1-H7p or Far1-D1p, with Far1-22p completely abolished its toxicity (Fig. 2 A). Far1-D1p is a mutant with two amino acid changes, Gly 646 to Asp (referred to as D1A) and Pro 671 to Leu (referred to as D1B; Valtz et al. 1995). The combination of Far1-22p with each of these single amino acid changes revealed that only the D1A mutation blocked the Far1-22p toxicity (Fig. 2 A). Similarly, Far1-22p overexpression in cdc24-m1 cells resulted in little to no toxicity. The effects of Far1p mutations and cdc24-m1 on Far1-22p toxicity were also observed in far1 strains (data not shown). In diploid cells expressing of Far1-D1A-22p or Far1-H7-22p, Cdc24GFP was still observed at growth sites in contrast to Far1-22p and Far1-D1B-22p which resulted in predominantly nuclear localization (Fig. 2 C). Immunoblot analyses indicated that the different Far1-22p mutants were all expressed and that their levels were similar in wild-type and cdc24-m1 cells (data not shown). Far1p localized to the nucleus in cdc24-m1 cells (see below) arguing against an indirect effect of cdc24-m1 on Far1p localization.
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To examine whether nuclear Cdc24p could be exported and subsequently targeted to sites of growth we used a cdc28 strain to accumulate Cdc24GFP in G1 cell nuclei at 37°C and followed its localization upon a shift to 25°C (Fig. 3 A). Within 15 min when Cdc24p nuclear fluorescence was still observable, Cdc24GFP was seen at the plasma membrane as a crescent before bud emergence. By 30 min, small buds with Cdc24p localized as a tight patch were observable in some cells and an increase in cytoplasmic fluorescence was apparent. After another 30 min, the majority of the cells were budded with Cdc24p localized to the tips and nuclear Cdc24p fluorescence was undetectable in most cells. These results suggest that nuclear export and localization of Cdc24p to the bud tip occur in similar time frames. Far1p degradation is likely to occur in the nucleus (Henchoz et al. 1997) providing a possible trigger for Cdc24p export upon G1 exit in the absence of pheromone.
In contrast, pheromone treatment results in Far1p export from the nucleus (Butty et al. 1998), suggesting that in these conditions a Cdc24p–Far1p complex might be exported from the nucleus. To examine pheromone-dependent nuclear exit, G1-arrested cdc28 cells were -factor treated (Fig. 3 B). Within 5 min, the level of Cdc24p in the nucleus decreased and Cdc24p appeared on the plasma membrane. As cells became pear-shaped a tight patch of shmoo tip Cdc24p was seen. In contrast, cells not treated with pheromone had little Cdc24p associated with the plasma membrane. To address the possibility that pheromone treatment results in the degradation of nuclear Cdc24p and instead newly synthesized Cdc24p localizes to the site of growth, similar experiments were carried out using the protein synthesis inhibitor cycloheximide (Fig. 3 C). In the presence of cycloheximide, pheromone triggered the export of Cdc24p from the nucleus and localization to growth sites on the plasma membrane. However, at longer incubation times, Cdc24p was no longer visible at the plasma membrane, perhaps due to the general toxicity of cycloheximide (data not shown). In the absence of cycloheximide, the number of cells with nuclear Cdc24GFP decreased approximately threefold after 15 min with pheromone, whereas a substantial increase in shmoo site localized Cdc24GFP was not observed until 30 min (Fig. 3c and Fig. d). Quantification of the levels of nuclear and cytoplasmic fluorescence also showed a decrease in the ratio of nuclear to cytoplasmic fluorescence with pheromone (Fig. 3 D). These results indicate that preexisting Cdc24GFP is exported from the nucleus upon pheromone addition and subsequently localizes to sites of growth.
To directly confirm that the nuclear Cdc24p relocalized to sites of growth we used a strain in which Cdc24GFP expression was regulated. Gal Cdc24GFP cdc28 cells were grown in galactose, shifted to 37°C, and after 1 h resuspended in glucose containing media to repress Cdc24p expression. Pulse labeling demonstrated that Cdc24GFP was no longer synthesized after 1 h in glucose (data not shown). Either the addition of pheromone or a temperature shift to 25°C resulted in the export of Cdc24GFP and localization to growth sites (Fig. 4). After 30 min in pheromone 50% of cells showed shmoo tip localization, whereas only 10% of the cells had nuclear fluorescence. Similarly after a 60-min shift to 25°C, >70% of cells showed a bud tip localization and only 10% showed nuclear fluorescence. In a second temperature shift experiment we observed Cdc24GFP, which was synthesized before the temperature shift, shuttled out of the nucleus after approximately 1 h, back into the nucleus after 2 h, and subsequently at 2 h 40 min out of the nucleus again. Each nuclear export was accompanied by a corresponding increase in growth site localization. These experiments demonstrate that the nuclear localized Cdc24p is recruited to the site of growth on the plasma membrane upon export and that Cdc24p can cycle in and out of the nucleus several times.
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msn5/ste21 on Cdc24p localization. Surprisingly, msn5/ste21 resulted in a threefold reduction in the number of cells with nuclear Cdc24p yet did not affect the localization of Cdc24p to the mother-daughter bud neck (Fig. 5 A and data not shown). Immunoblot analyses indicated that although cellular levels of Cdc24p were unchanged, Far1p levels in msn5/ste21 cells were lower (Fig. 5 B), providing an explanation for the reduction in nuclear Cdc24p. Consistent with this explanation, an increase in Far1p levels upon pheromone treatment (Fig. 5 B) led to an increase in nuclear Cdc24GFP (Fig. 5 A). The localization of Cdc24GFP to the shmoo tip in msn5/ste21 (Fig. 5 A) and cdc28 msn5/ste21 (data not shown) strains appeared reduced, suggesting that similar to Ste5p and Far1p, Cdc24p nuclear export is defective in msn5/ste21.
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-Far1p complex (Butty et al. 1998; Nern and Arkowitz 1999), might be due to the absence of nuclear Cdc24p. A nuclear localization signal was fused to Cdc24-m1GFP which resulted in an increase in nuclear localization, however, growth site localized Cdc24p was still observable (80% of cells showed nuclear and 30% growth site localization; n = 200 in wild-type and cdc24-m1 cells). NLS-Cdc24p, but not NLS-Cdc24-m1p complemented the cdc24-m1 mating defect (data not shown) demonstrating that the lack of nuclear localization is not responsible for this mating defect. Both of these NLS fusions complemented the growth defect of a cdc24 temperature-sensitive mutant (data not shown) indicating they were functional. Overexpression of a truncated version of Far1p lacking its amino-terminal 131 amino acids including two nuclear localization signals (Blondel et al. 1999) yet still functional for Cdc24p and Gβ binding (Butty et al. 1998; Nern and Arkowitz 1999), rescued the mating defect of far1 cells (data not shown) yet did not accumulate Cdc24GFP in the nucleus (0% nuclear and 33% at growth site compared with 24% nuclear and 39% growth site with full-length Far1p, n = 300). These results suggest that it is the cytoplasmic Cdc24p–Far1p complex that is necessary for mating. As Far1p was required for Cdc24p nuclear accumulation, we determined conversely, whether Cdc24p played a role in Far1p localization. In response to pheromone, Far1p relocates from the nucleus to the cytoplasm, however, its localization to the shmoo tip has not been reported (Butty et al. 1998). Far1GFP cell cycle–dependent nuclear localization and expression levels were similar in CDC24 and cdc24-m1 cells (Fig. 5 C and data not shown). Pheromone addition resulted in the redistribution of Far1GFP to the cytoplasm in both strains, however, CDC24 cells had an enrichment of Far1p at the plasma membrane in both round cells and shmoo tips. Far1p at the cell periphery was difficult to observe due to high cytoplasmic fluorescence. Experiments with Far1GFP identical to Gal Cdc24GFP experiments (Fig. 4) showed that Far1p was exported and appeared at the shmoo tip with similar kinetics as Cdc24p (Fig. 5 D). Strikingly, Far1GFP localization to the plasma membrane was greatly reduced in pheromone-treated cdc24-m1 cells (52% of CDC24 cells had Far1GFP at the membrane compared with 8% of cdc24-m1 cells; n = 80) indicating that Far1p requires CDC24 for efficient delivery to or stabilization at the shmoo growth site.
Taken together, our data are consistent with the nuclear import of a preformed Cdc24p–Far1p complex and the degradation of Far1p during vegetative growth triggering the export of Cdc24p. In contrast, upon exposure to mating pheromone and completion of the cell cycle, this Cdc24p–Far1p complex is exported providing a source of this complex for targeting to Gβ. Both Ste5p and Far1p shuttle in and out of the nucleus during vegetative growth and are exported from the nucleus upon pheromone treatment (Blondel et al. 1999; Mahanty et al. 1999). Our results demonstrate that similar nucleocytoplasmic shuttling occurs with the GDP/GTP exchange factor Cdc24p. Recently, overexpressed Cdc24p was shown to localize to the nucleus in a cell cycle–dependent fashion (Toenjes et al. 1999) in agreement with our results. Cells in which Cdc24p does not accumulate in the nucleus appear normal for vegetative growth, suggesting that this cycling per se does not have a critical function during budding. Cytoplasmic Far1p is functional for cell mating indicating that nuclear import and export of Cdc24p–Far1p is not required for mating. However, as the normal pheromone-dependent cell cycle arrest function of Far1p is in the nucleus (Blondel et al. 1999) and the Cdc24p–Far1p growth orientation function is in the cytoplasm, nuclear export of this complex is required for efficient mating. The nuclear localization of Cdc24p upon mating pheromone treatment, due to increased levels of Far1p, may prevent inappropriate localization and activation of Cdc24p during completion of the cell cycle before shmoo formation and at the same time allow a fast response to pheromone. It is likely that nucleocytoplasmic shuttling of G protein exchange factors is a conserved feature of this class of G protein regulators as S. pombe Cdc24 (scd1; Chen et al. 1999) and very recently the human Rho G protein exchange factor Ect2 also localize to the nucleus in a cell cycle–dependent fashion (Tatsumoto et al. 1999). This suggests that the intracellular distribution of exchange factors might also be regulated by interacting proteins analogous to Far1p.
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Acknowledgments |
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This work was supported by the Medical Research Council. A.N. was supported by a Marie Curie fellowship of the EC.
Note Added in Proof. Similar findings were recently reported in a study by Shimada et al. (Nat. Cell Biol. 2000. 2:117–124).
Submitted: 22 December 1999
Revised: 2 February 2000
Accepted: 8 February 2000
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Bender A. Pringle J.R. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1, Proc. Natl. Acad. Sci. USA, 86, 1989, 9976–9980.
Blondel M. Alepuz P.M. Huang L.S. Shaham S. Ammerer G. Peter M.. Nuclear export of Far1p in response to pheromones requires the export receptor Msn5p/Ste21p, Genes Dev, 13, 1999, 2284–2300.
Butty A.C. Pryciak P.M. Huang L.S. Herskowitz I. Peter M.. The role of Far1p in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating, Science, 282, 1998, 1511–1516.
Chen C. Li Chen Y. Hou J.M. Papadaki P. Chang E.. Moe1, a conserved protein in Schizosaccharomyces pombe, interacts with a Ras effector, Scd1, to affect proper spindle formation, Proc. Natl. Acad. Sci. USA, 96, 1999, 517–522.
Henchoz S. Chi Y. Catarin B. Herskowitz I. Deshaies R.J. Peter M.. Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast, Genes Dev, 11, 1997, 3046–3060.
Mahanty S.K. Wang Y. Farley F.W. Elion E.A.. Nuclear shuttling of yeast scaffold Ste5 is required for its recruitment to the plasma membrane and activation of the mating MAPK cascade, Cell, 98, 1999, 501–512.[Medline]
Nern A. Arkowitz R.A.. A GTP-exchange factor required for cell orientation, Nature, 391, 1998, 195–198.[Medline]
Nern A. Arkowitz R.A.. A Cdc24p, Far1p, Gβ protein complex required for yeast orientation during mating, J. Cell Biol, 144, 1999, 1187–1202.
Rose M.D. Winston F. Hieter P., Methods in Yeast GeneticsA Laboratory Course Manual, 1991 Cold Spring Harbor Press, Cold Spring Harbor, NY.
Tatsumoto T. Xie X. Blumenthal R. Okamoto I. Miki T. Human ECT2 is an exchange factor for Rho GTPases, phosphorylated in G2/M phases, and involved in cytokinesis, J. Cell Biol, 147, 1999, 921–927.
Toenjes K. Sawyer M. Johnson D.. The guanine nucleotide exchange factor Cdc24p is targeted to the nucleus and polarized growth sites, Curr. Biol., 9, 1999, 1183–1186.[Medline]
Valtz N. Peter M. Herskowitz I.. FAR1 is required for oriented polarization of yeast cells in response to mating pheromones, J. Cell Biol, 131, 1995, 863–873.
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,•) and open (
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) symbols indicate percentage cells (n = 200) with nuclear and growth site localized Cdc24p, respectively. Ratio of nuclear to cytoplasmic fluorescence intensity (n = 100) was determined for each time (values were not corrected for background cellular fluorescence) and errors bars signify SEM. Bars, 5 µm.
