INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Cyclin-dependent kinases (Cdks) are serine/threonine protein kinases that are required for key events in the cell division cycle. Cdk activity depends on conformational changes induced by physical association with cyclin proteins and subsequent cyclin-dependent targeting of Cdk activity to potential substrates (3, 49, 50, 56). A single Cdk can carry out multiple functions, dependent on the identity of its cognate cyclin partner. In budding yeast, the Cdk Cdc28p is required for all key cell cycle events. When bound by any one of three G₁ (Cln) cyclins, Cdc28p supports cell cycle entry (12). When bound by one of six B-type (Clb) cyclins, Cdc28p supports later cell cycle events, such as DNA replication and mitosis (54). For both G₁ Cln cyclins and B-type Clb cyclins, specific cyclins provide functional specificity to the Cdk complex independent of expression patterns and abundance of associated kinase activity (14, 30, 38).

Deletion of all three G₁-type cyclin CLN genes results in a cell cycle arrest before cell cycle initiation, as unbudded cells with 1C DNA content. CLN1, CLN2, and CLN3 are functionally redundant, since all double cln deletions result in viability. However, there are significant differences in the functional specificity of CLN2 (representative of the highly homologous CLN1 CLN2 gene pair) and CLN3. Functional analysis of CLN2 and CLN3 indicates that CLN3 is primarily required for the expression of genes during late G₁, including CLN1 and CLN2. This Cln3p activity requires the SCB transcription complex, consisting of Swi4p and Swi6p (34). Once expressed, Cln2p is believed to trigger events required for cell cycle progression, such as bud emergence and activation of Clb-Cdc28p complexes (7, 12, 15, 40). Bck2p also triggers SCB-dependent transcription in G₁, most likely working in parallel with Cln3p (17, 21). We have characterized the subcellular localization patterns of the G₁ cyclins Cln2p and Cln3p and find strikingly distinct localization patterns. These patterns are consistent with the idea that Cln2p and Cln3p confer functional specificity to the Cdk Cdc28p and support cell cycle entry by distinct mechanisms. Cln3p, thought to be the physiologically relevant cyclin for activation of transcription (19, 38, 70), is localized primarily to the nucleus (45). Cln2, thought to trigger other events, such as bud emergence (7, 12, 15, 40), is localized primarily to the cytoplasm (45).

Differences in functional specificity between Cln2p and Cln3p can be assayed in specific genetic backgrounds. For example, CLN2 is able to support viability in the absence of Swi4p, while CLN3 is not (38, 55, 57). This suggests that the ability of CLN3 to rescue a cln1 cln2 strain requires Cln3p- and Swi4p-dependent transcription. The PCL1 and PCL2 gene pair (encoding cyclin homologues that can bind to and activate the Pho85p Cdk) is also essential for Cln3p-dependent viability in the absence of cln1 and cln2 (38, 43, 57). PCL1 and PCL2 are transcribed under the control of Swi4p (43, 57), suggesting that at least some of the Swi4p requirement in the cln1 cln2 background may be due to the Pcl1p, Pcl2p requirement. This requirement may involve promoting functions important for morphogenesis (37).

Studies of higher eukaryotic cyclins have established the importance of spatial control for regulation of cyclin and/or Cdk activity. The most extensively studied are the mitotic cyclin B1 and the G₁ cyclin D1. When unphosphorylated, cyclin B1 localizes to the cytoplasm through physical association with the exportin CRM1. The physical association between cyclin B1 and CRM1 is abolished when cyclin B1 is phosphorylated, resulting in localization to the nucleus, possibly through interactions with the karyopherin - complex (46, 58, 82). Cyclin B1 may also enter the nucleus through physical interactions with cyclin F or independently through physical interactions with importin  (71). Phosphorylation of cyclin B1 has also been implicated in triggering nuclear import, independent of inhibition of binding to CRM1 (25, 83). Physical association of cyclin B1 with Cdk does not significantly contribute to nuclear import (46). In vitro studies show that both the cyclin B1-Cdk1 complex, mitogen-activated protein kinase, and casein kinase II are able to phosphorylate cyclin B1 in the region that associates with CRM1 (28, 42). The D-type cyclins function at early stages in the cell division cycle, responding to growth factor stimulation and facilitating the activation of cyclin-Cdk2 complexes required for entry into the phase of the cell cycle at which DNA replication occurs. The shift in localization of cyclin D1 from the nucleus to the cytoplasm at the onset of DNA replication is linked to the proteosomal degradation of cyclin D1 and requires phosphorylation by glycogen synthase kinase-3 on Thr-286. This shift in localization reflects phosphorylation-dependent export of cyclin D1 (18). Two different human B-type cyclins, B1 and B2, exhibit different subcellular localization (to microtubules and to the Golgi apparatus) (29). Interestingly, chimeras constructed that switch the localization signal of cyclin B1 and cyclin B2 also influence their ability to function, demonstrating that differences in subcellular localization influence the functional specificity of these cyclins (20).

In this paper, we analyze mechanisms that regulate localization of Cln2p and Cln3p. We address the role of Cdc28p in regulated localization of G₁ cyclins in budding yeast and characterize the distinct mechanisms used to import different G₁ cyclins into the nucleus.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

The relevant genotypes of the yeast strains used are shown in Table 1. All strains (except for the grr1 strain, MMY42 strain, ACY212, MMY103, MMY106, and controls) are congenic with BF264-15D (59). YPD (dextrose), YPG (galactose), and synthetic complete (SC) media for yeast growth were prepared as described previously (63). Yeast cells were transformed with plasmid DNA as described previously (24).

Plasmids. The 9× myc-epitope-tagged CLN3 CEN/ARS plasmid pMM99, Cln2^mycp integrating construct pMM56, and CEN/ARS plasmid pMM82 (CLN3::CLN2^myc) have been described previously (45). The 9× myc tag is engineered at the COOH terminus of the cyclin proteins in these constructs.

Indirect immunolocalization and GFP localization. Exponentially growing cultures expressing the myc-tagged Cln proteins were fixed in growth medium with formaldehyde (1:10 dilution) for 60 min at 30°C with rotation. When indicated, cells were fixed by "fast fixation." In this case, cells were harvested by filtration and fixed in buffer (0.1 M KH₂PO₄, 0.5 M MgCl₂, 40 mM KOH) with formaldehyde (1:10 dilution) for 5 min. The extended fixation time (up to 2 h) did not change localization patterns with the fast fixation method, and fast fixation gave essentially identical results to standard fixation.

Functional assays. Cell viability and volume assays were carried out as described previously (45). Mutant strains (Table 1) were transformed with the following plasmids: pMM82, pMM99, pMM98, pMM104, pMM105, pMM106, pMM107, pMM60, pMM61, pMM92, pMM93 and pRS416. All plasmids are episomal centromeric low-copy plasmids and carry the TRP1 gene. The data presented are representative of data from at least two independent experiments.

Protein extraction, immunoprecipitations, cellular fractionation, and immunoblotting. Total cellular protein lysates were obtained, immunoprecipitated, and immunoblotted as previously described (38, 45). Cellular fractionation was carried out exactly as previously described (45). The monoclonal antibody HA.11 was used for immunoprecipitation of HA-tagged Cdc28p. Polyclonal HA.11 antibody was used for Western blot detection of Cdc28^hap present in immunoprecipitates. The polyclonal antibody -myc A-14 (Santa Cruz Biotechnology) was used to detect the myc epitope in Western blot analysis. Detection was by enhanced chemiluminescence (ECL) with the Super Signal ECL kit (Pierce).

Elutriation. The diploid strain MMY42 contains CLN2^myc at one CLN2 locus and CLN2^PA at the other CLN2 locus; both expressed from the CLN2 promoter. MMY42 was grown to log phase (optical density at 660 nm [OD₆₆₀] ~0.9) in YEPD at 30°C (1-liter culture), harvested by filtration, resuspended in icewater, sonicated, and separated on the basis of cell size by centrifugal elutriation with a Beckman J6-M elutriating rotor with a 40-ml chamber. Elutriation was carried out with 0°C water, and the rotor chamber was maintained at 4°C throughout the elutriation. The rotor was maintained at the speed of 2,400 rpm, and the first fraction was taken at the flow rate of 70 ml/min. Four-hundred-milliliter fractions were collected with 10% increments in flow rates between fractions. The first fraction was saved when the OD₆₆₀ of the sample rose above 0.02. After collection of fractions, equivalent amounts (as determined by OD) of each fraction were concentrated by filtration, resuspended in ice-cold YPD medium, and immediately subjected to indirect immunofluorescence analysis and protein extraction. Unfixed samples were subjected to Coulter counter analysis to determine the size of the fractions and used to measure the OD₆₆₀. Subsequent immunoblotting analysis was carried out as described above.

Metabolic poisoning. Metabolic poisoning was carried out as described in reference 65. Exponential cultures (50 ml) were harvested by filtration, washed with an equal volume of water, and resuspended in 6 ml of metabolic poison (10 mM NaN3, 10 mM deoxyglucose in YEP medium lacking glucose). Cells were incubated at 30°C with rotation for 45 min. Cells were removed, collected by filtration, fast formaldehyde fixed (see above), and processed for indirect immunofluorescence (see above). The poison was washed out of the culture by filtration followed by washing with an equal volume of water. Cells were allowed to recover by resuspending the cells in YPD medium and incubation at 30°C with rotation. Samples were collected and fast formaldehyde fixed for indirect immunofluorescence after various times of recovery.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Localization of Cln2p is regulated by Cdc28p-dependent phosphorylation. We showed previously that Cln2p was localized primarily to the cytoplasm (45) (Fig. 1). Since Cdc28p binds to Cln2p, phosphorylates Cln2p, and also localizes to the cytoplasm (78; our unpublished data), we investigated the possibility that Cdc28p activity might be important for Cln2p cytoplasmic localization. We assayed the localization pattern of a Cln2^mycp mutant in which all seven potential Cdc28p phosphorylation sites have been mutated to alanine (Cln2-4t3s^mycp) (36, 62). Cln2-4t3sp binds Cdc28p, but the mutant Cln2p is not phosphorylated (36). myc-tagged CLN2-4T3S was integrated at the CLN2 locus in a wild-type strain. Wild-type Cln2p localizes primarily to the cytoplasm with clearly detectable "holes" of nuclear depletion, while the Cln2-4t3s^mycp mutant is no longer depleted in the nucleus and shows some nuclear accumulation (Fig. 1). We note that this nuclear accumulation is not complete, and a population of Cln2-4t3s^mycp remains in the cytoplasm (Fig. 1). It is unclear if the punctate nature of the Clnp staining in this assay results from fixation or reflects a relevant localization pattern in vivo. Consistent with these data, Cln2p also accumulates in the nucleus when CDC28 has been inactivated by using the temperature-sensitive allele cdc28-4 (Fig. 2); Cln2p phosphorylation is strongly reduced under these conditions (data not shown).

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Indirect immunolocalization of Cln2mycp, Cln2-4t3smycp, and Cln3mycp. Strains expressing wild-type Cln2mycp (MMY1) (A), mutant Cln2-4t3smycp (MMY4) (B), or wild-type Cln3mycp (MMY32) (C) were assayed for indirect immunolocalization as described in Materials and Methods. Images were collected, processed, and deconvolved by using the DeltaVision restoration system (Applied Scientific) as described in Materials and Methods. Shown are three sections taken at 0.2-µm intervals labeled 1, 2, and 3. The DAPI signal indicates the position of the nucleus (blue), the -myc signal indicates the position of the myc-epitope-tagged cyclin (red), and the merge combines these two images. The negative control (no myc-tagged cyclin) strain 1255-5C gave no detectable signal (data not shown).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Indirect immunolocalization of Cln2mycp. cdc28-4 and cdc34 mutant cells expressing integrated myc-epitope-tagged Cln2p from the CLN2 promoter were assayed by indirect immunofluorescence as described in Materials and Methods. Cells were grown to log phase at 23°C. Cultures were split, and half was incubated at 23°C and half was incubated at 37°C for 2 h. (A) Quantitation of Cln2 localization. The percentage of cells showing nuclear depletion (gray bars), signal throughout the cell (white bars), or nuclear accumulation (black bars) is shown. A cell was scored positive for nuclear depletion when the position of the nucleus (DAPI signal) could be estimated correctly due to a reduction in Cln2mycp signal. A cell was scored positive for nuclear accumulation when the position of the nucleus (DAPI signal) could be estimated correctly due to an increase in Cln2mycp signal. When the position of the nuclei could not be determined based on Cln2mycp localization, the cells were scored as having a signal throughout the cell. Note that this procedure is unbiased in that the position of Cln2 depletion is determined from immunostaining before the position of the nucleus is determined from DAPI staining. Cln2 depletion from the vacuole will not be scored as nuclear depletion by this procedure. Data for Cln2 localization in the presence of mutant cdc28-4 and mutant cdc34 incubated at 23 and 37°C are shown. No fewer than 200 cells were counted with the DeltaVision microscopy restoration system. (B and C) Indirect immunolocalization of Cln2mycp expressed in mutant cdc28-4 (B) and mutant cdc34 (C) strains. The first row shows the indirect immunofluorescence with monoclonal anti-myc antibody 9E10 (-myc), and the second row shows DAPI staining of DNA. Images were collected by standard microscopy.

View larger version (57K): [in this window] [in a new window]   FIG. 3.   Localization of Cln2mycp and Cln2-4t3smycp by subcellular fractionation. Fractions were prepared as described in Materials and Methods. Duplicate samples of each fraction were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% polyacrylamide) and analyzed by Western blotting. Fractions are indicated at the top of each set of blots. Fraction 1 corresponds to the crude cytoplasmic fraction, fraction 2 corresponds to the surface of the sucrose gradient, fraction 3 corresponds to the surface-to-2.01 M sucrose interface, fraction 4 corresponds to the 2.01-to-2.1 M-sucrose interface, fraction 5 corresponds to the 2.1-to-2.3 M sucrose interface, and fraction 6 corresponds to the remaining pellet of the sucrose gradient. (A) Immunoblot analysis of fractions from a strain expressing Cln2mycp from the CLN2 promoter (MMY1). The position of Cln2mycp is indicated by "Cln2." Corresponding immunoblot analysis of the Nop1p and Pgk1p fractionation is shown below the Cln2p blot. The nucleolar protein Nop1p detected with the monoclonal antibody D77 (4) indicates which fractions contain nuclear proteins. The cytoplasmic protein Pgk1p detected with the anti-Pgk1p polyclonal antibody (Molecular Probes) indicates which fractions contain cytoplasmic proteins. (B) Immunoblot analysis of fractions from a strain expressing Cln2-4t3smycp from the CLN2 promoter (MMY4). The Cln2-4t3smycp, Nop1p, and Pgk1p immunoblots are shown. A moderate level of nuclear breakage apparently occurred in both fractionation experiments, as indicated by the leakage of Nop1p into the intermediate and cytosolic fractions.

Regulated subcellular localization of Cln2p requires binding to Cdc28p. To ask if Cdc28p binding is necessary for Cln2p cytoplasmic localization, we made use of a Cln2p mutant (Cln2-KAEAp) with crippling mutations (K129A E183A) in the cyclin box region that mediates interaction with Cdc28p (27, 32, 38). The localization pattern of Cln2-KAEA^mycp differs from that of wild-type Cln2^mycp. Nuclear depletion is observed in approximately 60% of the cells expressing Cln2^mycp, while it is observed in approximately 10% of the cells expressing Cln2-KAEA^mycp (Fig. 4). Cln2p localization was also assayed in a strain expressing a mutant Cdc28p, Cdc28^csr1-1p (cdc28-Q188P). Cdc28^csr1-1p shows 100-fold reduced binding to wild-type Cln2p upon immunoprecipitation (39). Similar to the KA, EA cln2 mutation, the cdc28^csr1-1 mutation causes a drop in the percentage of cells with nuclear depletion of Cln2^mycp (Fig. 4). Residual binding between mutant Cdc28^csr1-1p and wild-type Cln2p may occur, accounting for the remaining cells (approximately 30%) showing nuclear depletion. This residual binding may be lost in the case of the Cln2-KAEA^mycp mutant (39). Using this mutant, we observed only a low level of nuclear depletion in the presence of either wild-type or mutant Cdc28^csr1-1p. These data suggest that the physical association between Cln2p and Cdc28p is required for efficient nuclear exclusion of Cln2^mycp.

View larger version (96K): [in this window] [in a new window]   FIG. 4.   Requirement for binding to Cdc28p for regulated Cln2mycp localization. (A) Strain 1255-5C containing plasmids that express Cln2mycp (pMM159), mutant Cln2-4t3smycp (pMM151), mutant Cln2-KAEAmycp (pMM167), or mutant Cln2-KAEA-4t3smycp (pMM166) was assayed for indirect immunolocalization as described in Materials and Methods. Images were collected, processed, and deconvolved with the DeltaVision restoration system (Applied Scientific) as described in Materials and Methods. All plasmids are episomal CEN (low copy number) and express the myc-tagged Cln2 protein from the CLN3 promoter. Shown are three sections taken at 0.2-µm intervals labeled 1, 2, and 3 for each set. The DAPI signal indicates the position of the nucleus (blue), the -myc signal indicates the position of the myc epitope-tagged cyclin (red), and the merge combines these two images. The negative control (no myc-tagged cyclin) strain 1255-5C containing plasmid pRS416 gave no detectable signal (data not shown). (B) Quantitation of Cln2 localization. Percentages of cells showing nuclear depletion (gray bars), signal throughout the cell (white bars), or nuclear accumulation (black bars) were determined as described in the legend to Fig. 2. Data for Cln2 localization in the presence of wild-type Cdc28p (CDC28) and Cdc28csr1-1p (CDC28csr1-1) are shown. No fewer than 200 cells were counted for each experiment. The data shown are the average of three independent transformants with standard deviation indicated by the error bar. Images in this figure were captured and processed in parallel.

Energy-dependent nuclear localization of Cln2p. We assayed cells expressing wild-type or mutant Cln2^mycp for energy-dependent localization by incubating the cells in metabolic poison and assaying Cln2p localization by indirect immunofluorescence. Exponential cultures were treated with metabolic poison as described previously (65). We find that after poison treatment, no difference in the localization pattern of wild-type Cln2^mycp is observed (Fig. 5). In contrast, the Cln2-4t3s^mycp showed a dramatic change in localization, failing to accumulate in the nucleus in the presence of the poison (Fig. 5). Additionally, nearly 50% of the cells show some nuclear depletion of Cln2-4t3s^mycp (Fig. 5). We find that the maximal shift in localization occurred at 45 min of incubation with poison and did not change with longer incubations (up to 2 h). We were able to reconstitute nuclear accumulation by washing out the poison and incubating the cells in rich medium at 30°C. The nuclear accumulation of Cln2-4t3s^mycp after the poison was washed out was rapid and maximized between 15 and 20 min (Fig. 5). Steady-state protein levels of Cln2^mycp did not change significantly when incubated with poison (data not shown), though the population of hypophosphorylated Cln2^mycp increased. Upon recovery, there was some reduction in Cln2^mycp levels combined with some recovery of Cln2^mycp phosphorylation. Cln2-4t3s^mycp abundance did not change significantly when incubated with poison or during recovery (data not shown). Overall, poison effects on Cln2p abundance seem unlikely to account for the localization results.

View larger version (61K): [in this window] [in a new window]   FIG. 5.   Energy-dependent localization of Cln2mycp. Strains expressing wild-type untagged Cln2 (1255-5C), wild-type Cln2mycp (MMY1), Cln2-4t3smycp (MMY4), or Cln2-KAEAmycp (pMM167 in strain 1255-5C) were treated with metabolic poison and assayed for localization as described in Materials and Methods. (A) Indirect immunofluorescence of strains growing exponentially (log) after 45 min of treatment with metabolic poison (poison) and 20 min after removal of poison and incubation in rich medium at 30°C (recovery). Cells express untagged Cln2 (no tag), wild-type Cln2mycp (Cln2), or hypophosphorylated Cln2 mutant (Cln2-4t3smyc). Negative controls (no tag) incubated with poison and after recovery gave background signal identical to data shown for exponentially growing cells. (B) Quantitation of Cln2mycp localization during treatment with metabolic poison. Percentages of cells showing nuclear depletion (gray bars), signal throughout the cell (white bars), or nuclear accumulation (black bars) are shown as follows: exponentially growing cells expressing wild-type Cln2mycp without poison treatment (Cln2  poison), cells expressing Cln2mycp after 45 min of treatment with poison (Cln2 + poison), cells expressing Cln2mycp after poison had been washed out and allowed to grow in rich medium for 20 min (Cln2 recovery 20), exponentially growing cells expressing mutant Cln2-4t3smycp without the addition of poison (Cln2-4t3s  poison), cells expressing Cln2-4t3smycp after 45 min of treatment with poison (Cln2-4t3s + poison), cells expressing Cln2-4t3smycp after poison had been washed out and allowed to grow in rich medium for 5, 10, and 20 min (Cln2-4t3s recovery 5, recovery 10, and recovery 20, respectively), cells expressing Cln2-KAEAmycp without addition of poison (Cln2-KAEA  poison), cells expressing Cln2-KAEAmycp after 45 min of treatment with poison (Cln2-KAEA + poison), and cells expressing Cln2-KAEAmycp after poison had been washed out and allowed to grow in rich medium for 20 min (Cln2-KAEA recovery 20). Quantitation was performed as described in the legend to Fig. 2. Essentially identical results were obtained with plasmid-based and genomic expression of Cln2p and Cln2-4t3sp. No fewer than 200 cells were counted for each experiment. The data shown are the average of two independent experiments with standard deviation indicated by the error bar. Images in this figure were captured and processed in parallel.

View larger version (41K): [in this window] [in a new window]   FIG. 6.   Immunolocalization of Cln2mycp and Cln2-4t3smycp in the absence of Ran activity. GSP1 deletion strains carrying a plasmid that expresses either the wild-type GSP1, mutant gsp1-1, or mutant gsp1-2 alleles and integrated myc epitope-tagged Cln2p from the CLN2 promoter were assayed by indirect immunofluorescence as described in Materials and Methods. Cells were grown to log phase at 23°C. Cultures were split, and half was incubated at 23°C and half was incubated at 37°C for 2 h. (A) Indirect immunolocalization of Cln2mycp and Cln2-4t3smycp expressed in wild-type GSP1 strain (top row), gsp1-1 strain (second row) and gsp1-2 strain (third row) incubated at 37°C. The first column shows the indirect immunofluorescence with monoclonal anti-myc antibody 9E10 (red), the second column shows DAPI staining of DNA (blue), and the third column shows the merge of these two images. Images were collected with the DeltaVision microscopy restoration system. (B) Quantitation of Cln2 localization. The percentage of cells showing nuclear depletion (gray bars), signal throughout the cell (white bars), or nuclear accumulation (black bars) is shown. Quantitation was performed as described in the legend to Fig. 2. Data for Cln2mycp and Cln2-4t3smycp localization in the presence of wild- type and mutant GSP1 incubated at 37°C are shown. No fewer than 200 cells were counted with the DeltaVision microscopy restoration system.

Cln2p nuclear depletion may be inefficient early in the cell cycle. We analyzed Cln2p phosphorylation and subcellular localization in a population of rapidly growing cells separated by centrifugal elutriation into fractions of different cell size. The smallest (newborn) cells obtained from the elutriation contain Cln2p that is significantly underphosphorylated compared to larger unbudded cells (Fig. 7A). This is true for 9× myc-epitope-tagged Cln2p, as well as for protein A-tagged Cln2p (in both cases integrated at the CLN2 locus). Indirect immunofluorescence shows little or no detectable depletion of Cln2p^myc from the nucleus of the smallest unbudded cells (Fig. 7B and C), consistent with the idea that Cln2p shuttles between the cytoplasm and nucleus. These data also suggest the possibility that shuttling is differentially regulated, with more nuclear Cln2p present very early in the cell cycle.

View larger version (60K): [in this window] [in a new window]   FIG. 7.   Cell-cycle-regulated nuclear depletion of Cln2p. Rapidly growing cultures of diploid strain MMY42 were chilled and subjected to centrifugal elutriation to obtain fractions of cells of increasing size as described in Materials and Methods. (A) Fractions were subjected to protein extraction and immunoblot analysis to detect 9× myc epitope-tagged or PA epitope-tagged Cln2p, each expressed from a different allele from the CLN2 promoter. Nop1p levels were monitored as a loading control. (B) Fractions were assayed by indirect immunofluorescence to detect Cln2mycp localization. The bar graph shows cells that were scored for the presence of nuclear depletion as described in the legend to Fig. 2. No nuclear accumulation was observed in these fractions. The percentage of budded cells is the percentage of cells with buds in the population of cells in each fraction. The percentage of the total is the percentage of cell mass present in a fraction of the total amount of cell mass as determined by OD of samples. (C) Representative immunofluorescence results of two MMY42 elutriations (sets 1 and 2). Cells were assayed by indirect immunofluorescence as described in Materials and Methods. The first row shows differential interference contrast (DIC) images (cells), the second row shows the indirect immunofluorescence with monoclonal anti-myc antibody 9E10 (-myc), and the third row shows DAPI staining of DNA. The untagged control from this experiment consisted of exponentially growing cells of strain W303d. The amounts of cells of fractions 1, 2, 3, and 4 were 66, 73, 78, and 89 fl, respectively, as determined by identification of peak channels by Coulter Counter analysis of cells.

Cln3p localization does not require Cdc28p activity or binding. In contrast to Cln2p localization, the localization of Cln3p does not appear to be influenced by Cdc28p activity. Cln3p remains nuclear when expressed in the cdc28-4 strain at permissive and nonpermissive temperatures (data not shown). Additionally, mutations within the cyclin box of Cln3p that eliminate detectable interaction with Cdc28p (M. Miller, unpublished observations) do not alter Cln3p nuclear localization (data not shown). Nuclear localization of both wild-type and cyclin box-defective Cln3p was also observed in the presence of Cdc28^csr1-1p mutant Cdc28p (data not shown); this Cdc28 mutation reduces immunoprecipitable Cln3p-Cdc28p complexes by about 10-fold (39). Thus, we were unable to detect any Cdc28p requirement for Cln3p localization.

Carboxy-terminal Cln3p bipartite NLS. The CLN3-1 truncation lacks the C-terminal 177 amino acids of Cln3 and shows a partial increase of cytoplasmic localization when compared to full-length Cln3p (45). These data suggest that sequences important for the nuclear localization are present in the carboxy-terminal tail. The last 22 amino acids of Cln3p contain a potential bipartite NLS (this was proposed as a candidate NLS previously, although it was not tested functionally [81]). Deletion of the last 22 amino acids of Cln3p (Cln3-22p) resulted in a primarily cytoplasmic localization, unlike the nuclear localization of Cln3^mycp (Fig. 8). Thus, the last 22 amino acids are required for nuclear localization of Cln3p.

View larger version (87K): [in this window] [in a new window]   FIG. 8.   Indirect immunolocalization of Cln3-22mycp. Wild-type (1255-5C) cells were transformed with plasmids pRS414 (vector), pMM99 (Cln3mycp), and pMM98 (Cln3-22mycp). All plasmids are episomal CEN (low copy number) and express the myc-tagged Cln3 protein from the CLN3 promoter. Transformants were assayed by indirect immunofluorescence as described in Materials and Methods. The first row shows DIC images (cells), the second row shows indirect immunofluorescence with monoclonal anti-myc antibody 9E10 (-myc), and the third row shows DAPI staining of DNA. Images in this figure were captured and processed in parallel.

View larger version (87K): [in this window] [in a new window]   FIG. 9.   Cln3-NLS-GFP localization. (A) Amino acid sequence of the bipartite-type Cln3 NLS. The mutant Cln3 nls AA substitutes the basic residues in the first and second basic clusters to alanine. The mutant Cln3 nls AK substitutes the first basic cluster with alanines, and the mutant Cln3 nls KA substitutes the second basic cluster with alanines. Mutated residues are underlined. (B) Immunofluorescent localization of the various Cln3 NLS-GFP in living cells. The top row shows DIC images (cells), and the bottom row shows GFP signal in an identical field of cells. Images in this figure were captured and processed in parallel.

Cln3-22p protein characterization. To determine if deletion of the Cln3 NLS alters steady-state expression levels, Western blot analyses were carried out on a wild-type strain expressing Cln3^mycp and Cln3-22^mycp. Cln3-22^mycp was expressed at levels as high as wild-type Cln3^mycp, and in 3 out of 5 experiments Cln3-22^mycp was expressed at higher levels than wild-type Cln3^mycp (Fig. 10B, lanes 1 and 2 and data not shown).

View larger version (53K): [in this window] [in a new window]   FIG. 10.   Comparison of myc-tagged Cln3 proteins. (A) Indirect immunolocalization of NLS-Cln3-22mycp. Wild-type (1255-5C) cells were transformed with plasmids pMM99 (Cln3mycp), pMM98 (Cln3-22mycp), and pMM104 (NLS-Cln3-22mycp). All plasmids are episomal CEN (low copy number) and express the myc-tagged Cln3 protein from the CLN3 promoter. Transformants were assayed by indirect immunofluorescence as described in Materials and Methods. The first row shows DIC images (cells), the second row shows indirect immunofluorescence with monoclonal anti-myc antibody 9E10 (-myc), and the third row shows DAPI staining of DNA. The vector control from this experiment (data not shown) gave no detectable signal. (B) Wild-type strain (1255-5c) transformed with plasmids pMM99 (Cln3myc, set 1), pMM98 (Cln3-22myc, set 2), pMM104 (NLS-Cln3-22myc, set 3), pMM105 (mnls-Cln3-22myc, set 4), pMM106 (NES-Cln3-22myc, set5), and pMM107 (mnes-Cln3-22myc, set 6). All plasmids are episomal CEN (low copy number) and express the myc-tagged Cln protein from the CLN3 promoter. Cellular lysates of two independent transformants for each clone were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide) and analyzed by Western blot analysis as described in Materials and Methods. (C) Coimmunoprecipitation assays were carried out as described in Materials and Methods. Strain 1559-3B-7 expressing HA-epitope-tagged Cdc28p was transformed with plasmids pRS414 (vector), pMM99 (CLN3myc), and pMM98 (cln3-22myc). The top panel shows the anti-HA immunoblot of immunoprecipitated Cdc28hap. The middle panel shows the anti-myc immunoblot of Cln3myc protein present in the immunoprecipitation. The bottom panel shows the anti-myc immunoblot of Cln3myc protein present in the total cellular lysate used for the immunoprecipitation.

Functional consequences of Cln3p mislocalization. The last 22 amino acids of Cln3p encode an NLS that is important for the nuclear localization of Cln3p. Functional analysis was carried out to determine if this NLS was significant for Cln3p function in vivo. Previously, we demonstrated that shifts in the localization patterns of Cln3p correlate with changes in the functional profile of Cln3p. This study was carried out with the Cln3-1p mutant, which lacks the carboxy-terminal 177 amino acids of Cln3p. In addition to missing the C-terminal 22 amino acids (NLS characterized above), this mutant lacks several PEST domains and demonstrates a dramatic stabilization phenotype (13, 45, 81). In contrast, the Cln3-22p mutant is a much smaller deletion, missing only the NLS and parts of a single PEST domain and causes significantly less stabilization of the protein than the deletion of the C-terminal 177 amino acids. In addition to determining if the C-terminal NLS defined in this study is a functionally significant NLS in vivo, we were also interested in whether the changes in the functional profile previously observed for the mislocalized Cln3-1p mutant (45) required hyperstabilization.

View larger version (46K): [in this window] [in a new window]   FIG. 11.   Functional assays for Cln3-22p. Mutant strains, each containing a GAL1::CLN for viability, were transformed with CLN plasmids. For each transformant, 10-fold serial dilutions were prepared from independent pools of transformants (5 to 10 colonies), and 3 µl of each dilution was plated onto both YPD (dextrose) and YPG (galactose) plates. Plates were incubated at 30°C, except where 38°C is indicated. The CLN plasmids used are pMM82 (CLN2myc), pMM99 (CLN3myc), pMM98 (cln3-22myc), pMM104 (NLS-cln3-22myc), pMM105 (mnls-cln3-22myc), pMM106 (NES-cln3-22myc), pMM107 (mnes-cln3-22myc), pMM60 (NLS-CLN2myc), pMM61 (mnls-CLN2myc), pMM92 (NES-CLN2myc), and pMM93 (mnes-CLN2myc). All plasmids are episomal CEN (low copy number) and express the myc-tagged Cln protein from the CLN3 promoter. Strains are listed in Table 1. Genotypes are as follows (lowercase denotes deletion; uppercase denotes wild type, and GAL1:: CLN gene not indicated): cln1 cln2 cln3 and cln1 cln2 cln3 bck2 (A); cln1 cln2 cln3 pcl1 pcl2 and cln1 cln2 CLN3 pcl1 pcl2 (B); cln1 cln2 CLN3 swi4 and cln1 cln2 cln3 swi4 (C). (cl1 clz cl3 swi4 data were reproduced for comparison to the cln1 cln2 CLN3 swi4 strain from reference 45.)

Localization-dependent cell size control by Cln3p. We also assayed for CLN activity by monitoring cell volume. Initiation of the cell cycle requires CLN activity for bud emergence and activation of Clbp-Cdc28p complexes. Cells with reduced CLN activity will have a delay in these events resulting in a population of relatively large cells due to a longer period of cell growth after cell division and before cell cycle initiation. Conversely, high CLN activity will result in a population of relatively small cells (11, 48, 53). Links between cell cycle progression and cellular growth may also exist in higher eukaryotic cells, suggesting that this process could be conserved (as reviewed in reference 10). Plasmids encoding the various mutant Cln3 proteins were introduced into a cln2 cln3 strain, and transformants were assayed for cell volume. (A cln2 cln3 strain was used to sensitize the assay to CLN function.) As expected, expression of Cln2^mycp and Cln3^mycp confers smaller cell volume than that of the vector control. Expression of NES-Cln3-22^mycp (but not the mnes controls) produces populations of cells with larger cell volumes, near the volume of control vector transformants (Fig. 12). These data show a strong reduction of CLN3 functional activity when Cln3p is exported out of the nucleus and into the cytoplasm through the combination of the Cln3p NLS deletion and addition of the PKI NES. Thus, as with the cln1 cln2 cln3 rescue assay, the cell volume assay for CLN activity leads to the conclusion that nuclear Cln3 may be required to efficiently drive cell cycle initiation.

View larger version (36K): [in this window] [in a new window]   FIG. 12.   Cell size assay for CLN gene function. CLN1 cln2 cln3 cells (1421-21D) were transformed with plasmids containing various CLN2 and CLN3 constructs (see the legend to Fig. 9). Cell size analysis was carried out as described in Materials and Methods. Data for two transformants are shown on each graph.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Different mechanisms control localization of the G₁ cyclins Cln2p and Cln3p. In addition to activating Cdks through conformational changes associated with cyclin-Cdk binding, the spatial context in which cyclin proteins function is an important component of Cdk regulation. In previous work, we found that the localization pattern of the G₁cyclin Cln2p is primarily cytoplasmic and that of the G₁ cyclin Cln3p is primarily nuclear (45). Here we have investigated the role that the Cdk Cdc28p plays in the control of distinct subcellular localization patterns of these cyclins. Using a mutant Cln2p in which all potential Cdc28p target sites have been mutated, we find a partial accumulation of Cln2p in the nucleus (Fig. 1). Inactivation of CDC28 also results in a partial accumulation of Cln2p in the nucleus as demonstrated by indirect immunofluorescence (Fig. 2). The nuclear accumulation of hypophosphorylated Cln2p does not reflect Cln2p stabilization, since stabilization of Cln2p via mutation of the CDC34 gene or deletion of the GRR1 gene does not alter Cln2p localization (Fig. 2 and data not shown). Taken together, these data are consistent with direct Cdc28p-dependent phosphorylation of Cln2 being required for nuclear depletion of Cln2p. Additionally, there is a requirement of Cln2p-Cdc28p complex formation in regulated Cln2p localization. Both nuclear depletion and nuclear accumulation are influenced by physical association with Cdc28p, as seen with mutations in Cln2p that are defective in binding Cdc28p and with mutations in Cdc28p that are defective for binding Cln2p (Fig. 4).