INTRODUCTION

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Key events in the eukaryotic cell cycle are regulated by the serine-threonine cyclin-dependent kinase (cdk). The binding of regulatory cyclin subunits to the cdk enzyme induces structural changes that are required for kinase activity. In addition to the activation of the cdk, cyclin molecules are thought to serve targeting functions, conferring functional specificity to different cyclin-cdk complexes (37). In Saccharomyces cerevisiae the cdk encoded by the CDC28 gene, Cdc28p, regulates the cell division cycle when bound to one of nine different cyclins: CLN1 to CLN3, and CLB1 to CLB6.

During the postmitotic growth period prior to DNA replication, haploid cells are able to respond to mating pheromone or initiate a round of cell division. Initiation of the cell division cycle follows a critical cell size threshold and requires the accumulation of Clbp-Cdc28p kinase activity, which is required for DNA replication and subsequent mitosis. Each of these events (cell size, mating pathway activation, and Clbp activation) are regulated by the Cln proteins.

Deletion of all three CLN genes results in a cell cycle arrest as unbudded cells with 1C DNA content. Genetic characterization of the CLN genes shows that CLN1, CLN2, and CLN3 are functionally redundant, since any one CLN gene is able to complement the arrest caused by the triple cln1 cln2 cln3 deletion. However, there are significant differences in expression, primary sequence, and functional specificity of CLN2 (representative of the highly homologous CLN1-CLN2 gene pair) and CLN3. CLN2 transcription is cell cycle regulated, peaking at late G₁ (59, 64). CLN3 transcription is relatively constitutive, with a two- to threefold peak during the time in the cell cycle where wild-type cells finish mitosis and begin G₁, prior to CLN2 expression (16, 33, 41, 59, 64). The sequence similarity between these two classes of CLN genes is low and confined to the cyclin box, a region of the cyclin involved in physical interactions with Cdc28p (19).

Functional analysis of CLN2 and CLN3 indicate that CLN3 is primarily required for the expression of genes during late G₁. The Swi4p-Swi6p complex, called SBF, is required for the CLN3-dependent expression of CLN1-CLN2 (26). The BCK2 gene product also triggers transcription of CLN1-CLN2. The BCK2-dependent activation of CLN1-CLN2 most likely occurs through the SBF complex, working in parallel with Cln3p to activate CLN1-CLN2 (9, 13). CLN3 is unable to complement the triple cln1 cln2 cln3 deletion in the absence of SWI4 or SWI6, indicating that this transcription complex is crucial for Cln3p-dependent viability. Cln2p-dependent viability in the triple cln1 cln2 cln3 deletion strain does not require Swi4p or Swi6p (27). The Mbp1p-Swi6p complex, called MBF, may also be responsive to Cln3p. Transcripts potentially regulated by Cln3p through the MBF include the CLB5 and CLB6 cyclins (50), as well as other genes involved in DNA replication. It is important to note that the Cln2p-Cdc28p complex is able to activate SBF- and MBF-dependent transcription, although not as efficiently as the Cln3p-Cdc28p complex (12, 56). For this reason, the Cln3p-Cdc28p complex is thought to be the physiologically relevant cdk complex regulating transcription in G₁.

Since Cln2p supports viability in the cln swi4 and cln swi6 strains, while Cln3p does not, it is possible that Cln3p induces expression of proteins that carry out one of several events normally triggered more directly by Cln2p. The gene pairs CLB5-CLB6 (50) and PCL1-PCL2 (34, 42) are essential for Cln3p-dependent viability in the triple cln1 cln2 cln3 deletion strain (27). Each of these genes encodes a cyclin homologue that can bind to and activate the cdk Cdc28p or Pho85p, respectively. PHO85 is also essential for viability in cln1 cln2 cells (14), and Pho85p contributes to phosphorylation of the Sic1p inhibitor of Clbp-Cdc28p complexes (40). The molecular basis for lethality of cln1 cln2 pcl1 pcl2 or cln1 cln2 pho85 strains is not well established.

The activation of Clb cyclins results from a combination of cellular events that are regulated by Clnp-Cdc28p protein kinase activity. As described above, Cln3p may regulate the MBF-dependent transcription of CLB5-CLB6. In addition to this, Clnp-Cdc28p-dependent phosphorylation leads to the SCF-dependent ubiquitination and degradation of Sic1p (15, 61). Sic1p is a stoichiometric inhibitor of Clbp-Cdc28p protein kinase activity (35, 49). Overexpression of Sic1p results in growth arrest, due to inactivation of Clbp-Cdc28p activity, and Cln2p is able to suppress this growth arrest (27, 58). Finally, the Clnp-Cdc28p activity may be involved in regulating the phosphorylation state of Hct1p. Unphosphorylated Hct1p promotes the activation of the anaphase-promoting complex toward Clb2p (22, 48, 62, 68). Anaphase-promoting complex activity leads to ubiquitination and subsequent degradation of Clb2p by the proteosome (67).

Events required for proper cell cycle progression that may be regulated by Cln2p, rather than Cln3p, include cell polarization (29) and bud emergence (3, 8). The ability of CLN3 to complement the triple cln1 cln2 cln3 deletion is strongly reduced in the absence of the BUD2 gene due to failure of bud emergence, indicating that this gene product is important for Cln3p-dependent morphogenesis. Cln2p-dependent viability in the triple cln1 cln2 cln3 deletion strain is not influenced by deletion of BUD2 (27). In wild-type cells, Bud2p is not essential and is involved in bud site selection (5).

Intrinsic qualitative differences between Cln2p and Cln3p (as opposed to simple quantitative or timing differences in expression or associated kinase activity) have been demonstrated through a comparison of CLN3::CLN2 (CLN2 under the control of the CLN3 promoter), CLN3, and cln2 mutants by using strains that distinguish genetically between Cln2p and Cln3p activity (27). However, this study did not establish a molecular basis for the functional specificity. We address here two possible mechanisms. First, the cyclin molecule might confer structural differences to the cdk active site, changing the substrate specificity of the kinase. Second, differences in subcellular localization might bring Clnp-Cdc28p complexes into contact with different subsets of Clnp-Cdc28p protein kinase substrates.

Studies of higher eukaryotic cyclin molecules suggest that localization is important for cyclin function. Cyclins E, A, D1, and B1 have been implicated in nuclear events and show nuclear localization when the cyclin-cdk activity is required for function (2, 4, 11, 20, 25, 31, 32, 43, 45). The correlation is extended by the fact that a cyclin D1 mutant that is unable to enter the nucleus is unable to activate DNA replication in fibroblasts (11). The ability of cyclin B1 to promote mitosis in frog eggs is abolished in mutant cyclin B1 that does not localize to the nucleus (30). Also, cyclin B1 that is targeted to the nucleus through addition of a nuclear localization signal (NLS) or through disruption of the cyclin B1 nuclear export signal (NES) shows a defect in DNA damage-induced G₂ arrest (23, 57). Different cyclins may be localized by distinct mechanisms. The redistribution of cyclin D1 to the cytoplasm is triggered by a glycogen synthase kinase 3-dependent phosphorylation event (10). The distinct localization pattern of cyclin B1, which accumulates in the nucleus during mitosis, results from a balance between CRM1-mediated nuclear export and nuclear import most likely mediated by the importin / complex (36, 44, 66).

In the study presented here, we analyze differential localization of Cln2p and Cln3p in Saccharomyces cerevisiae. The localization patterns are distinct, and our data support the idea that these distinct patterns contribute to cyclin functional specificity.

	MATERIALS AND METHODS

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The relevant genotypes of the yeast strains used are shown in Table 1. All strains are congenic with BF264-15D (46). YPDex, YPGal, and synthetic complete (SC) media for yeast growth were prepared as described earlier (51). Yeast cells were transformed with plasmid DNA as described previously (18).

Plasmids. The 9X myc epitope-tagged GAL1::CLN2^myc (AG4) and GAL1::CLN3^hamyc (AG2) were derived from the plasmids KH100 and KL002-4 (27) by A. Gartner. The 9X myc tag is engineered at the C terminus of the cyclin proteins in these constructs. The Cln2^myc integration plasmid (pMM56) was constructed by replacing the NotI cassette containing the 3× hemagglutinin (HA) epitope tag of MT104 with the NotI cassette containing the 9X myc epitope tag of AG2. This construct contains a CLN2 promoter driven CLN2^myc and integrates at the endogenous CLN2 locus. The GAL1::CLN3^hamyc integration plasmid (pMM64) was constructed by inserting the NotI cassette containing the 9X myc epitope tag of AG2 into the NotI site of pKL036. pKL036 results from the insertion of the SalI-SacII fragment of pKL002-4 (27) into the polylinker of pRS404 (52). The pMM64 construct was used for ectopic integration of the GAL1::CLN3^hamyc cassette at the URA3 locus.

Indirect immunolocalization. Exponentially growing cultures expressing the myc-tagged Cln proteins were fixed in growth medium with formaldehyde (1:10 dilution) for 90 min at 30°C with rotation. Cells were washed with phosphate-buffered saline (PBS), briefly sonicated, washed again in PBS, and then washed in sorbitol citrate buffer (0.1 M K₂HPO₄, 33 mM citric acid, 1.2 M sorbitol, 2 mM dithiothreitol [DTT]). Cells walls were removed by digestion with a 1:10 volume of glusalase, a 1:100 volume of a 20-mg/ml concentration of zymolyase, and 1 mM DTT for 2 h at 30°C with rotation. Cells were washed four times with sorbitol citrate buffer. Cells were fixed to polylysine-coated slides by placing the cell suspension in wells for 3 min and aspirating off the suspension until they were dry. The slides were placed in 100% Methonal for 5 min, 100% acetone for 5 min, and then air dried. Fixed cells were then rehydrated and blocked by incubation with 2% nonfat dry milk in PBS-Tween solution (1× PBS, 0.2% Tween 20) overnight at 4°C. Primary monoclonal antibody 9E10 (Santa Cruz Biotechnology) was diluted 1:200 in blocking solution, cleared for 2 min by centrifugation in a microfuge, and incubated on the cells for 2 h. The wells were washed quickly four times, followed by three 5-min washes. Secondary antibody (Cy3-conjugated anti-mouse immunoglobulin G; Jackson Immunochemicals) was diluted 1:200 in blocking solution, cleared, and incubated on the wells for 2 h. Cells were again washed, and mounting solution (10% 1× PBS in glycerol with 0.02 µg of DAPI [4',6'-diamidino-2-phenylindole] and 1 mg of phenylenediamine per ml) was added to the wells. The negative control for integrated myc-tagged Cln proteins was the wild-type strain 1255-5C. The negative control for plasmid-based expression of myc-tagged Cln proteins was the vector control, pRS414 (52).

Biochemical fractionation. Biochemical fractionation was carried out as described elsewhere (24, 47), with the following modifications. Culture volumes were scaled down to 1 liter, and cells were allowed to recover from spheroplasting for 1 h in YPDex (for CLN2^myc) or YPGal (for GAL1::CLN3^hamyc) before the cells were harvested and lysed by Polytron shearing. Western blots of different fractions from these preparations were probed with the anti-myc polyclonal antibody A-14 (Santa Cruz Biotechnologies) and anti-Pgk1p polyclonal antibody (Molecular Probes) to visualize the cytoplasmic fractions and with anti-Nop1p antibody (M. Rout) to visualize the nuclear fractions.

-factor synchronization. -Factor was added to 500 ml of exponentially growing cells to a final concentration of 0.6 mM until they were synchronized, as determined by morphology (2 h for CLN2^myc strain grown in YPDex). -Factor was washed out with 500 ml of medium, then briefly sonicated, and finally washed again. Washing consisted of collecting cells by filtration and resuspending cells in a 500 ml of medium. After the second wash, cells were resuspended in 30°C YPDex medium. Samples were collected every 15 min and assayed by indirect immunofluorescence and Western blot analysis. Synchrony was confirmed by determining the budding index.

Cell volume assay. Cell volume assays were carried out on a cln2 cln3 GAL1::CLN3 strain grown on glucose-containing medium that had been transformed with the following plasmids: pMM45, pMM55, pMM82, pMM60, pMM61, pMM92, pMM93, pMM83, pMM84, pMM85, pMM86, and pRS416. Subsequent experiments were done with this strain transformed with pMM99 and pRS416. All plasmids are episomal centromeric low-copy plasmids and carry the TRP1 gene. For each strain, three individual transformants were assayed as described earlier (27), with the following modifications. Overnight cultures grown in defined (SC) medium containing 3% galactose and lacking tryptophan (SCGal-trp) were used to inoculate SC medium containing 2% glucose lacking tryptophan (SCDex-trp) and grown overnight at 30°C to an optical density at 660 nm (OD₆₆₀) of approximately 1.0. These cultures were then diluted to an OD₆₆₀ of approximately 0.2 in SCDex-trp and allowed to grow at 30°C to an OD₆₆₀ of 0.8. Cells were then fixed in 1% formaldehyde and sonicated to disperse the clumps. Cell volume analysis was done with a Coulter Channelyzer 256. The data shown are representative of at least two independent experiments for each strain.

Cell viability assays. Mutant strains (see Table 1) were transformed with the following plasmids: pMM45, pMM55, pMM82, pMM60, pMM61, pMM92, pMM93, pMM83, pMM84, pMM85, pMM86, pMM99, and pRS416. For each transformant strain, 10-fold serial dilutions were prepared for two pools of transformants (5 to 10 colonies), and 5 µl of each dilution were plated onto a YPDex (dextrose) or YPGal (galactose) plate and assayed for growth at 30 and 38°C after 2 to 3 days. The maximum decrease in viability that can be determined from this assay is 1,000-fold. Plates were replica plated to SCGal-trp and SCDex-trp to confirm that colonies forming on YPDex and YPGal plates maintained the test plasmids. The data presented are representative of data from at least two independent experiments.

Protein extraction and immunoblotting. Total cellular protein lysates were obtained as follows: 10 ml of exponentially growing yeast cultures (OD₆₆₀ between 0.5 and 0.9) were quick chilled by pouring over ice. The remaining steps were done at 4°C. The cells were spun, and the pellet was resuspended in 1 ml of 1× TE (pH 7.4; 10 mM Tris, 1 mM EDTA) and transferred to a 1.5-ml microfuge tube. Cells were spun for 5 s in a microcentrifuge, and the pellets were resuspended in 100 µl of extraction buffer with inhibitors (0.6% sodium dodecyl sulfate [SDS]; 10 mM Tris, pH 7.4; 10% aprotinin; 1:1,000 [vol/vol] 0.5 M phenylmethylsulfonyl fluoride; 1:100 [vol/vol] 1 mg of leupeptin-pepstatin per ml; 1:10 [vol/vol] 100 mM NaPP_i, pH 7.3). An equivalent of 150 µl of glass beads was added, and the bead slurry was vortexed for 3 min. Then, 100 µl of 2× SDS sample buffer was added, and the lysates were analyzed by immunoblotting. Cellular lysates containing the 35-kDa Cln3^hamycp were also prepared by lysis in 1.85 N NaOH and 7.4% -mercaptoethanol, followed by trichloroacetic acid precipitation. Identical results were obtained by using both lysis protocols. Lysates were run on SDS-polyacrylamide gels and transferred to immunoblots as described previously (7). Immunoblotting was performed as described previously (27). The antibody used to detect the myc epitope was polyclonal -myc A-14 (Santa Cruz Biotechnology), and the antibody used to detect the HA epitope in the peptide kinase assays was polyclonal antibody 12CA5 (Babco). Detection was done by enhanced chemiluminescence (ECL) with the super signal ECL Kit (Pierce).

Immunoprecipitation and peptide kinase assays. Immunoprecipitations were performed as described earlier (27), with the following modifications. The HA epitope-tagged Cln strains (2195-5c for Cln2^hap and 1022 for Cln3^hap expressed from the GAL1 promoter) were scaled up to 500 ml and resuspended in a final volume of 300 µl of kinase buffer. Extraction buffer N (50 mM Tris-HCl, pH 7.5; 100 mM NaCl; 0.1 mM EDTA, pH 8.0; 10 mM NaF; 60 mM -glycerophosphate; 0.1% NP-40) was used instead of TNN buffer. Immunoprecipitates were washed with extraction buffer five times instead of three times. Then, 15 µl of the final suspension was used for each peptide kinase assay. The cell suspension was incubated with various concentrations of peptide substrate in a volume of 2 µl, with 2 µl of 50 mM ATP and 1 µl of [-³²P]ATP (NEN). The kinase reaction mixtures were incubated at 30°C for 5 min. The reactions were stopped by adding 18 µl of the reaction mixture to 2 µl of 0.5 M EDTA, followed by a 10-min incubation at 65°C. The reaction was then spotted onto phosphocellulose paper disks and processed as described elsewhere (53). D. Lawrence kindly provided the peptides. Each assay was performed in duplicate, and assays were done from at least three separate immunoprecipitations. The apparent K_m (± the standard error of the mean [SEM]) and V_max (± SEM) values were determined from initial rate experiments that included five different peptide concentrations over a 10-fold range encompassing the K_m. These data were plotted by using the Lineweaver-Burke procedure; the corresponding plots proved to be linear.

	RESULTS

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Enzyme kinetics of Cln2p-Cdc28p and Cln3p-Cdc28p kinase activities. To investigate the possibility that the functional specificity observed between Cln2p and Cln3p reflects differences in substrate specificity, we measured the phosphorylation kinetics of Cln2p-Cdc28p and Cln3p-Cdc28p with previously characterized peptide substrates (53). The amino acid sequence of these peptides is based on the minimal necessary sequence for substrate recognition by cyclin-dependent kinase, S/T-P-X-Z, where Z is typically basic (see Table 2 for peptide sequences). To allow analysis of Cln2p- and Cln3p-associated Cdc28p activity, immunoprecipitated complexes of HA epitope-tagged cyclin were used as the source of cyclin-associated kinase activity. The V_max is expressed relative to the amount of Cdc28p present in coimmunoprecipitation, as determined by Western blot analysis (see Materials and Methods).

Cellular localization of Cln2p. The functional specificity of Cln2p-Cdc28p and Cln3p-Cdc28p complexes may result from differential localization of cyclins, making distinct subsets of substrates or cyclin-cdk regulators accessible to different cyclin-Cdc28p complexes. Cellular localization of Cln2p and Cln3p were determined by using indirect immunofluorescence of 9X myc epitope-tagged proteins. We myc epitope tagged the carboxy terminus of CLN2 at the endogenous CLN2 locus (see Materials and Methods). The integration results in the expression of myc epitope-tagged Cln2p under the control of the endogenous CLN2 promoter (Cln2^mycp). The staining pattern for Cln2^mycp appears to be primarily cytoplasmic and somewhat punctate, with the nucleus appearing understained relative to the cytoplasm (Fig. 1A). This signal was specific to Cln2^mycp and occurred in unbudded and small budded cells, a finding consistent with the cell cycle-regulated expression pattern of CLN2. To confirm that the signal observed by indirect immunofluorescence of asynchronous cultures was due to Cln2^mycp, we assayed for localization by using a synchronized culture. In a culture synchronized by -factor block and release, cytoplasmic Cln2^mycp signal was detected by indirect immunofluorescence almost uniformly in the population at the same time (at or just prior to bud emergence) that Cln2^myc protein peaked, as determined by Western blot analysis (data not shown).

View larger version (7776K): [in this window] [in a new window]   FIG. 1.   Indirect immunolocalization of Cln2p and Cln3p. (A) Strains expressing Cln2mycp from the CLN2 promoter (strain MMY1) and Cln3hamycp from the GAL1 promoter (strain MMY2). (B) Cln3hamycp from the CLN3 promoter (1255-5C with pMM45). Strains were assayed by indirect immunofluorescence as described in Materials and Methods. The first row shows DIC images (cells); the second row shows indirect immunofluorescence where monoclonal anti-myc 9E10 (Santa Cruz Biotechnology) was used as the primary antibody (-myc), and the third row shows the DAPI staining of DNA. The myc-tagged cyclins expressed are indicated at the top of each set. The Cln2mycp signal appeared to be punctate and cytoplasmic. At a low frequency, spots of Cln2mycp signal are visible but do not lie within the area of the nucleus as defined by the DAPI stain. Nuclear Cln3-1hamycp staining was usually throughout the area of the nucleus, but on rare occasions (<0.5% of the cells with overexpressed Cln3-1hamycp) the staining appeared along the edge of the nucleus. Bar, 5 µm.

Cellular localization of Cln3p. The localization pattern of Cln3p was visualized by using the 9X myc epitope placed in frame at the carboxy terminus of the CLN3^hap. Since the expression level of Cln3p is significantly lower than the peak expression of cell cycle-regulated Cln2p, we first characterized the localization of overexpressed myc epitope-tagged Cln3p by using the galactose-inducible GAL1 promoter (GAL1::CLN3^hamyc). The GAL1::CLN3^hamyc construct was integrated at the URA3 locus in strain 1255-5c, and cells grown in the presence of galactose were assayed by indirect immunofluorescence. Overexpressed Cln3^hamycp is present throughout the cell, with a concentration of Cln3^hamycp in the nucleus of some cells (Fig. 1A). The cells that accumulate Cln3^hamycp in the nucleus consist primarily of budded cells.

Cellular fractionation of Cln2p and Cln3p. Western blot analysis with antibodies against the myc epitope indicate that a significant amount of the immunoreactive myc in lysates from cells expressing Cln3^hamycp comes from a species of approximately 35 kDa (Fig. 2). An estimated 18 kDa of this protein is due to the 3X HA and 9X myc epitopes, leaving 17 kDa of Cln3p. This product is consistent with a cleavage event internal to Cln3p, one approximately 150 amino acids from the carboxy-terminal end of the protein. The 35-kDa species is observed in both anti-myc immunoprecipitations and crude cellular lysates. It is present when Cln3p is expressed from the GAL1 and CLN3 promoters. A similar species is observed when the protein A epitope is engineered at the carboxy terminus of CLN3 (data not shown). We have been unable to detect a faster-migrating species with the 3X HA epitope-tagged Cln3p, possibly because of the smaller size of this epitope. Experiments that use anti-Cln3p serum to detect untagged Cln3p on Western blots show a nonfunctional species shorter than full-length Cln3p (7), the size of which might result from the loss of C-terminal sequences. It remains unclear if the cleavage of Cln3^hamycp resulting in the approximately 35-kDa species occurs in vivo or in vitro.

View larger version (44K): [in this window] [in a new window]   FIG. 2.   Immunoblot analysis of Cln3hamycp and Cln3-1hamycp. A wild-type strain (1255-5c) was transformed with plasmids pRS414 (vector, lane 1), pAG2 (Cln3hamycp expressed from the GAL1 promoter, lane 2), and pMM55 (Cln3-1hamycp, lane 3). All plasmids are episomal CEN (low copy number). Cellular lysates were separated by SDS-12% polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blotting as described in Materials and Methods. Proteins were visualized by using the polyclonal anti-myc A-14 antibody (Santa Cruz Biotechnology). Molecular size markers are listed to the left of the blot in kilodaltons.

View larger version (61K): [in this window] [in a new window]   FIG. 3.   Localization of Cln3p and Cln2p by subcellular fractionation. Fractions were prepared as described in Materials and Methods. Duplicate samples of each fraction were separated by SDS-12% PAGE and analyzed by Western blotting. Fractions are indicated at the top of each blot. Fraction 1 corresponds to the crude cytoplasmic fraction, fraction 2 corresponds to the surface of the sucrose gradient, fraction 3 corresponds to the surface-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. The total protein isolated from cells prior to polytron shearing is indicated in the lane marked 7. (A) Immunoblot analysis of fractions from a strain expressing Cln3hamycp from the GAL1 promoter (MMY2). The position of full-length Cln3hamycp is indicated by Cln3, and the 35-kDa species is indicated by Cln3*. Corresponding immunoblot analysis of the Nop1p and Pgk1p fractionation is shown below the Cln3p blot. The nucleolar protein Nop1p detected with the monoclonal antibody D77 (1) 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 Cln2mycp from the CLN2 promoter (MMY1). The Cln2p, Pgk1p, and Nop1p immunoblots are shown. A moderate level of nuclear breakage apparently occurred in this fractionation, as indicated by leakage of Nop1p into the intermediate and cytosolic fractions.

Cellular localization of Cln3-1p. If the 35-kDa Cln3^hamycp results from a cleavage within the C-terminal tail of Cln3p, then a mutant Cln3p lacking the C-terminal tail would be missing the presumed site of cleavage and would not produce the 35-kDa Cln3^hamycp. To test this idea, we 9X myc epitope tagged the truncated allele of CLN3^ha, CLN3-1^ha. CLN3-1 encodes a stable Cln3-1p that is more active than full-length Cln3p, as measured by cell size experiments, presumably because of the increase in steady-state levels of Cln3-1p (7, 60, 65). Protein lysates from a strain expressing Cln3-1^hamycp do not show faster-migrating species (Fig. 2, lane 3), a result consistent with the idea that a cleavage site falls within the C-terminal tail of Cln3. Indirect immunofluorescence of Cln3-1^hamycp shows a staining pattern similar to that of overexpressed Cln3^hamycp, with localization to the nucleus in budded cells and localization throughout the cell in unbudded cells (see Fig. 5). Comparison of Cln3-1p localization and Cln3p localization shows an increase in cytoplasmic staining with Cln3-1^hamycp (see Fig. 1 and 5).

Altering cellular localization of Cln3p. To determine the functional relevance of Clnp localization patterns, we attempted to redirect localization by engineering localization signals on the N terminus of Cln3p. To facilitate exit from the nucleus, the PKI NES was used. The NES consists of a leucine-rich sequence that is necessary and sufficient to mediate nuclear export (63). This export signal is functional in S. cerevisiae (17, 54). We also engineered the simian virus 40 large T NLS onto Cln3p. The NLS consists of a short lysine-rich sequence that is sufficient to mediate nuclear import of a heterologous protein in yeast cells (39). Point mutants of the NLS (mnls) and NES (mnes) that disrupt localization activity of these sequences were used to control for nonspecific effects of the additional sequences at the N terminus of the proteins. Indirect immunofluorescence shows no changes in the cellular localization of the proteins. Indirect immunofluorescence shows no changes in the cellular localization of the NLS-Cln3^mycp expressed from the CLN3 promoter compared to the Cln3^mycp or mnls-Cln3^mycp patterns. A slight increase in cytoplasmic staining was observed with the NES-Cln3^mycp compared to Cln3^mycp or mnes-Cln3^mycp (Fig. 4 and data not shown).

View larger version (61K): [in this window] [in a new window]   FIG. 4.   Indirect immunolocalization of NLS- and NES-Cln3mycp. Wild-type cells (1255-5c) were transformed with plasmids pRS414 (vector), pMM99 (Cln3mycp), pMM100 (NLS-Cln3mycp), pMM101 (mnls-Cln3mycp), pMM102 (NES-Cln3mycp), and pMM103 (mnes-Cln3mycp). 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 the indirect immunofluorescence with monoclonal anti-myc antibody 9E10 (-myc), and the third row shows DAPI staining of DNA. The myc-tagged cyclins expressed are indicated at the top of each set. Bar, 5 µm.

View larger version (52K): [in this window] [in a new window]   FIG. 5.   Indirect immunolocalization of NLS- and NES-Cln3-1hamycp. Wild-type cells (1255-5c) were transformed with plasmids pRS414 (vector), pMM55 (Cln3-1hamycp), pMM83 (NLS-Cln3-1hamycp), pMM84 (nmls-Cln3-1hamycp), pMM85 (NES-Cln3-1hamycp), and pMM86 (mnes-Cln3-1hamycp). All plasmids are episomal CEN (low copy number) and express the myc-tagged Cln 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 the indirect immunofluorescence with monoclonal anti-myc antibody 9E10 (-myc), and the third row shows DAPI staining of DNA. The myc-tagged cyclins expressed are indicated at the top of each set. Bar, 5 µm.

Altering cellular localization of Cln2p. Attempts to change the localization pattern of Cln2^mycp were carried out as described for Cln3^hamycp. Sequences comprising the NLS, mnls, NES, and mnes sequences were engineered at the N terminus of Cln2^mycp and assayed for localization by indirect immunofluorescence. Expression of the NLS-Cln2^mycp does not significantly change the localization pattern compared to mnls-Cln2^mycp or Cln2^mycp. Some nuclear accumulation of NLS-Cln2^mycp (but not of Cln2^mycp and mnls-Cln2^mycp) is observed when overexpressed from the GAL1 promoter, but cytoplasmic staining is still obvious (data not shown). Thus, while the NLS is functional, it is unable to confer efficient nuclear localization to Cln2^mycp. As for Cln3p, this may be due to efficient endogenous regulation of Cln2p localization. NES-Cln2^mycp shows no evident difference in localization patterns from wild-type Cln2^mycp and mnes-Cln2^mycp, although the cytoplasmic localization of Cln2p already observed would make any increased cytoplasmic accumulation difficult to detect (data not shown).

Localization-mediated functional specificity of Cln2p, Cln3-1p, and Cln3p. Strains containing the NLS- or NES-CLN2^myc and the NLS- or NES-CLN3-1^hamyc constructs were assayed for CLN function. Both CLN2 and CLN3-1 are expressed from the constitutive CLN3 promoter to control for differences in the expression of CLN2 and CLN3. Western blot analysis of the NES/mnes/NLS/mnls-Cln3-1^hamycp and NES/mnes/NLS/mnls-Cln2^mycp show similar steady-state protein levels (Fig. 6). In all of the assays described here, we assigned functional defects to changes in localization exclusively based on differences between Cln proteins that contain the wild-type (NLS or NES) and mutant (nls or nes) localization signals. The mnls and mnes control for nonspecific alterations in Clnp function that may result from the addition of sequences to the N terminus of the protein. In cases where indirect immunofluorescence shows little to no detectable alterations in localization patterns, the mnls and mnes will allow us to attribute any detectable functional defects to the localization activity of the NLS or NES.

View larger version (40K): [in this window] [in a new window]   FIG. 6.   Comparison of myc-tagged Cln proteins. Wild-type strain (1255-5c) was transformed with plasmids pRS414 (vector, lane 1), pMM45 (Cln3, lane 2), pMM55 (Cln3-1, lane 3), pMM83 (NLS-Cln3-1, lane 4), pMM84 (mnls-Cln3-1, lane 5), pMM85 (NES-Cln3-1, lane 6), pMM86 (mnes-Cln3-1, lane 7), pMM82 (Cln2, lane 8), pMM60 (NLS-Cln2, lane 9), pMM61 (mnls-Cln2, lane 10), pMM92 (NES-Cln2, lane 11), and pMM93 (mnes-Cln2, lane 12). All plasmids are episomal CEN (low copy number) and express the myc-tagged Cln protein from the CLN3 promoter. Cellular lysates were separated by SDS-12% PAGE and analyzed by Western blotting as described in Materials and Methods.

View larger version (31K): [in this window] [in a new window]   FIG. 7.   Cell size assay for CLN gene function. CLN1 cln2 cln3 cells (1421-21D) were transformed with plasmids containing various CLN2 and CLN3 constructs (see Fig. 5 legend). Cell size analysis was carried out as described in Materials and Methods. The data for three transformants are shown on each graph. The x and y axis scales for all of the graphs in this figure are identical.

View larger version (91K): [in this window] [in a new window]   FIG. 8.   Viability assay for NLS- and NES-CLN2 and CLN3-1 strains. Mutant strains, each containing a GAL1::CLN for viability, were transformed with CLN plasmids. For each transformant strain, 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 YPDex and YPGal plates. Plates were incubated at 30 or 38°C as indicated. CLN plasmids used are those listed in the legend to Fig. 5. (A) cln and cln bck2 strains. (B) cln swi4 and CLN3 cln1,2 GAL::SIC1 strains. (C) CLN3 cln1,2 clb5,6 strain. (D) cln pcl1,2 and cln bud2 strains. (E) CLN3 cln1,2 swi4 strain.

cln bck2

cln clb5,6

cln swi4

cln bck2

cln swi4

2 swi4

cln swi4

cln clb5

cln bud2

cln pcl1

cln pcl1,2

cln swi4

View larger version (73K): [in this window] [in a new window]   FIG. 9.   Viability assays and Western blot for NLS- and NES-CLN3 strains. (A and B) Mutant strains, each containing a GAL1::CLN for viability, were transformed with plasmids pRS414 (vector), pMM45 (CLN3), pMM83 (NLS-CLN3), pMM84 (mnls-CLN3), pMM85 (NES-CLN3), and pMM86 (mnes-CLN3). For each transformant strain, 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 YPDex and YPGal plates. Plates were incubated at 30 or 38°C as indicated. (A) cln pcl1,2 strain. (B) cln swi4 strain. (C) Wild-type strain 1255-5C was transformed with the plasmids listed above. Cellular lysates from two independent transformants were prepared in parallel for each plasmid-bearing strain (except pRS414). All plasmids are episomal CEN (low copy number) and express the myc-tagged Cln protein from the CLN3 promoter. Cellular lysates were separated by SDS-10% PAGE and analyzed by Western blotting as described in Materials and Methods. Proteins were visualized by using the polyclonal anti-myc A-14 antibody (Santa Cruz Biotechnology). Lanes: 1, vector; 2, CLN3; 3, NLS-CLN3; 4, mnls-CLN3; 5, NES-CLN3; 6, mnes-CLN3. The position of full-length Cln3hamycp is indicated by Cln3, and the 35-kDa species is indicated by *Cln3.

cln swi4

cln swi4

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Enzyme kinetics of Cln2p-Cdc28p and Cln3p-Cdc28p. No significant differences in the binding affinity, catalysis rate, or overall efficiency of the Cln2p-Cdc28p and Cln3p-Cdc28p complexes were observed in our analysis. These data are consistent with an overall similarity of the active site of Cdc28p when activated by the two different cyclins. We note that these studies utilize a limited set of three short peptides. For this reason, these data are not definitive, and kinetic differences may exist with additional peptide substrates. However, the fact that the enzyme kinetics in this limited study showed no differences between Cln2p- and Cln3p-associated Cdc28 kinase activity prompted us to look for other mechanisms to explain the cyclin functional specificity.

Subcellular localization of Cln2p and Cln3p. Cln2p and Cln3p have distinct localization patterns. Cln2^mycp is localized primarily to the cytoplasm, as demonstrated by indirect immunofluorescence and biochemical fractionation of cells. While the majority of Cln2^mycp is clearly cytoplasmic, it is likely that some proportion of Cln2p also localizes to the nucleus, since limiting the ability of Cln2p to reside in the nucleus with an NES inhibits some Cln2p functions (Fig. 7 and 8). The point mutant mnes has no effect, indicating that this is probably specific to nuclear export, not to addition of a nonspecific sequence to Cln2p. In contrast to the primarily cytoplasmic localization of Cln2^mycp, Cln3^mycp accumulates in the nuclei of large budded cells. Nuclear accumulation of Cln3p appears gradual, in that we cannot correlate a morphological event (such as bud emergence or nuclear division) with efficient Cln3p nuclear localization. Constitutively expressed Cln2p remains cytoplasmic in large budded cells (data not shown), while Cln3p localizes to the nuclei. These data indicate distinctly regulated cellular localization of Cln2p and Cln3p, providing a possible mechanism to confer substrate specificity to Clnp-Cdc28p complexes.

Cln3p function and cellular localization. The mechanism by which Cln3p activity results in the activation of late G₁ transcripts, such as those regulated by the SBF or MBF transcription factors, is not understood. However, one might speculate that Cln3p acts directly on the components of the transcriptional machinery, possibly within the nucleus of the cell. We demonstrate that the efficient nuclear localization of Cln3-1p is required for proper CLN function in the cln bck2 strain, which is specifically defective in SBF-dependent transcription (27). These data suggest the likelihood that Cln3p acts directly within the nucleus to trigger transcription of late G₁ transcripts. This raises the speculation that direct interactions occur between Cln3p and the transcriptional machinery; testing this idea further is beyond the scope of this study.

Functional significance of Cln2p and Cln3p localization. If the localization patterns of Cln2p and Cln3p reflect significant regulatory mechanisms, then mislocalization of the cyclins should alter CLN function. We found NES-dependent decreases in Cln2p and Cln3-1p activity in both cln complementation and cell size assays, indicating that the presence of Cln proteins in the nucleus is critical for some CLN functions (see Fig. 7 and 8). We also saw consequences of the addition of NES to Cln2p in most of the mutant strains tested (Fig. 8). These data suggest that the localization of Cln protein to the nucleus of the cell is important for the efficient function of both Cln2p and Cln3p. The lack of effect of the NLS on Cln2p function in most assays is difficult to evaluate given that the NLS has very little cytologically detectable effect on Cln2p localization. Additionally, as described below, specific NES- and NLS-Cln3-1p-dependent changes in Cln protein activity were observed in the series of mutant strains tested. These results suggest that there are CLN-dependent cytoplasmic and CLN-dependent nuclear events that are important for cell cycle initiation.

cln clb5

cln pcl1

cln bud2

cln swi4