Distinct subcellular localization patterns contribute to functional specificity of the Cln2 and Cln3 cyclins of Saccharomyces cerevisiae.

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 3β-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 α/β 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.