Academic journal article Genetics

Degradation of the Mitotic Cyclin Clb3 Is Not Required for Mitotic Exit but Is Necessary for G1 Cyclin Control of the Succeeding Cell Cycle

Academic journal article Genetics

Degradation of the Mitotic Cyclin Clb3 Is Not Required for Mitotic Exit but Is Necessary for G1 Cyclin Control of the Succeeding Cell Cycle

Article excerpt

THE eukaryotic cell cycle is regulated by cyclin-dependent kinases (CDKs) bound to cyclins (Bloom and Cross 2007). In Saccharomyces cerevisiae, the Cdc28 CDK binds to nine cyclins: G1 cyclins Cln1-3; S phase B-type cyclins Clb5,6; and mitotic cyclins Clb1-4. These cyclins have sharply different biological functions: cln1,2,3 null strains have an absolute defect in cell cycle initiation ("Start") but no defect in any post-Start events (DNA replication, spindle assembly, nuclear division, and cytokinesis) (Richardson et al. 1989; Cross 1990). In contrast, clb1,2,3,4 null strains execute Start and DNA replication, but fail to execute cell division (Fitch et al. 1992; Richardson et al. 1992).

Specificity of cyclin function can derive from differential regulation at many levels: cyclin abundance or subcellular localization, response to inhibitors, degree of activation of Cdc28 kinase activity, and cyclin-specific substrate targeting by docking motifs (Loog and Morgan 2005; Bloom and Cross 2007; Kõivomägi et al. 2011). These diverse controls may be coordinated to regulate the overall temporal pattern of specific CDK activity. On the other hand, deletion of many cyclin genes leads to, at most, minor defects. Thus, cyclin specificity is a strong, but not absolute, determinant of function (Roberts 1999; Bloom and Cross 2007).

B-type cyclins are essential for entry into mitosis; subsequent mitotic exit (cytokinesis, telophase, and resetting the systemtoG1 in newborn cells) requires mitotic cyclin degradation (Murray and Kirschner 1989; Murray et al. 1989; King et al. 1996). Degradation requires cyclin ubiquitination by the anaphase-promoting complex (APC), targeted by the cyclin destruction box (D box) or KENboxmotifs (Glotzer et al. 1991; Pfleger and Kirschner 2000). Consistent with the requirement for mitotic cyclin degradation for mitotic exit, precise genomic removal of the D box and KEN boxes from the budding yeast mitotic cyclin Clb2 caused a first-cycle block to mitotic exit (Wäsch and Cross 2002).

The ability of mitotic B-type cyclins to both induce mitotic entry and block mitotic exitmaytightly couplemanyaspects of cell cycle progression to once-per-CDK-cycle (Nasmyth 1996). As B-type cyclin-CDK activity rises, mitotic entry is induced, but exit is suppressed; upon B-type cyclin degradation, no further mitotic entry events occur, but mitotic exit is allowed (Nasmyth 1996). Systematic variation in "locked" levels of the Clb2 mitotic cyclin led to the need to revise this "ratchet" model to include a key role for the regulated Cdc14 phosphatase (Drapkin et al. 2009). Cdc14 activation, in turn, is under partially autonomous oscillatory control, requiring a mechanism for oscillator coordination (Lu and Cross 2010).

The CLB1/2 and CLB3/4 gene pairs are highly similar, but the CLB3/4 vs. CLB1/2 divergence is ancient (Archambault et al. 2005). Of CLB1-4, clb2 deletion led to the most extreme phenotypes; CLB3 has mitotic functions partially overlapping with CLB2 (Fitch et al. 1992; Richardson et al. 1992). Clb3 and Clb2 are similarly abundant through the cell cycle (Cross et al. 2002), but differ in activity toward diverse substrates (Kõivomägi et al. 2011).

Clb3 is degraded upon mitotic exit in parallel with Clb2 (Cross et al. 2002). Removal of the Clb2 D box results in failure of mitotic exit and consequent lethality (Wäsch and Cross 2002). Here, we characterize the requirement for the Clb3 D box for proteolytic regulation and for cell cycle control.

Materials and Methods

Strains and plasmids

Standard methods were used for transformation, mating, and tetrad analysis. All strains were derivatives of W303. All strains with CLB3Ddb were generated using HO-induced exact gene replacement of the CLB3 allele (Cross and Pecani 2011).

Construction of clb1 clb2 CLB3Ddb required some more complex procedures. We crossed a clb1 clb2:GALL-CLB2- URA3 clb6:kanMX strain with a MATainc GAL-HO CLB3Ddb- URA3-CLB3 clb4:HIS3 strain on a YEPD plate to keep GAL-HO inactive, then dissected tetrads on galactose medium to simultaneously maintain viability of segregants bearing clb1 clb2:GALL-CLB2, and induce cleavage of the HO cut site to obtain CLB3Ddb recombinants. …

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