Compartmentalization of the functions and regulation of the mitotic cyclin Clb2 inS. cerevisiae

Abstract

Orderly progression through the eukaryotic cell cycle is a complex process involving both regulation of cyclin dependent kinase activity and control of specific substrate-Cdk interactions. In Saccharomyces cerevisiae, the mitotic cyclin Clb2 has a central role in regulating the onset of anaphase and in maintaining the cellular shape of the bud by inhibiting growth polarization induced in G1. However, how Clb2 and the partially redundant cyclin Clb1 confer specificity to Cdk1 in these processes still remains unclear. Here, we show that Clb2 mutants impaired in nuclear import or export are differentially affected for subsets of Clb2 functions while remaining fully functional for others. Our data support a direct role of the cytoplasmic pool of Clb1,2-Cdk1 in terminating cytoskeleton and growth polarization, independently of G1 cyclin transcriptional regulation. By contrast, the nuclear form of the cyclin is required for timely initiation of anaphase. Clb2 localization influences its stage-specific degradation as well. We report that Clb2 trapped in the cytoplasm is stabilized during anaphase but not at the time of mitotic exit. Altogether, our results demonstrate that the subcellular localization of the mitotic cyclin Clb2 is one of the key determinants of its biological function.

New insights into the regulation of anaphase by mitotic cyclins in budding yeast

“…This could be due to a direct regulation of astral microtubule or to the important defect in cell morphology and actin cytoskeleton due to the absence of Clb2. 66,67 In summary, this work indicates that in budding yeast, mitotic cyclins play a key role in growth suppression of kMTs and might actively inhibit new bipolar spindle formation during anaphase. Deregulated mitotic activity has been found in a variety of cancer cells.…”

Spatially distinct functions of Clb2 in the DNA damage response

“…42 The apical-isotropic switch terminates polarized bud growth after bud emergence, and this event is also under the control of M-CDK activity. 7,8 Consistent with the idea that Clb2 contributes an important function to the bud growth pattern, clb2Δ mutants frequently exhibit some elongated cells.…”

“…The clb1 ΔNLS allele was replaced at the chromosomal CLB1 locus using the same strategy as that used for the gene replacement of clb2 ΔNLS and clb2 nes described in reference. 42 Details about the procedure are available upon request. Yeast cultures were grown in YEP medium (1% yeast extract, 2% Bacto Peptone, 0.005% uracil, and 0.01% adenine) supplemented with 2% of either of the following carbon source: dextrose, galactose, or raffinose.…”

“…Removal of the bipartite nuclear localization signal (NLS) in Clb2 ΔNLS causes the mutant cyclin to predominantly accumulate in the cytoplasm at the expense of the nuclear pool. 39,40,42 Conversely in a clb2 nes background, the cytoplasmic pool of Clb2 is drastically reduced owing to a point mutation in the main nuclear export sequence of the protein. 41,42 In the cdc13-1 context, both clb2 ΔNLS and clb2 nes yielded a robust G 2 /M checkpoint arrest phenotype when monitored by FACS analysis in a G 1 release experiment (Fig.…”

G1 cyclin driven DNA replication

“…26 Still, it cannot be ruled out that mitotic Cdk1 activity may be necessary both in the nucleus and the cytoplasm for correct chromosome segregation. 27 We thus constructed a strain carrying inducible copies of Cln2(7A)-2xNLS and untagged Cln2 (7A).…”