The budding yeast polo-like kinase Cdc5 regulates the Ndt80 branch of the meiotic recombination checkpoint pathway

Abstract

Defects in chromosome synapsis and/or meiotic recombination activate a surveillance mechanism that blocks meiotic cell cycle progression to prevent anomalous chromosome segregation and formation of aberrant gametes. In the budding yeast zip1 mutant, which lacks a synaptonemal complex component, the meiotic recombination checkpoint is triggered, resulting in extremely delayed meiotic progression. We report that overproduction of the polo-like kinase Cdc5 partially alleviates the meiotic prophase arrest of zip1, leading to the formation of inviable meiotic products. Unlike vegetative cells, we demonstrate that Cdc5 overproduction does not stimulate meiotic checkpoint adaptation because the Mek1 kinase remains activated in zip1 2μ-CDC5 cells. Inappropriate meiotic divisions in zip1 promoted by high levels of active Cdc5 do not result from altered function of the cyclin-dependent kinase (CDK) inhibitor Swe1. In contrast, CDC5 overexpression leads to premature induction of the Ndt80 transcription factor, which drives the expression of genes required for meiotic divisions, including CLB1. We also show that depletion of Cdc5 during meiotic prophase prevents the production of Ndt80 and that CDK activity contributes to the induction of Ndt80 in zip1 cells overexpressing CDC5. Our results reveal a role for Cdc5 in meiotic checkpoint control by regulating Ndt80 function.

FIGURE 1:

CDC5 overexpression partially suppresses the checkpoint-dependent meiotic delay of zip1 but does not improve spore viability. (A) Overexpression of CDC5, but not that of other FEAR/MEN genes, partially alleviates the sporulation defect of the zip1 mutant. Dityrosine fluorescence after 3 d on a sporulation plate is shown as an indicator for the formation of mature asci. The sporulation efficiency, assessed by microscopic counting of asci, is also presented. Strains are BR2495 (wild type) and MY63 (zip1) transformed with vector alone (YEp352) or with the indicated plasmids: pPD2 (2μ-CDC14), pJC29 (2μ-CDC5), pSJ56 (2μ-TEM1), pSJ57 (2μ-DBF2), and pSJ103 (2μ-CDC15). (B) Suppression of zip1 meiotic arrest by CDC5 overexpression. Time course of meiotic nuclear divisions; the percentage of cells containing more than two nuclei is represented. Strains and plasmids used are wild type (DP396/pRS426), wild type + 2μ-CDC5 (DP396/pJC29), zip1 + 2μ-CDC5 (DP386/pJC29), zip1 + 2μ-cdc5-kd (DP386/pSS127), and zip1 (DP386/pRS426). (C) CDC5 overexpression partially alleviates the meiotic delay of the dmc1 mutant. Strains are wild type (BR1919-2N/pRS426), dmc1 (DP456/pRS426), and dmc1 2μ-CDC5 (DP456/pJC29). (D) CDC5 overexpression does not suppress the spore viability defect of zip1 and zip1 swe1. Spore viability as determined by asci dissection is plotted. Averages and standard deviations from two to six experiments, in which independent colonies were dissected, are represented. Strains are wild type (DP396/pRS426), wild type + 2μ-CDC5 (DP396/pJC29), zip1 (DP386/pRS426), zip1 2μ-CDC5 (DP386/pJC29), zip1 swe1 (DP393/pRS426), and zip1 swe1 2μ-CDC5 (DP393/pJC29). The total number of spores scored for each strain is indicated (n).

FIGURE 2:

Suppression of the zip1 meiotic delay by CDC5 overexpression does not result from checkpoint adaptation. (A) Analysis of Mek1 phosphorylation as an indicator for activation of the meiotic recombination checkpoint effector kinase. Note that, for a better comparison, the experiment was performed in ndt80-arrested cells to avoid possible differences that could be due to the different meiotic progression of the various mutants. PGK was used as a loading control. Strains are ndt80 (DP424), ndt80 zip1 (DP428), ndt80 zip1 spo11 (DP728), ndt80 zip1 mec1 (DP680), and ndt80 zip1 mek1 (DP674). (B) Western blot analysis of Mek1 activation throughout meiosis in wild type (DP396/pRS426), zip1 (DP386/pRS426), and zip1 2μ-CDC5 (DP386/pJC29). Ponceau S staining of the membranes is shown as a loading control.

FIGURE 3:

Bypass of zip1 meiotic delay by high levels of active Cdc5 does not result from altered Swe1 function. (A) Schematic representation of the two regulatory branches targeted by the meiotic recombination checkpoint to restrain CDK activity, thus preventing meiosis I entry. Note that the positive and negative arrows connecting Mek1 to Swe1 and Ndt80, respectively, do not necessarily imply direct action. (B) Time course of meiotic nuclear divisions; the percentage of cells containing more than two nuclei is represented. Strains are wild type (DP396/pRS426), zip1 swe1 + 2μ-CDC5 (DP393/pJC29), zip1 swe1 (DP393/pRS426), zip1 swe1 + 2μ-cdc5-kd (DP393/pSS127), and zip1 (DP386/pRS426). (C) Swe1-dependent inhibitory phosphorylation of Cdc28 is not affected by Cdc5 overproduction. Western blot analysis of phosphorylation of Cdc28 at tyrosine 19 and total Cdc28 (PSTAIRE) throughout meiosis in wild type (DP396/pRS426), zip1 (DP386/pRS426), and zip1 2μ-CDC5 (DP386/pJC29). Extracts from a zip1 swe1 strain (DP393), which lacks Cdc28^Tyr19 phosphorylation, were used as control for specificity of the antibody.

FIGURE 4:

Cdc5 functions on the NDT80-CLB1 branch of the meiotic recombination checkpoint. Time course of meiotic nuclear divisions; the percentage of cells containing more than two nuclei is represented. Strains and plasmids used in the experiments are as follows: (A) Wild type (DP396/pRS426), zip1 + 2μ-CDC5 (DP386/pJC29), zip1 + 2μ-CDC5-CLB1 (DP386/pSS121), zip1 + 2μ-CLB1 (DP386/pR2045), and zip1 (DP386/pRS426). (B) Wild type (DP396/pRS426), zip1 swe1 + 2μ-CDC5-CLB1 (DP393/pSS121), zip1 swe1 + 2μ-CDC5 (DP393/pJC29), zip1 swe1 + 2μ-CLB1 (DP393/pR2045), zip1 swe1 (DP393/pRS426), and zip1 (DP386/pRS426).

FIGURE 5:

CDC5 overexpression in zip1 cells leads to earlier induction of Ndt80 production. (A) Western blot analysis of Cdc5 and Ndt80 production throughout meiosis in wild type (DP396/pRS426), zip1 (DP386/pRS426), and zip1 2μ-CDC5 (DP386/pJC29). Tubulin was used as a loading control. The asterisk marks a presumptive degradation product of Ndt80 (Tung et al., 2000; Shubassi et al., 2003). (B) Western blot analysis of Ndt80 production in zip1 swe1 (DP393/pRS426) and zip1 swe1 2μ-CDC5 (DP393/pJC29). PGK is shown as a loading control. (C) Quantification of the relative meiotic induction at the 36-h time point of the CLB1 mRNA in wild type (DP396/pRS426), zip1 (DP386/pRS426), zip1 2μ-CDC5 (DP386/pJC29), and ndt80 (DP424/pRS426).

FIGURE 6:

Production of active and stable Ndt80 depends on Cdc5. (A) Time-course analysis of meiotic nuclear divisions in wild-type (BR1919-2N) and cdc5-dg (DP402) strains. The percentage of cells containing more than two nuclei is represented. Meiotic cultures incubated at 25°C were split in two 15 h after meiotic induction (arrow), and doxycycline was added to one-half of the cultures (+dox), which were shifted to 33°C. The other half remained at 25°C without doxycycline. (B) Western blot analysis of Cdc5 and Ndt80 production in the cdc5-dg meiotic cultures described in A. Extracts from 18-h meiotic cultures of ndt80 (DP424; center lane) and wild type (BR1919-2N; right lane) were also included as controls. The asterisk in the Ndt80 blot indicates the Ndt80 form reportedly resulting from degradation (Tung et al., 2000; Shubassi et al., 2003). The double asterisk in the Cdc5 blot marks a nonspecific band produced in midmeiosis recognized by the anti-Cdc5 antibody. PGK was used as a protein loading control.

FIGURE 7:

CDK inhibition further compromises Ndt80 induction in zip1 cells. (A) Time-course analysis of meiotic nuclear divisions. The percentage of cells containing more than two nuclei is represented. Cultures were split in two 12 h after meiotic induction (arrow) to allow premeiotic S phase to occur, and 1 μM of 1NM-PP1 (dissolved in DMSO) was added to one-half of the culture. The same amount of DMSO alone was added to the other half. (B) Western blot analysis of Cdc5 and Ndt80 production in the meiotic cultures described in A. PGK was used as a loading control. Strains are cdc28-as1 (DP430/pRS426), zip1 cdc28-as1 (DP431/pRS426), and zip1 cdc28-as1 2μ-CDC5 (DP431/pJC29).

FIGURE 8:

Schematic representation of a proposed model for Cdc5-dependent regulation of Ndt80. In unperturbed meiosis (left), basal levels of Cdc5 would contribute to trigger the Ndt80 autoactivation loop that, once engaged, also induces CDC5 expression itself as a middle sporulation gene and other genes required for meiotic divisions (i.e., CLB1). In contrast, when synapsis or recombination is defective (i.e., zip1 mutant; right) the checkpoint sensors relay the signal to the Mek1 effector kinase, which, in turn, directly or indirectly, affects the two main regulatory branches of the checkpoint. Our results indicate that Cdc5 acts specifically on the Ndt80 branch. It is possible that when the checkpoint is triggered, Cdc5 function is inhibited to prevent activation of Ndt80, thus contributing to the meiotic cell cycle delay. Alternatively, Cdc5-independent inhibition of Ndt80 by the checkpoint is also plausible. See Discussion for additional details.