Nuclear localization of the meiosis-specific transcription factor Ndt80 is regulated by the pachytene checkpoint

Abstract

In budding yeast, the Ndt80 protein is a meiosis-specific transcription factor that is essential for the exit of pachytene and progression into nuclear divisions and spore formation. The pachytene checkpoint responds to defects in meiotic recombination and chromosome synapsis and negatively regulates the activity of Ndt80. The activity of Ndt80 was suggested to be regulated at both transcriptional and posttranslational levels; however, the mechanism for posttranslational regulation of Ndt80 was unclear. From a study of ndt80 in-frame deletion mutations, we have identified a dominant mutation NDT80-bc, which is able to completely bypass the pachytene checkpoint. The NDT80-bc mutation relieves the checkpoint-mediated arrest of the zip1, dmc1, and hop2 mutants, producing spores with low viability. The NDT80-bc mutant provides direct evidence for the posttranslational control of Ndt80 activity. Furthermore, the data presented show that Ndt80 is retained in cytoplasm in the zip1 mutant, whereas Ndt80-bc is found in the nucleus. We propose that the nuclear localization of Ndt80 is regulated by the pachytene checkpoint through a cytoplasmic anchor mechanism.

FIGURE 1:

NDT80-bc suppresses the zip1 sporulation defect. Nuclear divisions and spore formation were monitored and compared among wild type, NDT80-bc/NDT80-bc, and NDT80-bc/NDT80 (A and B); wild type, zip1, zip1 NDT80-bc/NDT80-bc, and zip1 NDT80-bc/NDT80 (C and D); zip1 NDT80-bc/NDT80-bc, zip1 NDT80-bc/ndt80Δ, and zip1 NDT80-bc/NDT80 (E and F). These experiments were repeated with similar results.

FIGURE 2:

The expressions of Ndt80 and Ndt80-bc are similar in zip1 cells. Production of Ndt80 in zip1 (A) and zip1 NDT80/NDT80-bc (C) cells were monitored through meiosis by Western blot analysis with anti-HA antibodies. Production of Ndt80-bc in zip1 NDT80-bc/NDT80-bc (B) and zip1 NDT80/NDT80-bc (D) cells were analyzed through meiosis with anti-myc antibodies. Tubulin was also analyzed and used as a loading control on each blot (*). The ratios in expression of Ndt80 or Ndt80-bc to tubulin are plotted (E).

FIGURE 3:

Differential localizations of Ndt80 and Ndt80-bc. NDT80-HA/NDT80-bc-myc cells were stained with DAPI (A, E, and I), anti-HA antibodies (B, F, and J), and anti-myc antibodies (C, G, and K), to detect nuclei, Ndt80, and Ndt80-bc, respectively. Merged images are shown in (D), (H), and (L). Three categories of differential localization of Ndt80 and Ndt80-bc (A–D, E–H, and I-L) were as indicated and described in Results. Scale bar, 2 μm.

FIGURE 4:

Quantification of subcellular localizations of Ndt80 and Ndt80-bc. The subcellular localizations of Ndt80 and Ndt80-bc in NDT80-HA/NDT80-bc-myc cells (A) and zip1/zip1 NDT80-HA/NDT80-bc-myc cells (B) were examined by anti-HA and anti-myc antibodies at indicated time points after inoculation into sporulation medium. Only mononucleated cells with both HA and myc signals were scored and classified into three categories as shown in Figure 3: I. both Ndt80 and Ndt80-bc are in the cytoplasm (white bars). II. Ndt80 is in cytoplasm, but Ndt80-bc is in the nucleus (gray bars). III. both Ndt80 and Ndt80-bc are in the nucleus (black bars). The data shown in (A) and (B) are reorganized to present the individual fractions of nuclear localization for Ndt80 (gray bars) and Ndt80-bc (black bars) in NDT80-HA/NDT80-bc-myc (C) and in zip1/zip1 NDT80-HA/NDT80-bc-myc (D) cells. Averages of five repeats for each strain at each time point are presented. A total of at least 249 cells were scored at each time point for each strain.

FIGURE 5:

Model of posttranslational regulation of Ndt80 activity. (A) In wild-type cells, Ndt80 protein is able to activate the expression of middle sporulation genes (MSGs) after meiotic recombination has been completed. (B) In zip1 cells, Ndt80 is directly targeted by the pachytene checkpoint and unable to activate transcription of MSG. (C) When Ndt80 is overproduced in zip1 cells, some Ndt80 might escape the control of checkpoint protein and induce the expression of MSG. (D) In zip1 NDT80-bc cells, the checkpoint cannot target the Ndt80-bc, and the transcription of MSG is induced.