Phosphorylation and maximal activity of Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80 is dependent on Ime2

Abstract

The Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80 is responsible for the induction of a class of genes referred to as middle sporulation genes. Among the members of this family are the B-type cyclins and other genes whose products are required for meiotic chromosome division and spore morphogenesis. Inactivation of NDT80 leads to a failure to induce the middle sporulation genes and a subsequent arrest in pachytene. The expression of NDT80 is itself highly regulated. The initial transcription of NDT80 is dependent upon the protein kinase Ime2; once Ndt80 protein accumulates, it activates its own promoter, thus generating an autoactivation loop. In addition to being transcriptionally regulated, Ndt80 protein is posttranslationally regulated. Phosphorylation of Ndt80 occurs coincident with its activation as a transcription factor. If expressed prematurely in meiosis, Ndt80 accumulates initially in an unmodified form that is subsequently modified by phosphorylation. In contrast, Ndt80 expressed in ime2 mutant strains does not become modified and has a reduced ability to activate transcription of its target genes. Ime2 can also phosphorylate Ndt80 in vitro, further supporting a direct role for Ime2 in the phosphorylation of Ndt80. These data indicate that Ime2 plays a novel and previously unexpected role in promoting chromosome dissemination and progress through meiotic development by activating Ndt80.

FIG.6.

The C terminus of Ndt80 encodes a transactivation domain. (A) The endogenous NDT80 in a diploid yeast strain was replaced by a version of NDT80 encoding either full-length Ndt80 or the indicated C-terminal deletion (Δ27, Δ67, or Δ167). These strains also harbored an SPS4-LacZ reporter gene. The ability of these mutant versions of NDT80 to promote middle sporulation gene expression was assayed by the β-galactosidase activity induced after 6 h in SPM and is expressed in Miller units. The ability of the mutant versions of NDT80 to promote and scored for the formation of asci. (B) IME2 is not required for the function of the NDT80 trans activation domain. The C terminus of NDT80 or the indicated C terminally deleted versions of NDT80 were fused to the Gal4 DNA binding domain and expressed in vegetatively growing cells under the regulation of a CUP1 promoter. β-Galactosidase activity expressed from an integrated GAL1-LacZ reporter gene was measured and is displayed in Miller units. Similarly, the entire Ndt80 C terminus (amino acids 426 and 627) was fused to LexA and expressed under the regulation of a CUP1 promoter in IME2/IME2 or ime2/ime2 diploid cells. Both strains harbor an integrated 8×Lex^o-LacZ reporter gene. β-Galactosidase activity induced by the LexA fusions was assayed after the cells had been in SPM for 5 h. (C) Ime2 phosphorylates Ndt80 lacking its transactivation domain. An Mbp-Ndt80 fusion lacking 167 amino acids from its C terminus was produced in E. coli and used as a substrate for immunoprecipitated Ime2Δ175. Lanes 1 to 4 show an autoradiogram (Autorad) of the kinase assay, while lanes 5 to 8 show Coomassie staining of the same gel. Ime2Δ175 kinase activity was assayed against Mbp-Ndt80Δ167 (lanes 1 and 5), Mbp1 (lanes 2 and 6), or no added substrate (lanes 3 and 7). Molecular weight markers are included in lane 8.

FIG. 1.

Phosphorylation of Ndt80 is dependent upon IME2. The expression of Ndt80 during sporulation was monitored by Western blot assay in wild-type SK1 diploids (A and B), a clb5 clb6 mutant diploid (C), an ime2 mutant diploid (D and E), and an ime2 sic1 double mutant (F). Ndt80 was expressed under the regulation of its endogenous promoter (A) or ectopically expressed under the regulation of an IME2 promoter (B, C, and D) or a copper-inducible CUP1 promoter (E and F). The same amount of protein extract was loaded in each lane, and the time points at which samples were taken from the sporulating cultures are indicated in hours postinduction of sporulation. To induce Ndt80 expression in cells that carry NDT80 under the regulation of the CUP1 promoter, CuSO₄ was added to the medium to a final concentration of 100 μM at time zero. The abundance of Ndt80 expressed under the regulation of an IME2 promoter or a CUP1 promoter was compared (G). Equal amounts of protein isolated from ime2 IME2-NDT80 and ime2 CUP1-NDT80 homozygous diploid strains 6 h after the induction of sporulation were probed for the abundance of Ndt80. Tubulin served as a loading control. (H) The species of Ndt80HA that display retarded mobility when expressed under the regulation of an IME2 promoter in IME2/IME2 cells are due to phosphorylation. DSY1312 IME2-NDT80HA was induced to sporulate, and after 6 h in SPM, cells were harvested and Ndt80HA was isolated by immunoprecipitation. The sample was divided into three aliquots. One sample was untreated, the second was incubated with 100 U of CIP, and the third was incubated with CIP in the presence of 5 mM β-glycerophosphate to inhibit the CIP activity.

FIG. 2.

Ectopic expression of Ndt80 does not profoundly accelerate the induction of middle sporulation gene transcription. RNA samples (10 μg) obtained at the indicated time points from wild-type NDT80-expressing cells (A) or wild-type cells that also express NDT80 under the regulation of an IME2 promoter, IME2-NDT80 (B), were probed for the middle sporulation gene transcripts SPS1 and SPS2. RNA samples (10 μg) from a homozygous ime2 mutant expressing NDT80 from its endogenous promoter, NDT80 (C), an IME2 promoter, IME2-NDT80 (D), or a CUP1 promoter, CUP1-NDT80 (E), were probed for the middle sporulation gene transcripts SPS1 and SPS2. The ACT1 transcript served as a loading control.

FIG. 3.

Overproduction of Ndt80 allows activation of middle sporulation gene promoters in an ime2 mutant but does not promote spore formation. Wild-type cells (IME2/IME2) or ime2 mutants (ime2/ime2) harboring an SPS4-LacZ reporter gene were transformed with vector plasmid YCplac111 (vector), a high-copy plasmid carrying NDT80 (YEpNDT80), YCplac111 expressing NDT80 under the regulation of an IME2 promoter (IME2-NDT80), or YCplac111 expressing NDT80 under the regulation of a CUP1 promoter (CUP1-NDT80). The transformed cells were induced to sporulate, and 6 h following the induction of sporulation, β-galactosidase activity was assayed and expressed as Miller units (filled boxes). In cells that carry CUP1-NDT80, the expression of Ndt80 was induced at time zero by the addition of CuSO₄ to the medium to a final concentration of 100 μM. After 24 h in SPM, the cultures were microscopically examined for the percentage of cells that had formed asci (open boxes).

FIG. 4.

Ime2 kinase accumulates early in meiosis and persists throughout MI and MII. (A) Ime2-Myc was immunoprecipitated at the indicated times from a culture of sporulating yeast cells, and the kinase activity was assayed by using histone H1 as a substrate. (B) Samples of the culture were stained with the DNA binding dye DAPI, and the number of cells that had completed MI was assayed by fluorescence microscopy. Two hundred cells were counted at each time point, and cells displaying more than one DAPI-staining body were counted as past MI.

FIG. 5.

Ime2 phosphorylates Ndt80 in vitro. (A) A C terminally truncated but functional version of Ime2 (Ime2Δ175) was immunoprecipitated from sporulating yeast cells, and its kinase activity was assayed in vitro. Lanes 1 to 4 show an autoradiogram (Autorad) of the kinase reactions, while lanes 5 to 8 show Coomassie staining of the same gel. Ime2Δ175 kinase activity was assayed against E. coli-expressed Mbp-Ndt80 (lanes 1 and 5), Mbp (lanes 2 and 6), or no added substrate (lanes 3 and 7). The substrate, Mbp-Ndt80, was also assayed for intrinsic kinase activity (lanes 4 and 8). Molecular weights of markers (lane 9) are indicated in thousands. Mbp-Ndt80 was not phosphorylated by a kinase-dead mutant form of Ime2 (Ime2K⁹⁷R) that was immunoprecipitated from yeast. Mbp-Ndt80 was treated with Ime2K⁹⁷R (lane 10) or Ime2Δ175 (lane 11), and the kinase assays were separated on a 10% polyacrylamide gel and visualized by autoradiography. (B) Ndt80 lacking 167 amino acids from its C terminus was expressed under the regulation of an IME2 promoter in otherwise wild-type diploid cells. Expression of the truncated protein was monitored by Western blot analysis.

FIG. 7.

Phosphorylation of Ndt80 by Ime2 is not required for Ndt80 to enter the nucleus. An NDT80-GFP fusion was expressed under the regulation of an IME2 promoter in an IME2/IME2 strain or an ime2/ime2 mutant strain or from a CUP1 promoter in an IME2/IME2 strain or an ime2/ime2 mutant strain. Samples of the cultures were taken at 0 and 4 h after induction of sporulation, and unfixed cells were examined by fluorescence microscopy to determine the localization of Ndt80-GFP fusions. Two hundred cells per sample were scored. CUP1-NDT80 was induced at time zero by addition of CuSO₄ to a final concentration of 100 μM.

FIG.8.

Chromatin immunoprecipitation (ChIP) indicates that IME2 is required for Ndt80 to effectively interact with a middle sporulation gene promoter. (A) NDT80-HA was expressed under the regulation of a CUP1 promoter that was induced at the zero time point by addition of 100 μM CuSO₄ (left side) or an IME2 promoter (right side) in either an IME2/IME2 diploid strain (top) or an ime2/ime2 diploid strain (bottom). The strains were inoculated in SPM, and samples of the culture were fixed in formaldehyde at the indicated time points. Following immunoprecipitation with anti-HA antibodies, whole-cell extracts (WCE) and immunoprecipitates (IP) were assayed by PCR for the presence of NDT80 promoter DNA (upper band in all panels) or the ARS305 sequence as a control (lower band). Ndt80-HA accumulation was assayed by probing samples of each extract with anti-HA antibodies. To control for loading, the blots were also probed with an anti-PSTAIRE antibody that recognizes both Cdc28 and Pho85. (B) DSY1289 (IME2/IME2) and DSY1299 (ime2/ime2) were assayed for Tbp1 binding to the TATA box sequence of the ACT1 promoter. Both strains were induced to sporulate in the presence of 100 μM CuSO₄. At the indicated time points, cells were fixed with formaldehyde and the extracts were subjected to immunoprecipitation with anti-Tbp1 serum or preimmune serum. Both the immunoprecipitates (IP) and extracts (WCE) were assayed for the presence of ACT1 promoter DNA or the control DNA ARS305. Samples of each extract were assayed for the presence of Tbp1 by Western blotting with anti-Tbp1 antiserum. As in panel A, PSTAIRE antibody was used as a control for sample loading.