Fission yeast Ste9, a homolog of Hct1/Cdh1 and Fizzy-related, is a novel negative regulator of cell cycle progression during G1-phase - PubMed (original) (raw)

K Kitamura et al. Mol Biol Cell. 1998 May.

Free PMC article

Abstract

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G1-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G1-phase. The role of ste9(+) in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G1-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G1/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10(ts), cells arrested in G1-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G1-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G1 progression possibly by controlling the amount of the mitotic cyclin in the G1-phase.

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Figures

Figure 1

Figure 1

Molecular cloning and characterization of ste9+. (A) Restriction enzyme map of ste9+. The top shaded box indicates the genomic region around ste9+. The arrow indicates the Ste9 open reading frame. Each WD-repeat is indicated by the black box. Restriction sites: H, _Hin_dIII; S, _Sal_I; Bc, _Bcl_I; C, _Cla_I; Xh, _Xho_I; Pv, _Pvu_II. The second line shows the disrupted ste9 allele. The thin lines in the lower part indicate various truncated genomic fragments. Their ability to rescue the ste9 mutation is indicated by (+) and (−). The nucleotide sequence of ste9+ has been submitted to the DDBJ/EMBL/GenBank databases under accession number AB001285. (B) Alignments of Ste9 and Hct1/Cdh1. Identical amino acids between two proteins are indicated by vertical lines (‖). Residues of similar property are indicated by asterisks (*).

Figure 2

Figure 2

The ste9 mutants cannot arrest in the G1-phase upon nitrogen starvation. (A) Changes in cell number (open symbols) and percent of septated cells (filled symbols) of cells cultured in EMM2−N. Wild-type (L972, circle) and Δste9 (KJ100–8C, square) strains were used. (B) Wild-type (L972, left) or Δste9 (KJ100–8C, center) strains were cultured in EMM2−N for the duration indicated and analyzed by flow cytometry. The Δrum1 (KJ153–1A, right) is a control for the G1 arrest-defective strain.

Figure 3

Figure 3

Synthetic lethality between ste9 and wee1 mutations. (A) Viability for wee1–50 Δcig2 (KJ109–5A, open square), Δste9 wee1–50 Δcig2 (KJ111–1A, filled circle), and Δste9 wee1–50 Δcig2 cells in the presence of HU (open triangle). Log-phase cultures at 23°C were shifted to 35°C at 0 h. HU was added after 2 h of incubation at a final concentration of 12 mM. (B) DNA content of wee1–50 Δcig2 (left) and Δste9 wee1–50 Δcig2 (right) at the restrictive temperature. Cells were shifted to 35°C at 0 h.

Figure 4

Figure 4

The ste9 mutant initiates mitosis from the G1-phase in the absence of S-phase. In all experiments, KJ32–2A (cdc10–129) and KJ32–1A (ste9-B36 cdc10–129) were used. (A) The double mutant of ste9 cdc10 cannot grow even at the semipermissive temperature for _cdc10_ts (32°C). Cells of cdc10 (left) or ste9 cdc10 (right) were cultured for 3 d at 32°C and 37°C. (B) Flow cytometric analysis of cdc10 (left) or ste9 cdc10 (right) at the semipermissive temperature. (C) Changes of the septation index. Asynchronous culture of cdc10 (open square) or ste9 cdc10 (filled circle) at the permissive temperature was shifted to 36°C. At the time indicated, cells were stained with calcofluor, and septated and nonseptated cells were counted. (D) DNA content of cdc10 (left) and ste9 cdc10 (right) mutants at 36°C. HU was added at the same time as the cells were shifted to 36°C. Addition of HU at 2 h at the restrictive temperature resulted in the same DNA profile (our unpublished observations). (E) “Cut” phenotype in the ste9 cdc10 double mutant at the restrictive temperature. Cells were incubated at 36°C for 6 h and stained with DAPI and calcofluor. (F) Lethal mitosis in the ste9 cdc10 double mutant occurred in the absence of S-phase. HU (final concentration 12 mM) was added to the cells of cdc10 (open bar) and ste9 cdc10 (black bar), which were then incubated at 28°C or 36°C for 4 h. Aliquots were plated onto YE and incubated at 25°C, and the number of colonies was counted after 3 d. Viability is expressed as the percent ratio of colony numbers of cells after 4 h of incubation relative to that of untreated cells at the beginning of the experiments (0 h). Sensitivity to HU of the cdc10 single mutant at 28°C was not examined, but the cdc10 cells were viable at 36°C in the presence of HU.

Figure 4

Figure 4

The ste9 mutant initiates mitosis from the G1-phase in the absence of S-phase. In all experiments, KJ32–2A (cdc10–129) and KJ32–1A (ste9-B36 cdc10–129) were used. (A) The double mutant of ste9 cdc10 cannot grow even at the semipermissive temperature for _cdc10_ts (32°C). Cells of cdc10 (left) or ste9 cdc10 (right) were cultured for 3 d at 32°C and 37°C. (B) Flow cytometric analysis of cdc10 (left) or ste9 cdc10 (right) at the semipermissive temperature. (C) Changes of the septation index. Asynchronous culture of cdc10 (open square) or ste9 cdc10 (filled circle) at the permissive temperature was shifted to 36°C. At the time indicated, cells were stained with calcofluor, and septated and nonseptated cells were counted. (D) DNA content of cdc10 (left) and ste9 cdc10 (right) mutants at 36°C. HU was added at the same time as the cells were shifted to 36°C. Addition of HU at 2 h at the restrictive temperature resulted in the same DNA profile (our unpublished observations). (E) “Cut” phenotype in the ste9 cdc10 double mutant at the restrictive temperature. Cells were incubated at 36°C for 6 h and stained with DAPI and calcofluor. (F) Lethal mitosis in the ste9 cdc10 double mutant occurred in the absence of S-phase. HU (final concentration 12 mM) was added to the cells of cdc10 (open bar) and ste9 cdc10 (black bar), which were then incubated at 28°C or 36°C for 4 h. Aliquots were plated onto YE and incubated at 25°C, and the number of colonies was counted after 3 d. Viability is expressed as the percent ratio of colony numbers of cells after 4 h of incubation relative to that of untreated cells at the beginning of the experiments (0 h). Sensitivity to HU of the cdc10 single mutant at 28°C was not examined, but the cdc10 cells were viable at 36°C in the presence of HU.

Figure 5

Figure 5

Cdc13, a mitotic B-cyclin, is not decreased in the G1-arrested ste9 mutant. Extracts were prepared from cdc10 (lanes 1–4) or ste9 cdc10 (lanes 5–8) strains. Cells were cultured at 36°C for 0 h (lanes 1 and 5), 2 h (lanes 2 and 6), 3 h (lanes 3 and 7), and 4 h (lanes 4 and 8). Western blotting was carried out using anti-Cdc13 antibody (upper panel) or anti-PSTAIRE antibody for the loading control (lower panel). The location of Cdc13 is indicated by the arrow. The lower bands (indicated by asterisk) are unknown protein cross-reacted with the anti-Cdc13 antibody.

Figure 6

Figure 6

Overexpression of Ste9 prevents mitosis and induces rereplication of the genome. (A) Changes in the Cdc13 level and the H1-histone kinase activity during Ste9-overexpression. Cells harboring the integrated nmt-ste9+ gene were cultured to mid-log phase in the presence of thiamine. At 0 h, the cells were washed and then reinoculated into fresh medium without thiamine (ON) or with thiamine (OFF). Cell extracts were prepared from the cultures at the times indicated. Note that induction from the nmt promoter requires more than 10 h (Maundrell, 1990). Immunoblotting was carried out by anti-Cdc13 antibody or by anti-PSTAIRE antibody (upper two panels). Arrow and asterisk indicate the locations of Cdc13 and cross-reacted proteins, respectively. H1-histone kinase activities of uninduced (OFF) and induced (ON) cultures were also measured (lower panel). (B) Overexpression of Ste9 inhibits cell division. Cell numbers of induced (ON) and uninduced (OFF) cultures were counted at the indicated time. (C) Changes in septation index in the same cultures described in panel B. (D) Changes in the DNA content during Ste9 overexpression. Samples were taken at the indicated time for flow cytometry. The DNA profiles of the uninduced culture at each time were the same as that of 0 h. (E) The ste9 mutation prevents rereplication of DNA due to heat inactivation of the temperature-sensitive Cdc2 protein. Log-phase cells were shifted to EMM2−N and cultured at 36°C for 6 h. Cells were plated at 0 h (open bar) and at 6 h (black bar) onto YE medium containing phloxine B. Numbers of red colonies (diploid cells) and pink colonies (haploid cells) were counted, and the percent of diploid cells was calculated. Strains: cdc2-M26 (KJ141–8A); Δste9 cdc2-M26 (KJ124–3A); Δchk1 cdc2-M26 (KJ155–1A).

Figure 7

Figure 7

Functional relationship between Ste9 and Rum1. (A) Changes of Rum1 protein level after starvation. The ste9-B36 Δcig2 rum1+-3HA strain (KJ238–7B) was transformed with a vector or the ste9+-plasmid. Both strains cultured in EMM2 were shifted to nitrogen-free medium. At the indicated time, samples were taken for immunoblotting. (B) DNA content of ste9+ or _ste9_− cells in the absence of cig2 function. Samples from the same cultures described in panel A were analyzed by flow cytometry. (C) Mutual suppression of synthetic lethality by either rum1+- or ste9+-plasmid. Cells of Δste9 wee1–50 Δcig2 (KJ111–1A, upper panel) or Δrum1 wee1–50 (KJ152–9A, lower panel) were transformed with the vector, pAL(ste9), and pREP1(rum1). Each transformant was streaked onto EMM2 plates containing 5 μM thiamine and incubated for 3 d at 34.5°C. (D) Suppression of the synthetic lethality at semipermissive temperature for cdc10 cells. Cells of Δste9 cdc10-V50 (KJ218–2B) were transformed with the vector, pAL(ste9), and pAL(rum1). Transformants were incubated at 30°C for 3 d (upper panel). Cells of Δrum1 cdc10–129 (Sp224) were transformed with the same plasmids, and then incubated at 32°C for 3 d (lower panel).

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References

    1. Amon A, Irniger S, Nasmyth K. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell. 1994;77:1037–1050. - PubMed
    1. Brandeis M, Hunt T. The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase. EMBO J. 1996;15:5280–5289. - PMC - PubMed
    1. Broek D, Bartlett R, Crawford K, Nurse P. Involvement of p34cdc2 in establishing the dependency of S phase on mitosis. Nature. 1991;349:388–393. - PubMed
    1. Bueno A, Richardson H, Reed S, Russell P. A fission yeast B-type cyclin functioning early in the cell cycle. Cell. 1991;66:149–159. - PubMed
    1. Caligiuri M, Beach D. Sct1 functions in partnership with cdc10 in a transcriptional complex that activates cell cycle ’start’ and inhibits differentiation. Cell. 1993;72:607–619. - PubMed

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