A positive regulator of mitosis, Sok2, functions as a negative regulator of meiosis in Saccharomyces cerevisiae - PubMed (original) (raw)

A positive regulator of mitosis, Sok2, functions as a negative regulator of meiosis in Saccharomyces cerevisiae

G Shenhar et al. Mol Cell Biol. 2001 Mar.

Abstract

The choice between meiosis and alternative developmental pathways in budding yeast depends on the expression and activity of transcriptional activator Ime1. The transcription of IME1 is repressed in the presence of glucose, and a low basal level of IME1 RNA is observed in vegetative cultures with acetate as the sole carbon source. IREu, a 32-bp element in the IME1 promoter, exhibits upstream activation sequence activity depending on Msn2 and -4 and the presence of acetate. We show that in the presence of glucose IREu functions as a negative element and that Sok2 mediates this repression activity. We show that Sok2 associates with Msn2. Sok2 functions as a general repressor whose availability and activity depend on glucose. The activity of Sok2 as a repressor depends on phosphorylation of T598 by protein kinase A (PKA). Relief of repression of Sok2 depends on both the N-terminal domain of Sok2 and Ime1. In the absence of glucose and the presence of Ime1 Sok2 is converted to a weak activator. Overexpression of Sok2 or mild expression of Sok2 with its N-terminal domain deleted leads to a decrease in sporulation. Previously it was reported that overexpression of Sok2 suppresses the growth defect resulting from a temperature-sensitive PKA; thus Sok2 has a positive role in mitosis. We show that Candida albicans Efg1, a homolog of Sok2, complements sok2 Delta in repressing IREu. Our results demonstrate that Sok2, a positive regulator of mitosis, and Efg1, a positive regulator of filamentation, function as negative regulators of meiosis. We suggest that cells use the same regulators with opposing effects to ensure that meiosis will be an alternative to mitosis.

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Figures

FIG. 1

FIG. 1

IREu serves as a URS element in SD and as a UAS element in SA. Strain Y422 carrying various lacZ plasmids either on a CEN vector (YCp plasmid) or integrated in the genomic LEU2 gene (YIp plasmid) was grown in PSP2 to 107 cells/ml. Cells were washed once in water and resuspended in SPM. Samples were taken to extract proteins and measure lacZ levels after 0 (SA), 3, and 6 h in SPM. In addition, proteins were extracted from 107 cells/ml grown in glucose-containing media (SD). The level of β-Gal is given in Miller units. The results are the averages of three or four independent transformants. Standard deviations were less than 10%. The sequence of IREu and its homology to the known SCB and STRE elements are given. Dotted boxes, sequences of IME1; open box, nested deletion; SCB-STRE box, IREu element.

FIG. 2

FIG. 2

Positive and negative regulators of IREu. Expression of the IREu-his4-lacZ chimeric gene integrated at LEU2 by 107 cells/ml grown in either SD or SA was measured. The level of β-Gal is given in Miller units. The results are the averages of three or four independent transformants. Standard deviations were less than 10%. The following isogenic strains were used: Y1161 (wt; column 1), Y1132 (_msn2_Δ _msn4_Δ; column 2), Y1162 (_sok2_Δ; column 3), Y1170 (_msn2_Δ _msn4_Δ _sok2_Δ; column 6), Y1086 (_ime1_Δ; column 8), Y1162 carrying on a 2μm SOK2 plasmid (YEp2432; column 4) or sok2T598A (YEp2452; column 5) and Y1170 carrying on a 2μm sok2T598A plasmid (YEp2573; column 7).

FIG. 3

FIG. 3

Expression of Sok2 is reduced in acetate media. Proteins extracted from 107 cells/ml were subjected to immunoblot analysis using antibodies directed against HA. (a) Strain Y422 carrying on a 2μm plasmid either pSOK2-3xHA-SOK2 (YEp2432) (lanes 1 and 2) or pCDC28-3xHA-SOK2 (YEp2486) (lanes 3 and 4). Cells were grown in either SD (lanes 1 and 3) or SA (lanes 2 and 4). (b) Strains. Y1064 (wt) (lanes 5 to 8) and its isogenic Y1078 (cdc25-2) (lanes 9 to 12) carrying pSOK2-3xHA-Sok2 (YEp2432). Cells were grown in either SD (lanes 5, 6, 9, and 10) or SA (lanes 7, 8, 11, and 12) at 25°C (lanes 5, 7, 9, and 11) or shifted to 37°C for 4 h (lanes 6, 8, 10, and 12).

FIG. 4

FIG. 4

Sok2 is a transcriptional repressor whose activity depends on PKA. Proteins were extracted from cells grown in either SD or SA to 107 cells/ml. The level of β-Gal was measured, and relative levels are given. The results are the averages of three or four independent transformants. Standard deviations were less than 10%. Strains used are Y1064 (wt; columns 1, 2, and 4), Y1078 (cdc25-2; column 3), and Y1075 (_ime1_Δ; column 5) carrying UASGAL1-UASHIS4-his4-lacZ (YIp2218) integrated at LEU2. These strains were mated to wt (Y1065), cdc25-2 (Y1093), and _ime1_Δ (Y1076) strains, respectively. They also carried the following 2μm TRP1 plasmids: pADH1-gal4(bd) (pAS2 [14]) (column 1), pADH1-Gal4bd-SOK2 (YEp2314) (columns 2, 3, and 5), and pADH1-gal4(bd)-sok2T598A (YEp2530) (column 4).

FIG. 5

FIG. 5

Sok2 is a phosphoprotein. Proteins were extracted from cells incubated with 32P for 30 min. Following immunoprecipitation with antibodies directed against HA, proteins were separated by SDS–10% PAGE and exposed to X-ray film. The strain used is Y422 carrying a vector (pRS426 [40]; lane 1; control), pCDC28-3x-HA-SOK2 (YEp2486; lane 2), or pCDC28-3xHA-sok2T598A (YEp2558; lane 3).

FIG. 6

FIG. 6

Sok2 associates with Msn2. Coimmunoprecipitation of GST-Msn2 and HA-Sok2 is shown. Proteins were extracted from logarithmic cultures grown in SD. Anti-HA or anti-GST immune complexes were prepared from strain Y422 carrying plasmids YEp2382 (HA-Sok2) (lanes 1, 3, 4, and 6) and YEp2536 (GST-Msn2) (lanes 2, 3, 5, and 6). Proteins were separated by SDS–8% PAGE, and immunoblotting was done with anti-HA for the anti-GST immune complexes and with anti-GST for the anti-HA immune complexes. Following the stripping of bound antibodies, a second immunoblotting was performed using anti-GST and anti-HA, respectively. (A) probing with anti- GST; (B) probing with anti-HA. IP, immunoprecipitation.

FIG. 7

FIG. 7

The N-terminal domain of Sok2 is required to relieve repression in SA. Shown is the expression of IREu-his4-lacZ chimeric gene integrated at LEU2. Cells were grown in SD or SA to 0.5 × 107 (wt) and 1 × 107 cells/ml (cdc25-2 strain) at 25°C and shifted to 37°C for 4 h. The level of β-Gal is given in Miller units. The results are the averages of three or four independent transformants. Standard deviations were less than 10%. The isogenic strains used were Y1161 (wt) and Y1087a (cdc25-2) carrying on a 2μm plasmid a vector (YEpLac112 [9]) or pSOK2-sok2(248-785) (YEp1784).

FIG. 8

FIG. 8

The role of PKA in determining the choice between mitosis and meiosis. Glucose increases the level of cAMP and consequently the activity of PKA. High activity of PKA leads to phosphorylation of several substrates, including Msn2 and -4 and Sok2. Msn2 and -4 are negative regulators of mitosis and positive regulators of meiosis. We suggest that nonphosphorylated Msn2 and -4 activate meiosis and repress mitosis, whereas PKA-phosphorylated Msn2 and -4 are neither inhibitors of mitosis nor activators of meiosis. The opposite relations are observed for Sok2, which is a negative regulator of meiosis and a positive regulator of mitosis. A PKA-phosphorylated Sok2 inhibits meiosis and promotes mitosis, whereas a nonphosphorylated Sok2 inhibits mitosis and promotes meiosis.

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