Cdc5 interacts with the Wee1 kinase in budding yeast

Abstract

Development of a multicellular organism requires that mitosis and morphogenesis be coordinated. These processes must also be synchronized during the growth of unicellular organisms. In the yeast Saccharomyces cerevisiae, mitosis is dependent on the prior growth of a daughter cell in the form of a bud. Overexpression of wild-type Polo-like kinase Cdc5 or a catalytically inactive form resulted in the formation of multinucleate cells in budding yeast. Immunofluorescence analysis of these multinulceate cells showed that mitosis and bud formation were no longer linked. Others have shown that Swe1 is required for coupling mitosis to bud formation during a perturbed cell cycle. When the normal pathway of bud formation is perturbed, Swe1 functions to delay mitosis through negative regulation of Clb/Cdk. In cells lacking Swe1, multinucleate cells are formed in response to delays in bud formation. Affinity purification, two-hybrid analysis, and mutant characterization results suggested that Cdc5 and Swe1 interact. From these results, we conclude that multinucleate formation in response to Cdc5 overexpression is linked to titration of Swe1 function. These results also suggest that Cdc5 may be a negative regulator of Swe1.

FIG. 1

Cells overexpressing CDC5 or cdc5N209A are multinucleate. To induce Cdc5 (A) or cdc5-N209A (B), 2% galactose was added to mid-logarithmic-phase cultures of strains JC256 (GAL-CDC5) and C1039 (GAL-cdc5N209A GFP-TUB1) for the indicated times. To induce GFP-Tub1 expression, the same cells were then resuspended in SC-methionine plus galactose for 2 h. The cells were fixed and stained with DAPI to visualize nuclei. It should be noted that the frequency of multinucleate cells as visualized here does not reflect the true distribution in these cultures. Rather, these particular fields were chosen in order to demonstrate the variety of multinucleate cells in the culture. Bars, 10 μm.

FIG. 2

Cdc5 interacts with Swe1. (A) Two-hybrid assay performed with pDB-Cdc5(1–705), pDB-Cdc5(340–705), pAD-Swe1(173–819), and pAD-Swe1(400–819). pDB (Ga14 DNA binding domain [DB] fusion vector) and pAD (Ga14 activation domain [AD] fusion vector) were used as negative controls. The HIS3 and ADE2 reporter strain PJ69-4a was used, and transformants were tested for growth on medium lacking uracil, leucine, histidine, and adenine. + and − indicates growth and no growth, respectively, after 5 days at 30°C. (B) GST and GST-Swe1 were incubated with yeast extract containing Cdc5 fused to ProA, derived from strain C199 (CDC5-ProA). After 2 h of incubation, beads were washed extensively and the bound proteins (lanes B) were resolved by SDS-PAGE and analyzed by immunoblotting; 10% of the extract added to each incubation was loaded in the input lane (I).

FIG. 3

Mitosis is unlinked from bud formation in cdh1/hct1 swe1 cells. (A) Visualization of elongated and disassembled spindles in unbudded and small-budded multinucleate cdh1/hct1 swe1 cells. Cultures of C968 (cdh1/hct1 swe1 MET3-GFP-TUB1) were grown to mid-log phase in YPD and transferred to SC lacking methionine for 1 h to induce GFP-Tub1. Arrows mark a multinucleate unbudded cell with an elongated spindle (i), a multinucleate small-budded cell with a disassembled spindle (ii), and a cell undergoing a normal mitosis (iii). (Inset) A rare cell type (0.1% of cdh1/hct1 swe1 GFP-TUB1 cells) in which the mother and its attached daughter are both undergoing mitosis. (B) The cdh1/hct1 swe1 double mutant grows slower than the single mutants at 37°C. Strains C895 (cdh1/hct1 swe1), C798 (swe1), C810 (cdh1/hct1), and W303a (wild type [WT]) were streaked on YPD plates and incubated at 30 or 37°C for 5 days. (C) Video sequence of spindle elongation and disassembly during mitosis in strain C968 (cdh1/hct1 swe1 MET3-GFP-TUB1). Cultures of C968 were grown to mid-log phase in YPD and transferred to SC lacking methionine for 2 h to induce GFP-Tub1. Following induction, they were placed on a slide with a thin agarose pad. A Z series of eight focal planes was collected over 8 s and projected onto a single two-dimensional image. Z series were collected every 2 min for 3 h. An unbudded cell undergoing spindle elongation (interval from 34 to 68 min) and disassembly (interval from 68 to 74 min) is shown (middle cell in the cartoon). The cell above and the cell below the middle cell in the cartoon elongated and aligned their spindles through the bud neck in a wild-type manner. Bars: (A) 10 μm; (C) 2.5 μm.

FIG. 4

cdc5N209A overproduction suppresses Swe1-dependent phenotypes. (A) Strains C508 (hsl1 GAL-cdc5N209A [DIC and FACS]) and C618 (hsl7 GAL-cdc5N209A [FACS only]) were grown to mid-logarithmic phase in raffinose. Galactose was added to 2% for the times indicated. Strains MAY1 (hsl1 [DIC and FACS]), MAY2 (hsl1 swe1 [DIC and FACS]), and W303a (wild type [WT] [FACS only]) were included for comparison. The cells were viewed by DIC (top); they were also stained with propidium iodide and analyzed by FACS (bottom) to evaluate cellular DNA contents. (B) Constitutive expression of SWE1 is toxic and induces elongated buds. Both of these phenotypes were suppressed by cdc5-N209A overproduction. Strains C474 (GAL-Swe1) and C499 (GAL-SWE1 GAL-cdc5-N209A) were streaked on rich plates containing either 2% glucose (repressing conditions) or 2% galactose (inducing conditions) for 5 days (left). Galactose was added to mid-logarithmic-phase cultures of the same strains for 4 h to induce Swe1 (GAL-SWE1) or Swe1 and Cdc5 (GAL-SWE1 GAL-cdc5-N209A) and viewed by DIC (right). (C) Strains C798 (swe1), C745 [cdc5(msd2-1)] and C667 [swe1 cdc5(msd2-1)] were incubated on rich medium at 23 or 30°C for 5 days. (D) Strain JC278 (GAL-cdc5N209A) was grown to mid-logarithmic phase in raffinose. Galactose was added to 2% for the times indicated. The cells were viewed by DIC (left); they were also stained with propidium iodide and analyzed by FACS (middle) to evaluate cellular DNA contents. The same strain was also streaked on rich plates containing either 2% glucose (repressing conditions) or 2% galactose (inducing conditions) for 5 days (right). Bars: 10 μm.

FIG. 5

Swe1 is found at one or two distinct spots in cells overexpressing CDC5 or cdc5N209A. (A) Strain C573 (GAL-CDC5-HA SWE1-GFP) was grown to mid-logarithmic phase in raffinose. Galactose was added to 2% for 2 h. (B) Strain C572 (GAL-cdc5-N209A-HA SWE1-GFP) was grown to mid-logarithmic phase in raffinose. Galactose was added to 2% for 2 h. Bars: 10 μm.

FIG. 6

Swe1 is found associated with cdc5-N209A at the SPBs of cells overexpressing cdc5-N209A. Strain CH572 (GAL-cdc5N209A-HA SWE1-GFP) was grown to mid-logarithmic phase in raffinose. Galactose was added to 2% for 2 h. The cells were fixed and stained for cdc5-N209A-HA (A) or Tub1 (B). Swe1-GFP was visualized by direct fluorescence. Bars: 10 μm.

FIG. 7

Swe1 is modified in response to Cdc5 or cdc5N209A overproduction. (A) Strains CH473 (GAL-CDC5 SWE1-ProA) and CH459 (GAL-cdc5N209A SWE1-ProA) were grown in raffinose and shifted to 2% galactose. Samples for immunoblot analysis were taken just before (−) and 3 h after (+) addition of galactose. (B) Strains CH507 (hsl1 GAL-CDC5) and CH508 (hsl1 GAL-cdc5N209A) were grown in raffinose and shifted to 2% galactose. Samples for immunoblot analysis were taken just before (−) and 3 h after (+) addition of galactose. (C) Strain JC256 (GAL-CDC5) was grown to mid-logarithmic phase in raffinose. Galactose was added to 2% for the times indicated. The cells were stained with propidium iodide and analyzed by FACS to evaluate cellular DNA contents.