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First published online 5 August 2008
doi: 10.1242/jcs.026286

Journal of Cell Science 121, 2824-2832 (2008)
Published by The Company of Biologists 2008
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Cdk5 kinase regulates the association between adaptor protein Bem1 and GEF Cdc24 in the fungus Ustilago maydis

Isabel Alvarez-Tabarés* and José Pérez-Martín

Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología-CSIC, 28049 Madrid, Spain


Figure 1
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  		Fig. 1. U. maydis Cdc24 and Bem1. (A) Scheme of the various protein domains in Cdc24-like proteins from Saccharomyces cerevisiae (ScCdc24, Accession number NP009359.1), Schizosaccharomyces pombe (SpScd1, Accession number NP594221.1) and Ustilago maydis (UmCdc24, Accession number XP758569.1). E values for similarity between the predicted domains are indicated. (B) Schematic representation of the domain architecture of Bem1 proteins in different yeast. ScBem1: Saccharomyces cerevisiae Bem1, Accession number NP009759.1; SpScd2: Schizosaccharomyces pombe Scd2, Accession number NP593744.1; UmBem1: Ustilago maydis Bem1, Accession number XP761858.1. The E values for the predicted domains are indicated within the boxes. (C) Serial tenfold dilutions of wild-type (wt; FB1), bem1nar1 (UMP83) and cdc24nar1 (UMI20) cells were spotted onto solid minimal medium (MMNO3, non-repressive conditions) and solid rich medium (YPD, repressive conditions). Repression of cdc24 affected the ability of the cells to produce colonies whereas repression of bem1 did not. (D) DAPI/calcofluor (D/CF) staining of cells of the indicated genotypes after incubation under repressive conditions (ammonium-containing medium) for 12 hours. bem1nar1 and cdc24nar1 cells lost their polarity, became rounded and displayed septa at the equator of the cell. Note that in bem1nar1 cells, nuclear division continued (arrowheads indicate nuclei) despite the defect in cell separation. Scale bars: 10 µm.

 

Figure 2
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  		Fig. 2. Subcellular localization and genetic interactions of Bem1 and Cdc24. (A) Consecutive cyan and yellow images of bem1-CFP cdc24-YFP (UMI67) cells were captured to reveal colocalization of Bem1 and Cdc24 at the cell poles. (B) cdc24nar1 bem1-GFP (UMI100) and bem1nar1 cdc24-GFP (UMI80) cells were incubated under repressive conditions (YPD) for 12 hours. Cdc24-GFP association with the cell tips was dependent on Bem1, whereas Bem1-GFP association with the cell tips was independent of Cdc24. Arrowheads indicate bem1-GFP localization at the cell tip in cdc24nar1 cells. (C) DAPI/calcofluor (D/CF) staining of Pcrg1:bem1 (UMP68) and Pcrg1:cdc24 (UMP80) cells grown in inductive conditions (YPA) for 12 hours. Overexpression of bem1 and cdc24 results in hyperpolarized bud growth. (D) cdc24nar1 Pcrg1:bem1 (UMI46) and bem1nar1 Pcrg1:cdc24 (UMI73) cells were incubated in YPA for 12 hours and stained with DAPI/calcofluor (D/CF). The hyperpolarized growth induced by bem1 overexpression was dependent on cdc24. Overexpression of cdc24 results in hyperpolarized growth even when bem1 was repressed. Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Interaction between Bem1 and Cdc24. (A) For immunoprecipitation (IP) analysis, whole cell extracts (WCEs) were prepared from the indicated strains. HA and MYC immunoprecipitates were isolated and blotted with antibodies that recognize the HA and MYC epitopes. Bem1 and Cdc24 coimmunoprecipitated. (B) Bimolecular fluorescence complementation (BiFC) assay. No YFP signal was detected in the negative controls NYFP-bem1 (UMI102) and cdc24-CYFP (UMI126) cells, whereas a tip-associated YFP signal was clearly seen in NYFP-bem1 cdc24-CYFP (UMI127) indicating that Bem1 and Cdc24 interacted at the cell tip. Scale bar: 10 µm.

 

Figure 4
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  		Fig. 4. Relationship between Cdk5 and Bem1. (A) cdc24-GFP (UMI29) and cdk5ts cdc24-GFP (UMI30) cells were grown in CMD at 34°C for 4 hours. Cell tip association of Cdc24-GFP is dependent on Cdk5 function. (B) bem1-GFP (UMI64) and cdk5ts bem1-GFP (UMI65) were grown as in A. Note that Bem1-GFP localization in the tip was independent of Cdk5 function. (C) cdk5ts (SONU99), Pcrg1:bem1 cdk5ts (UMP78) and Pcrg1:cdc24 cdk5ts (UMP82) cells were grown in YPA at 34°C for 6 hours and 21 hours. cdk5ts cells were lysed after 21 hours at 34°C. Overexpression of bem1 and cdc24 in cdk5ts cells rescued the polarity loss defect typical of cdk5ts cells. (D) Data from C after 6 hours of growth. bem1 and cdc24 overexpression in cdk5ts cells resulted in 90% and 96% of polarized cells, respectively. 100 cells were scored in each of 2 independent experiments. Results are mean ± s.d. Scale bars: 15 µm.

 

Figure 5
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  		Fig. 5. Interaction between Cdk5, Bem1 and Cdc24. (A) For immunoprecipitation analysis, whole cell extracts (WCEs) were prepared from cdk5-3MYC bem1-VSV cdc24-3HA (UMI83) and cdk5ts-3MYC bem1-VSV cdc24-3HA (UMI82) cells incubated for 4 hours at 34°C. HA, MYC and VSV immunoprecipitates were isolated and blotted with antibodies to recognize the HA, MYC and VSV epitopes. Bem1 associated with Cdk5 independently of Cdk5 function. However, the formation of the Bem1-Cdc24 complex was dependent on Cdk5 function. (B) Bimolecular fluorescence complementation (BiFC) assay. Tip-associated YFP signal is clearly seen in NYFP-bem1 cdc24-CYFP (UMI127) cells but no YFP signal is detected in NYFP-bem1 cdc24-CYFP cdk5ts (UMI138) cells grown for 4 hours at 34°C. Scale bars: 15 µm.

 

Figure 6
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  		Fig. 6. Cdk5 and Bem1 interact at the cell tip and within the nucleus. (A) Consecutive green and red images of cdk5-GFP bem1-RFP (UMI95) cells were captured. Cdk5-GFP and Bem1-RFP colocalize at the cell tip. (B) Bimolecular fluorescence complementation (BiFC) assay. No YFP signal was detected in the negative controls NYFP-bem1 (UMI102), cdk5-CYFP (UMP106) and NYFP-cdc24 (UMI113) cells whereas a cytoplasmic, nuclear and tip-associated YFP signal is clearly seen in NYFP-bem1 cdk5-CYFP (UMI103) cells. Interestingly, fluorescence signal was not detected in NYFP-cdc24 cdk5-CYFP (UMI114) cells. Scale bars: 15 µm (A), 10 µm (B).

 

Figure 7
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  		Fig. 7. Bem1 is able to localize transiently within the nucleus. (A,B) bem1-GFP (UMI64) and cdc24-GFP (UMI29) cells growing in CMD were mock treated with ethanol (EtOH) or treated with LMB (100 ng/ml). At the indicated times, a portion of each culture was removed for photography. (C) Quantification of nuclear accumulation of Bem1-GFP and Cdc24-GFP upon treatment with ethanol and LMB shown in A and B. 120 cells were counted for each time point and strain in each of two independent experiments. Scale bar: 10 µm.

 

Figure 8
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  		Fig. 8. Bem1 has a functional NES. (A) Bem1 protein has a putative nuclear export sequence (NES). Point mutations to destroy this NES were introduced in Bem1 creating Bem1-NES{Delta}. The NES of PKI was cloned in Bem1-NES{Delta} resulting in Bem1-NES{Delta}+NES. These three constructs were tagged at the C-terminus with GFP. (B) bem1-GFP (UMI64), bem1-NES{Delta}-GFP (UMI136) and bem1-NES{Delta}+NES-GFP (UMI137) cells were grown in CMD. Bem1-GFP localizes at the cell tips. Bem1-NES{Delta}-GFP accumulates within the nucleus and is also observed in the cytoplasm. A faint signal of Bem1-NES{Delta}+NES-GFP is distributed in the cytoplasm without any specific accumulation. Note that bem1-NES{Delta}-GFP cells presented loss of polarity but this was rescued by introducing the extra PKI NES (bem1-NES{Delta}+NES-GFP cells). Scale bar: 10 µm.

 

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© The Company of Biologists Ltd 2008