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First published online 3 January 2006
doi: 10.1242/jcs.02754

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Mdy2, a ubiquitin-like (UBL)-domain protein, is required for efficient mating in Saccharomyces cerevisiae

Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, Geb. 26.12, 40225 Düsseldorf, Germany

View larger version (29K): [in a new window]   Fig. 1. Deletion of MDY2 results in a reduction in mating efficiency. (A) Quantitative assay for the mating performance of different mdy2 deletants. Values are means of two independent experiments, and represent the percentage of diploids in the cultures. (B) Shmoo index. Cultures of mdy2 mutant (HZH686) and wild-type (W303-1A) strains were grown to log phase and treated with -factor (final concentration: 5 µM). Aliquots were taken after different times of incubation and the numbers of shmoos in samples of at least 300 cells were counted. (C) Halo assay for -factor production. 0.6% top agar was mixed with 105 indicator cells (MATa sst1). Aliquots (approximately 4x106 cells) of wild-type W303-1B (MAT) and W303-1A (MATa) wild-type and mdy2 mutant cells were spotted directly onto the top agar. The plate was incubated at 30°C for 2 days and photographed. 1. MAT mdy2 mutant cells (HZH683). 2. MAT wild-type cells (W303-1B). 3. MATa wild-type cells (W303-1A).

View larger version (50K): [in a new window]   Fig. 2. Assay for zygote formation. (A) Mating pair formation. Cultures of opposite mating type of mdy2 mutant (MATa and MAT) and wild-type strains were grown to log phase. Equal numbers of cells (3x106) of each mating type were mixed with each other. The cell pellets were then transferred onto a NC membrane on an YPD plate and incubated at 30°C. Samples were taken after various times and the numbers of mating pairs in samples of at least 800 cells were counted. The percentage of mating pairs at each time point is shown. (B) Zygote formation. The same samples were used as in A and the nuclei were stained with DAPI, and the numbers of zygotes was counted. The percentage zygote formation is shown. (C) Classification of mating pairs. Cultures of mdy2 mutant (MATa and MAT) and wild-type strains of opposite mating type were grown to log phase. Equal numbers (3x106 cells) of each mating type were mixed, pelleted, transferred onto a NC membrane on an YPD plate and incubated at 30°C. Samples were taken after 3.5 hours incubation and stained with DAPI to identify zygotes and follow the course of karyogamy. The percentage of cells in the different stages of zygote formation was determined in samples containing at least 200 zygotes.

View larger version (45K): [in a new window]   Fig. 3. Distribution of nuclei in zygotes 3.5 hours after initiation of mating. Cultures of wild-type (HZH638 and HZH639) and mdy2 mutant (HZH686 and HZH683) strains were grown to log phase. MATa and MAT cells were mixed and incubated on YPD plates for 3.5 hours to allow zygotes to form. Cells were fixed, stained with DAPI (A), and the distance between the nuclei was measured using the centre of each nucleus as reference (n=85 unbudded zygotes for each strain). Bar, 5 µm.

View larger version (25K): [in a new window]   Fig. 4. Distribution of nuclei in wild-type and mdy2 mutant zygotes 5.5 hours after initiation of mating. The mating mixtures were incubated for 5.5 hours on YPD plates, and the cells were stained with DAPI. Fields of cells containing zygotes were photographed at random for analysis, and the positions of the nuclei were determined. We categorized zygotes according to nuclear positions into one of three classes: pre-nuclear fusion zygotes (class I, top), post-nuclear fusion zygotes (class II, centre), and `zygotes' in which the haploid nuclei had undergone mitotic division (class III, bottom). 100 zygotes were counted for each cell type.

View larger version (34K): [in a new window]   Fig. 5. Determination of nuclear position in wild-type and mutant shmoos. The positions of nuclei in shmoos were classified into three groups: (i) in/at the shmoo neck (top), (ii) in the centre of the cell (centre) or (iii) opposite the mating projection (bottom). The percentage of cells in each class was calculated. n>250 shmoos for each strain. (A) Wild-type (HZH638) and mdy2 mutant strains (HZH686) were induced to form shmoos by treatment with -factor (5 µM) for 2.5 hours. Cells were fixed, stained with DAPI, and then scored for nuclear position. (B) Cultures of wild-type (W303-1A) and mdy2 mutant (HZH686) strains harbouring pGAL-GFP-NUP116 plasmids were grown to log phase and induced in 3% raffinose and 1% galactose medium for 2 hours, and then 5 µM -factor was added for another 3 hours. After brief fixation, wild-type (WT; W303-1A) and mdy2 cells were scored for GFP-Nup116 localization (n>300).

View larger version (43K): [in a new window]   Fig. 6. The mdy2 mutant shows defects in microtubule orientation and mislocalizes Kar9 in response to pheromone. (A) Cells containing pMET-GFP-TUB1 (pGX388) plasmids were grown to log phase and induced in methionine-free medium for 2 hours; then 5 µM -factor was added for another 3 hours. After brief fixation, wild-type (WT; HZH638) and mdy2 (HZH386) cells were scored for GFP-Tub1 localization (n>100). Cells were scored as having (from top to bottom): a single bundle of cytoplasmic tubules directed to the shmoo tip, a single bundle of cytoplasmic microtubules going to the shmoo tip plus other bundles oriented away from the shmoo tip, no cytoplasmic microtubules going to the shmoo tip and a spray of microtubules localized on the opposite side to the shmoo. (B) Cells containing pGAL-GFP-KAR9 were grown in 3% raffinose and 1% galactose for 2 hours, and then pheromone was added for another 3 hours. After brief fixation, wild-type (WT; W303-1A), mdy2 cells were scored for GFP-Kar9 localization (n>250). Cells were scored as having (from top to bottom): a single cortical dot, a line of localization, multiple dots forming a cap near the shmoo tip, or dispersed dots elsewhere in the cell body, possibly on misoriented microtubules.

View larger version (30K): [in a new window]   Fig. 7. Analysis of MDY2 overexpression. (A) Effect of MDY2 overexpression on mating efficiency. Wild-type strain (W303-1A) was transformed with an empty 2 µ vector (pRS426), mdy2 mutant (HZH686) were transformed with 2 µ MDY2 plasmids (pRS426-MDY2). The quantitative mating assay was conducted as described in Materials and Methods. The percentage of diploid cells formed is shown. The data are the means of two independent assays. (B) Halo assay for effect of MDY2 overexpression on -factor production. 1. MAT mdy2 mutant cells (HZH683) with an empty 2 µ vector (pRS426). 2. MAT wild-type cells (W303-1B) with 2 µ MDY2 plasmids (pRS426-MDY2). 3. MATa Wild-type cells (W303-1A). 4. MAT mdy2 mutant cells (HZH683) with 2 µ MDY2 plasmids (pRS426-MDY2). (C) Shmoo index. Wild-type strain W303-1A was transformed with the empty 2 µ vector (pRS426) and the mdy2 mutant (HZH686) was transformed with the 2 µ MDY2 plasmid (pRS426-MDY2) or pRS426. The cultures were grown in SD medium to log phase and then treated with 5 µM -factor. Samples were taken at the indicated times and the numbers of shmoos in samples of at least 300 cells were counted.

View larger version (45K): [in a new window]   Fig. 8. Localization of Mdy2. (A) Vegetatively growing cells. The mutant mdy2 (HZH686) was transformed with a plasmid encoding a functional GFP-Mdy2 fusion protein or with the empty vector as a control. Expression of the fusion gene was induced by incubating the cells in 3% raffinose and 1% galactose, and analysed by fluorescence and phase-contrast microscopy. (B) Induction with pheromone. mdy2 mutant cells were transformed with plasmids expressing a functional GFP-Mdy2 fusion protein or with the empty vector as a control. Cells were induced as above, then treated with 5 µM -factor for 2 hours, and analysed by fluorescence and phase-contrast microscopy. (C) Expression of GFP-MDY2 under its own promoter. Localization of GFP-Mdy2 expressed under the control of its own promoter. Cultures of the mdy2 mutant (HZH686) harbouring MDY2p-GFP-MDY2 (pZH152) were grown to log phase. Half of the culture was then harvested and the localization of GFP-Mdy2 fusion protein was investigated. (D) The other half was treated with 5 µM -factor for 2 hours before fusion protein localization was investigated.

View larger version (37K): [in a new window]   Fig. 9. The Mdy2-6His fusion shows no C-terminal cleavage. (A) Alignment of predicted Mdy2 sequences from residues 74-173 with S. cerevisiae ubiquitin and human GdX. Identical residues are shaded in dark grey; similar residues are shaded in light grey. (B) Expression of MDY2 constructs with C-terminal 6xHis tag is independent of -factor. Supersensitive sst2-1 (HGX132) cells carrying the functional GST-Mdy2-H6 construct (N-terminal GST tag and a C-terminal His6 tag) were assayed with 1 µM -factor and compared with identical untreated cells and cells carrying the GST vector as a negative control. The expression levels were measured by anti-6xHis immunoblot analysis. To control for correct expression, the blot was stripped and reprobed with anti-GST antibody. No anti-His signal could be detected in the GST-negative control (lane 1) and cells not expressing GST at all (lane 4). (C) Localization of Mdy2H6 by indirect immunofluorescence. mdy2 mutant (HZH686) cells, containing functional GST-Mdy2H6 on a CEN plasmid were prepared as described in Materials and Methods and stained with anti-His antibody (Qiagen) and affinity-purified goat-anti-mouse conjugated to fluorescein (Jackson ImmunoResearch). Micrographs show FITC fluorescence (-His), DAPI fluorescence and a differential interference contrast (DIC) image. Cells were photographed with Zeiss AxioCam and AxioVision.