1. Cell Biology

Protein phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae

  1. Dilara Kocakaplan
  2. HΓΌseyin KarabΓΌrk
  3. Cansu Dilege
  4. Idil KirdΓΆk
  5. Seyma Nur Bektas
  6. Ayse Koca Caydasi  Is a corresponding author
  1. Department of Molecular Biology and Genetics, Koç University, Turkey
https://doi.org/10.7554/eLife.72833
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Cite this article
  1. Dilara Kocakaplan
  2. HΓΌseyin KarabΓΌrk
  3. Cansu Dilege
  4. Idil KirdΓΆk
  5. Seyma Nur Bektas
  6. Ayse Koca Caydasi
(2021)
Protein phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae
eLife 10:e72833.
https://doi.org/10.7554/eLife.72833
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7 figures, 2 tables and 1 additional file

Figures

Figure 1
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Bud14 deletion rescues growth of cells with impaired mitotic exit.

(A) bud14βˆ† cells rescue the synthetic lethality of lte1βˆ† spo12βˆ† cells. (B) bud14βˆ† cells rescue the lethality of lte1βˆ† cells at 18Β°C. (C) Comparison of growth rescue of mitotic exit network temperature-sensitive (MEN-ts) mutants upon deletion of BUD14 and BFA1. Serial dilutions of indicated strains were spotted on indicated plates and grown at given temperatures. 5-Fluoroorotic acid (5-FOA) plates negatively select for the URA3-based plasmids (pRS316 containing the LTE1 in A and B, pRS316 containing the wild-type gene copy of the corresponding MEN mutant in C). Thus, only cells that have lost these plasmids can grow on 5-FOA plates where genetic interactions can be observed.

Figure 2 with 1 supplement
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bud14βˆ† cells are spindle position checkpoint (SPOC) deficient.

(A, B) Endpoint analysis of SPOC integrity of indicated yeast strains in kar9βˆ† (A) and dyn1βˆ† (B) background. Cells were fixed with ethanol and stained with DAPI. Cells with normally aligned nuclei, mispositioned nuclei, and multinucleated cells that failed to correctly position their spindle before mitotic exit were counted by microscopy and their SPOC deficiency index percentages were plotted, where SPOC deficiency index = % cells with multinucleation Γ· % cells with mispositioned nuclei Γ— 10. Graphs are average of three independent experiments. A minimum of 100 cells were counted from each strain in each experiment. Error bars show standard deviation. Two-tailed Student’s t-test was applied. **p<0.01, ***p<0.001. (C) Single-cell analysis of SPOC integrity in indicated strains. Duration of anaphase in cells with misaligned and normally aligned spindles was calculated as explained in the text and plotted as dotplots. Data points in red indicate the cells in which the spindle did not break down during the time-lapse movie. In this case, plotted values are the time duration during which these cells were observed in anaphase through the time-lapse movie. Consequently, the actual anaphase duration is greater than the value plotted in red. This fact is emphasized in the red part of the y-axis, indicated above the red dashed line, with addition of the β€˜>’ symbol before the y-axis values. Green dash line indicates the mean value of the anaphase duration in cells with normally aligned spindles. One-way ANOVA with uncorrected Fisher’s LSD was applied for statistical analysis. ***p<0.001, ****p<0.0001. n: sample size. All pairwise comparisons and descriptive statistics are shown in the corresponding source data files, whereas only comparisons of normal and misaligned spindles are shown in the figure.

Figure 2β€”source data 1

Numerical data and statistics for Figure 2A.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig2-data1-v2.xlsx
Figure 2β€”source data 2

Numerical data and statistics for Figure 2B.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig2-data2-v2.xlsx
Figure 2β€”source data 3

Numerical data and statistics for Figure 2C.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig2-data3-v2.xlsx
Figure 2β€”figure supplement 1
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Bud14 does not influence Kin4 function.

(A) Serial dilutions of indicated strains were spotted on glucose-containing (YPD) and galactose-containing (YP Raf/Gal) agar plates. Gal1-Kin4 overexpression is induced on galactose-containing plates. (B) Logarithmic growing cultures bearing Kin4-GFP and Spc42-eqFP were treated with nocodazole for 2 hr and Kin4 spindle pole body (SPB) and cortex localization was analyzed by microscopy. rts1βˆ† cells were used as a control in which Kin4 fails to localize to SPBs and cortex. Graph is an average of three independent experiments. Error bars are standard deviation. At least 100 cells were counted from each sample in each experiment. Scale bar: 2 Β΅m. (C) SPB localization of Kin4-GFP was analyzed in logarithmic growing lte1βˆ† and lte1βˆ† bud14βˆ† cultures. Spc42-eqFP and mCherry-Tub1 served as SPB and spindle markers. Only cells in anaphase were considered. Graphs were plotted as described in B. (D) Logarithmic growing cultures bearing Kin4-6HA and the indicated gene deletions were treated with nocodazole for 2.5 hr. Kin4 mobility shift was analyzed by western blotting using anti-HA antibodies. rts1βˆ† served as a control for hyperphosphorylated Kin4.

Figure 2β€”figure supplement 1β€”source data 1

Numerical data for Figure 2β€”figure supplement 1B.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig2-figsupp1-data1-v2.xlsx
Figure 2β€”figure supplement 1β€”source data 2

Numerical data for Figure 2β€”figure supplement 1C.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig2-figsupp1-data2-v2.xlsx
Figure 3 with 1 supplement
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Function of Bud14 in actin regulation is dispensable for spindle position checkpoint (SPOC).

(A) Endpoint analysis of SPOC deficiency index in bud14βˆ† kar9βˆ† cells carrying URA3-based empty plasmid (empty) or BUD14-containing URA3-based plasmids (BUD14 and bud14-5A). Graphs are average of three independent experiments. A minimum of 100 cells were counted from each strain in each experiment. Error bars show standard deviation. *p<0.05 according to Student’s t-test. (B) Serial dilutions of indicated strains bearing URA3-based empty plasmid (not indicated on figure) or BUD14-containing URA3-based plasmids (BUD14 and bud14-5A) were spotted on SC-URA plate and grown at indicated temperatures. (C) Endpoint analysis of SPOC deficiency index in indicated cell types. Graphs are average of three independent experiments. A minimum of 100 cells were counted from each strain in each experiment. Error bars show standard deviation. ***p<0.001 according to Student’s t-test. (D, E) Serial dilutions of indicated strains bearing LTE1 on URA3-based pRS316 plasmid were spotted on 5-fluoroorotic acid (5-FOA) and SC plates and grown at indicated temperatures. 5-FOA negatively selects for URA3-containing plasmids, thus cells lose their pRS316-LTE1 plasmids on 5-FOA plates and genetic interactions can be observed on this plate.

Figure 3β€”source data 1

Numerical data and statistics for Figure 3A.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig3-data1-v2.xlsx
Figure 3β€”source data 2

Numerical data and statistics for Figure 3C.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig3-data2-v2.xlsx
Figure 3β€”figure supplement 1
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Expression and Glc7 binding of Bud14 mutants.

(A) Analysis of BUD14 expression in cells bearing pMK60 (pRS416-endogenous-BUD14-promoter-GFP-BUD14), pDKY001 (pRS416-endogenous-BUD14-promoter-GFP-bud14-5A), pHK002 (pRS416-endogenous-BUD14-promoter-GFP-bud14-F379A), pDKY003 (pRS416-endogenous-BUD14-promoter-GFP-bud14-Ξ”SH3) Bud14 was detected using anti-GFP antibodies. (B) Interaction of Glc7 with Bud14 and Bud14 mutants in yeast two hybrid system.

Figure 3β€”figure supplement 1β€”source data 1

Labeled uncropped blot images for Figure 3β€”figure supplement 1A.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig3-figsupp1-data1-v2.pdf
Figure 3β€”figure supplement 1β€”source data 2

Raw scans of the blot images for (A) Figure 3β€”figure supplement 1β€”source data 1, anti-GFP blot, upper panel, and (B) Figure 3β€”figure supplement 1β€”source data 1, anti-tublin blot, lower panel.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig3-figsupp1-data2-v2.zip
Figure 4 with 1 supplement
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Glc7-Bud14 interaction is required for spindle position checkpoint (SPOC) regulatory function of Bud14.

(A) Serial dilutions of indicated strains bearing URA3-based empty plasmid (not indicated on figure) or BUD14-containing URA3-based plasmids (BUD14 and bud14-F379A) were spotted on SC-URA plate and grown at indicated temperatures. (B) SPOC deficiency indexes of indicated strains carrying URA3-based empty plasmid (empty) or BUD14-containing URA3-based plasmids (BUD14 and bud14-F379A). Graphs are average of three independent experiments. A minimum of 100 cells were counted from each strain in each experiment. Error bars show standard deviation. *p<0.05 according to Student’s t-test. (C) Serial dilutions of indicated strains were spotted on 5-fluoroorotic acid (5-FOA) plate and grown at indicated temperatures. Cells that contain LTE1 on a URA3-based plasmid are indicated with pRS316-LTE1. (D) SPOC deficiency indexes of indicated strains. *p<0.05 according to Student’s t-test.

Figure 4β€”source data 1

Numerical data and statistics for Figure 4B.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig4-data1-v2.xlsx
Figure 4β€”source data 2

Numerical data and statistics for Figure 4D.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig4-data2-v2.xlsx
Figure 4β€”figure supplement 1
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Assessment of glc7-12 inactivation at different temperatures.

(A) Serial dilutions of indicated strains were spotted on YPD and YPD-containing 0.01 M hydroxyurea (HU) and grown at indicated temperatures. The plates then were incubated at given temperatures for 2–3 days. (B) Analysis of cell morphology of GLC7 and glc7-12 cells at indicated temperatures. Liquid cultures were grown to log-phase at 23Β°C, shifted to indicated temperatures and further grown for 4 hr. Cells were fixed with ethanol and stained with DAPI. Cells were counted by microscopy and percentages of cells with large buds and single DAPI were plotted. A minimum of 100 cells were counted from each strain.

Figure 5 with 2 supplements
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Yeast two hybrid analysis of Bud14 and Bud14 mutants with spindle position checkpoint (SPOC) proteins.

(A) Bud14 interacts with Bfa1 but not with Bub2 or Kin4. (B) Bud14-Bfa1 interaction is dependent on Bub2. (C) Bfa1-Bud14 interaction is reduced in Bud14βˆ†SH3 mutant. SGY37 was co-transformed with indicated plasmids. Empty plasmids served as a control for any self-activation. Kel1 served as a positive control. Cells were grown for 2 days on selective agar plates before overlay. Blue color formation was monitored as an indication of protein-protein interaction.

Figure 5β€”figure supplement 1
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Analysis of Glc7 interaction with spindle position checkpoint (SPOC) components.

Yeast two hybrid assays of Glc7 with Bfa1, Bub2, and Kin4. Bud14 was included as a positive control.

Figure 5β€”figure supplement 2
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SH3 domain of Bud14 is important for Bud14 function in spindle position checkpoint (SPOC).

(A) Endpoint analysis of SPOC deficiency index in bud14βˆ† kar9βˆ† cells carrying URA3-based empty plasmid (empty) or BUD14-containing URA3-based plasmids (BUD14, bud14-Ξ”SH3). Values for controls (empty and BUD14) are identical to those shown in Figure 3A as experiments were performed together. Graphs are average of three independent experiments. A minimum of 100 cells were counted from each strain in each experiment. Error bars show standard deviation. *p<0.05 according to Student’s t-test. (B) Serial dilutions of indicated strains bearing URA3-based empty plasmid (not indicated on figure) or BUD14-containing URA3-based plasmids (BUD14 and bud14-Ξ”SH3) were spotted on SC-URA plate and grown at indicated temperatures. Controls (lanes 1 and 2) are identical to those shown in Figure 3B as all drops come from the same agar plate.

Figure 5β€”figure supplement 2β€”source data 1

Numerical data for Figure 5β€”figure supplement 2A.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig5-figsupp2-data1-v2.xlsx
Figure 6 with 3 supplements
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More Bfa1 localizes to daughter spindle pole body (dSPB) in the absence of BUD14.

(A) Bfa1-GFP signal intensity quantifications at the SPBs of BFA1-GFP mCherry-TUB1 or BFA1-GFP mCherry-TUB1 bud14βˆ† cells with normal aligned anaphase spindles were plotted on the right. Black and gray lines in the dotplots are the mean value and standard deviation, respectively. Representative still images of cells are shown on the left. (B) Bfa1-GFP signal intensities at the dSPB were plotted in BUD14 and bud14βˆ† cells bearing empty plasmid (blue and red), as well as bud14βˆ† cells bearing a ADH-BUD14-containing plasmid (green). (C) Bfa1-GFP signal intensities at the dSPB were plotted in bud14βˆ† cells bearing empty plasmid (red), ADH1-BUD14-containing plasmid (green) and ADH1-bud14-F379A-containing plasmid (yellow) (D) Analysis of Bfa1-GFP asymmetry at the SPBs of kar9βˆ†, kar9βˆ† kin4βˆ†, and kar9βˆ† bud14βˆ† cells with correctly aligned and misaligned anaphase spindles. Box and Whisker plot shows the ratio of Bfa1-GFP signal intensities at the SPB1 and SPB2, where SPB1 always corresponds to SPB with the greater Bfa1-GFP signal. The box represents first and third quartile while the whiskers show 10–90 percentile of the data. The horizontal line in the box indicates the median of the data. Only comparisons of normal and misaligned spindles are shown in the figure. Representative still images of cells are shown on the right. n: sample size. Scale bar: 2 Β΅m. One-way ANOVA with uncorrected Fisher’s LSD was applied for all statistical analyses. **p<0.01, ***p<0.001, ****p<0.0001. All pairwise comparisons are shown in the corresponding source data files.

Figure 6β€”source data 1

Numerical data and statistics for Figure 6A.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig6-data1-v2.xlsx
Figure 6β€”source data 2

Numerical data and statistics for Figure 6B.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig6-data2-v2.xlsx
Figure 6β€”source data 3

Numerical data and statistics for Figure 6C.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig6-data3-v2.xlsx
Figure 6β€”source data 4

Numerical data and statistics for Figure 6D.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig6-data4-v2.xlsx
Figure 6β€”figure supplement 1
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Bfa1, Bub2, and Tem1 localization in bud14βˆ† cells during anaphase and metaphase.

(A, B) Bub2-GFP and Tem1-GFP representative images (A) and signal intensity quantifications at the spindle pole bodies (SPBs) (B) of BFA1-GFP mCherry-TUB1 or BFA1-GFP mCherry-TUB1 bud14βˆ† cells with normal aligned anaphase spindles. Black and gray lines in the dotplots are the mean value and standard deviation, respectively. Representative still images of cells are shown in (A). (C) Bfa1-GFP, Bub2-GFP, and Tem1-GFP signal intensities at the SPBs of cells with metaphase spindles (spindle length = 1.5–2 Β΅m). Graph is plotted as described in (B). (D) Analysis of Bfa1-GFP signal intensities at the SPBs of kar9βˆ†, kar9βˆ† kin4βˆ†, and kar9βˆ† bud14βˆ† cells with correctly aligned and misaligned anaphase spindles. The graph was plotted as in (B). Pairwise comparisons were performed using one-way ANOVA with uncorrected Fisher’s LSD. ***p<0.001, ****p<0.0001, n: sample size. Scale bar: 2 Β΅m.

Figure 6β€”figure supplement 1β€”source data 1

Numerical data and statistics for Figure 6β€”figure supplement 1A.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig6-figsupp1-data1-v2.xlsx
Figure 6β€”figure supplement 1β€”source data 2

Numerical data and statistics for Figure 6β€”figure supplement 1B.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig6-figsupp1-data2-v2.xlsx
Figure 6β€”figure supplement 1β€”source data 3

Numerical data and statistics for Figure 6β€”figure supplement 1C.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig6-figsupp1-data3-v2.xlsx
Figure 6β€”figure supplement 1β€”source data 4

Numerical data and statistics for Figure 6β€”figure supplement 1D.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig6-figsupp1-data4-v2.xlsx
Figure 6β€”figure supplement 2
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Analysis of Mob1 spindle pole body (SPB) localization.

(A) Percentage of metaphase cells (spindle length = 1.5–2 Β΅m) with Mob1-GFP signal at the SPBs of the indicated cells. Graphs are average of three independent experiments. A minimum of 100 cells were counted from each strain in each experiment. Error bars show standard deviation. (B) Representative still images showing Mob1-GFP localization in metaphase cells. (C, D) Dotplot of Mob1-GFP signal intensity at the SPBs of indicated strains carrying MOB1-GFP mCherry-TUB1 in metaphase (C) and anaphase (D). Black lines represent the mean value, and gray lines show standard deviation. (E) Representative still images of anaphase cells. Scale bar: 2 Β΅m. One-way ANOVA with uncorrected Fisher’s LSD was used for pairwise comparisons. ****p<0.0001.

Figure 6β€”figure supplement 2β€”source data 1

Numerical data and statistics for Figure 6β€”figure supplement 2C.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig6-figsupp2-data1-v2.xlsx
Figure 6β€”figure supplement 2β€”source data 2

Numerical data and statistics for Figure 6β€”figure supplement 2D.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig6-figsupp2-data2-v2.xlsx
Figure 6β€”figure supplement 3
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Bfa1-GFP localization during spindle misalignment.

Selected frames from time-lapse series of BFA1-GFP SPC42-eqFP mCherry-TUB1-bearing kar9βˆ† (A), kar9βˆ† bud14βˆ† (B), and kar9βˆ† kin4βˆ† (C) gene deletions. Cell boundaries are outlined with a dashed line. Blue arrows indicate the spindle pole bodies (SPBs). Time points from the beginning of the time lapse are indicated on the frames. Scale bar: 2 Β΅m.

Figure 7 with 2 supplements
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Glc7-Bud14 promotes Bfa1 dephosphorylation in anaphase.

(A) Bfa1-3HA Gal1-UPL-Tem1-containing lte1βˆ† and lte1βˆ† bud14βˆ† cells grown in galactose-containing medium were released from alpha factor-imposed G1 arrest (t = 0) into an alpha factor-free medium supplemented with glucose to achieve Tem1 depletion, and samples were collected at indicated time points. Bfa1-3HA mobility shift was analyzed via western blotting using anti-HA antibodies. Red arrow indicates hyperphosphorylated forms of Bfa1-3HA, whereas blue arrow indicates hypophosphorylated forms of Bfa1-3HA. Percentage of cells with single nucleus (1 DAPI) and two separate nuclei (2 DAPI) were plotted as a marker for cell cycle progression. (B) Indicated time points of each cell type from the experiment shown in (A) were loaded side-by-side for better comparison of Bfa1-3HA mobility. (C) Bfa1-3HA mobility in Gal1-UPL-TEM1 or cdc5-10-bearing LTE1 BUD14 or lte1βˆ†bud14βˆ† cells. Percentage of cells with two separate nuclei (% 2 DAPI) are indicated as a measure of cells in anaphase. Cells were first arrested in G1, then released from the G1 arrest and cultured for 90 min before sample collection. Gal1-UPL-Tem1 cells were treated as in (A) to achieve the anaphase arrest, whereas anaphase arrest of cdc5-10 cells was achieved through growth at 37Β°C. (D) cdc15-as-bearing cells bearing BUD14 or Gal1-BUD14 at the Bud14 endogenous locus were grown to log-phase in raffinose-containing medium, treated with 1NM-PP1 for 2,5 hr followed by galactose addition (t0) to the medium. Samples were collected at 0 hr, 2 hr, and 3 hr after galactose addition and Bfa1 mobility was analyzed. Percentage of cells with two separate nuclei (% 2 DAPI) is indicated as a measure of cells in anaphase. (E) cdc15-as bud14βˆ† cells with BUD14-9myc, Gal1-BUD14-9myc, or Gal1-bud14-F379A-9myc integrated at the chromosomal leu2 locus were grown to log-phase in raffinose-containing medium, treated with 1NM-PP1 for 3 hr, followed by galactose addition (t0) to the medium. Samples were collected at 0 hr, 1 hr, 2 hr, and 3 hr after galactose addition and Bfa1 mobility was analyzed. Percentage of cells with two separate nuclei (% 2 DAPI) is indicated as a measure of cells in anaphase. (F) Quantification of relative levels of hypersphosphorylated Bfa1 from the experiment shown in (E). Band intensity ratio of slow-migrating forms to fast-migrating forms of Bfa1 is plotted. (G) In vitro phosphatase assay of immunoprecipitated Glc7-TAP on IgG beads is incubated with Bfa1-3HA purified from BFA1-3HA Gal1-UPL-TEM1 kin4βˆ† cells in the presence or absence of 1.5 Β΅M okadaic acid. As a no Glc7-TAP control, IgG beads incubated with cell lysates of ESM356-1 were used. Glc7-TAP levels were detected using anti-TAP antibodies. Bfa1-3HA was detected using anti-HA antibodies. Red and blue arrows indicate slow-migrating and fast-migrating forms of Bfa1-3HA, respectively. Quantification of relative levels of hypersphosphorylated Bfa1 is shown on the right. Each color represents a different independent experiment. One-way ANOVA with uncorrected Fisher’s LSD was applied for statistical analysis. **p<0.01, ****p<0.0001.

Figure 7β€”source data 1

Labeled uncropped blot images for Figure 7A and B.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig7-data1-v2.zip
Figure 7β€”source data 2

Raw scans of the x-ray films for Figure 7β€”source data 1b, (A) anti-HA blot, (B) anti-Clb2 blot, (C) anti-tubulin blot, and (D) anti-HA blot.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig7-data2-v2.zip
Figure 7β€”figure supplement 1
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Analysis of Bfa1 mobility in lte1βˆ† and lte1βˆ† kin4βˆ† cells.

(A) Bfa1-3HA Gal1-UPL-Tem1-containing lte1βˆ† and lte1βˆ†kin4βˆ† cells grown in galactose-containing medium were released from G1 arrest (t = 0) into an alpha factor-free medium supplemented with glucose. Samples were collected at indicated time points. Bfa1-3HA mobility shift was analyzed via western blotting. (B) Indicated time points of each cell type from the experiment shown in (A) were loaded side-by-side for better comparison of Bfa1-3HA mobility. (C) Percentage of cells with single nucleus (1 DAPI) and two separate nuclei (2 DAPI) as well as budding index of the experiment shown in (A) were plotted as a marker for cell cycle progression.

Figure 7β€”figure supplement 1β€”source data 1

Labeled uncropped blot images for Figure 7β€”figure supplement 1.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig7-figsupp1-data1-v2.pdf
Figure 7β€”figure supplement 1β€”source data 2

Raw scans of the blot images for (A) anti-HA blot.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig7-figsupp1-data2-v2.zip
Figure 7β€”figure supplement 2
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Kin4 overexpression toxicity in bud14βˆ† cells and phosphatase assay using Bud14-TAP.

(A) Serial dilutions of indicated strains were spotted on glucose-containing (YPD) and galactose-containing (YP Raf/Gal) agar plates. Gal1-Bud14 overexpression is induced on galactose-containing plates. (B) In vitro phosphatase assay of immunoprecipitated Bud14-TAP. Bud14-TAP was pulled on IgG Sepharose beads from the lysates of BUD14-TAP GLC7-9myc cells. Beads were incubated with Bfa1-3HA purified from BFA1-3HA Gal1-UPL-TEM1 kin4βˆ† cells in the presence or absence of okadaic acid. Beads incubated with ESM356-1 cell lysates were used as a negative control. Bud14-TAP levels were detected using anti-TAP, Glc7-9myc levels were detected using anti-myc, and Bfa1-3HA was detected using anti-HA antibodies. Quantification of relative levels of hypersphosphorylated Bfa1 is shown on the right. Each color represents a different independent experiment. One-way ANOVA with uncorrected Fisher’s LSD was applied for statistical analysis. **p<0.01.

Figure 7β€”figure supplement 2β€”source data 1

Labeled uncropped blot images for Figure 7β€”figure supplement 2B.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig7-figsupp2-data1-v2.pdf
Figure 7β€”figure supplement 2β€”source data 2

Raw scans of the blot images for (A) anti-HA blot and (B) anti-TAP and anti-Myc blots.

https://cdn.elifesciences.org/articles/72833/elife-72833-fig7-figsupp2-data2-v2.zip

Tables

Table 1
Yeast strains used in this study.
Strain nameDescriptionReference
AKY038ESM356 Bfa1-GFP-kanMX6 Spc42-eqFP- hphNT1 mCherry-Tub1-URA3This study
AKY043ESM356 BFA1-GFP-kanMX6 Spc42-eqFP-hphNT1 kar9βˆ†::klTRP1 pRS316-KAR9Caydasi and Pereira, 2009
AKY1533ESM356 leu2::LEU2-pGal1-KIN4This study
AKY1574ESM356 leu2::LEU2-pGal1-KIN4 bub2βˆ†::hphNT1This study
AKY2526FAY145 lte1βˆ†::kanMX6 pRS316-LTE1 spo12βˆ†::natNT2Caydasi et al., 2017
AKY260ESM356 kar9βˆ†::HIS3MX6 pRS316-KAR9Caydasi et al., 2017
AKY2916FAY145 lte1βˆ†::kanMX6 pRS316-LTE1 spo12βˆ†::natNT2 bud14βˆ†::HIS3MX6This study
AKY2917YPH499 GFP-Tub1-URA3 dyn1βˆ†::klTRP1 bud14βˆ†::HIS3MX6This study
AKY2918YPH499 GFP-Tub1-URA3 dyn1βˆ†::klTRP1 kin4βˆ†::hphNT1 bud14βˆ†::HIS3MX6This study
AKY315ESM356 kar9βˆ†::HIS3MX6 pRS316-KAR9 bfa1βˆ†::klTRP1Caydasi et al., 2017
AKY321ESM356 kar9βˆ†::HIS3MX6 pRS316-KAR9 kin4βˆ†::klTRP1Caydasi et al., 2017
AKY346YPH499 kar9βˆ†::klTRP1 pRS316-KAR9 GFP-Tub1-ADE2Caydasi et al., 2010b
AKY351YPH499 kar9βˆ†::klTRP1 pRS316-KAR9 kin4βˆ†::HIS3MX6 GFP-Tub1-ADE2Caydasi et al., 2017
AKY4001ESM356 leu2::LEU2-pGal1-KIN4 bud14βˆ†::klTRP1This study
AKY4006ESM356 KIN4-GFP-HIS3MX6 Spc42-eqFP-natNT2 bud14βˆ†::klTRP1 lte1βˆ†::hphNT1 mCherry-Tub1-URA3This study
AKY4007ESM356 Bub2-GFP-kanMX6 Spc42-eqFP-hphNT1 mCherry-Tub1-URA3This study
AKY4008ESM356 Tem1-GFP-kanMX6 Spc42-eqFP-hphNT1 bud14βˆ†::HIS3MX6 mCherry-Tub1-URA3This study
AKY4009ESM356 Bub2-GFP-kanMX6 Spc42-eqFP-hphNT1 bud14βˆ†::klTRP1 mCherry-Tub1-URA3This study
AKY4011ESM356 Bfa1-GFP-kanMX6 Spc42-eqFP-hphNT1 bud14βˆ†::klTRP1 mCherry-Tub1-URA3This study
AKY4012ESM356 Bfa1-GFP-kanMX6 Spc42-eqFP-hphNT1 mCherry-Tub1-URA3This study
AKY4013ESM356 Tem1-GFP-kanMX6 Spc42-eqFP-hphNT1 mCherry-Tub1-URA3This study
AKY4016ESM356 KIN4-GFP-HIS3MX6 Spc42-eqFP-natNT2 lte1βˆ†::URA3 mCherry-Tub1-kITRP1This study
AKY4028FAY145 lte1βˆ†::kanMX6 bud14βˆ†::HIS3MX6This study
AKY4036FAY145 lte1βˆ†::kanMX6 bud14βˆ†::HIS3MX6 pRS416-endogenous BUD14-promoter- GFP-BUD145AThis study
AKY4038FAY145 lte1βˆ†::kanMX6 bud14βˆ†::HIS3MX6 pRS416-endogenous BUD14-promoter- GFP-BUD14βˆ†SH3This study
AKY404ESM356 KIN4-GFP-HIS3MX6 Spc42-eqFP-natNT2 rts1βˆ†::klTRP1
AKY4040ESM356 Spc42-eqFP-hphNT1 BUD14-GFP-his3MX6 mCherry-Tub1-URA3This study
AKY4068YPH499 kar9βˆ†::klTRP1 pRS316-KAR9 GFP-Tub1-ADE2 bud14βˆ†::HIS3MX6 kin4βˆ†::hphNT1This study
AKY4078PAY704 MATa ade2-1 his3-11,15 leu2-3,112 ura3-1 can1-100 ssd1-d2 glc7::LEU2 trp1-1::GLC7::TRP1 kar9βˆ†::hphNT1This study
AKY4079PAY701 MATa ade2-1 his3-11,15 leu2-3,112 ura3-1 can1-100 ssd1-d2 glc7::LEU2 trp1-1::glc7-12::TRP1 kar9βˆ†::hphNT1This study
AKY4087ESM356 cdc15::CDC15-as1(L99G)-URA3 Bfa1-3HA- kITRP1 pGal1-Bud14-natNT2This study
AKY4088ESM356 Mob1-GFP-kanMX6 Spc42-eqFP-hphNT1 kin4βˆ†::natNT2 bud14βˆ†::HIS3MX6This study
AKY4091YPH499 cdc5-10 Bfa1-3HA-kITRP1 lte1βˆ†::natNT2 bud14βˆ†::HIS3MX6This study
AKY4094YPH499 cdc14-2 pRS316-CDC14 bfa1Ξ”::klTRP3This study
AKY4095YPH499 dbf2-2 pRS316-DBF2 bfa1Ξ”::klTRP3This study
AKY4102ESM356 Mob1-GFP-kanMX6 Spc42-eqFP-hphNT1 kin4βˆ†::natNT2 bud14βˆ†::HIS3MX6 mCherry-Tub1-LEU2This study
AKY415ESM356 KIN4-6HA-hphNT1 rts1βˆ†::klTRP1
BKY032ESM356 BFA1-GFP-kanMX6 Spc42-eqFP-hphNT1 kar9βˆ†::klTRP1 pRS316-KAR9 kin4βˆ†::HIS3MX6
DGY001ESM356 KIN4-6HA-hphNT1 bud14βˆ†::klTRP1This study
DGY004ESM356 KIN4-GFP-HIS3MX6 Spc42-eqFP-natNT2 bud14βˆ†::klTRP1This study
DKY056YPH499 bud14βˆ†::kITRP3This study
DKY057YPH499 cdc14-2 pRS316-CDC14 bud14Ξ”::klTRP3This study
DKY058YPH499 mob1-67 pRS316-MOB1 bud14Ξ”::klTRP3This study
DKY060YPH499 dbf2-2 pRS316-DBF2 bud14Ξ”::klTRP3This study
DKY061YPH499 cdc15-1 pRS316-CDC15 bud14Ξ”::klTRP3This study
DKY063ESM356 KIN4-GFP-HIS3MX6 Spc42-eqFP611-natNT2 bud14βˆ†::klTRP1This study
DKY069FAY145 lte1βˆ†::kanMX6 pRS316-LTE1 bud14βˆ†::HIS3MX6This study
DKY070PAY704 MATa ade2-1 his3-11,15 leu2-3,112 ura3-1 can1-100 ssd1-d2 glc7::LEU2 trp1-1::GLC7::TRP1This study
DKY071PAY701 MATa ade2-1 his3-11,15 leu2-3,112 ura3-1 can1-100 ssd1-d2 glc7::LEU2 trp1-1::glc7-12::TRP1This study
DKY074PAY701 glc7::LEU2 trp1-1::glc7-12::TRP1 lte1βˆ†::HIS3MX6This study
DKY075PAY704 glc7::LEU2 trp1-1::GLC7::TRP1 lte1βˆ†::HIS3MX6This study
DKY078PAY704 glc7::LEU2 trp1-1::GLC7::TRP1 lte1βˆ†::HIS3MX6 pRS316-LTE1This study
DKY079PAY701 glc7::LEU2 trp1-1::glc7-12::TRP1 lte1βˆ†::HIS3MX6 pRS316-LTE1This study
DKY080PAY701 glc7::LEU2 trp1-1::glc7-12::TRP1 lte1βˆ†::HIS3MX6 pRS316-LTE1 spo12βˆ†::hphNT1This study
DKY081ESM356 BFA1-GFP-kanMX6 Spc42-eqFP hphNT1 kar9βˆ†::klTRP1 pRS316-KAR9 bud14βˆ†::HIS3MX6This study
DKY101ESM356 GAL1-UPL-TEM1::kITRP1 Bfa1-3HA-hphNT1This study
DKY113SGY37-VIII,3 bub2βˆ†::hphNT1This study
DKY115ESM356 Bfa1-3HA-hphNT1 kin4βˆ†::natNT2 pWS103 (pGal1-UPL-Tem1-kITRP1)This study
DKY118ESM356 GAL1-UPL-TEM1::kITRP1 Bfa1-3HA-hphNT1 lte1βˆ†::natNT2This study
DKY123YPH499 cdc5-10 Bfa1-3HA-hphNT1This study
DKY125ESM356 GAL1-UPL-TEM1::kITRP1 Bfa1-3HA-hphNT1 kin4βˆ†::natNT2 bud14βˆ†::HIS3MX6This study
DKY126ESM356 GAL1-UPL-TEM1::kITRP1 Bfa1-3HA-hphNT1 lte1βˆ†::natNT2 bud14βˆ†::HIS3MX6This study
DKY131FAY145 lte1βˆ†::kanMX6 pRS316-LTE1 bud14βˆ†::HIS3MX 6GFP BUD14 CEN URAThis study
DKY132ESM356 bud14βˆ†::klTRP GFP BUD14 CEN URAThis study
DKY135FAY145 lte1βˆ†::kanMX6 bud14βˆ†::HIS3MX6 pRS416-ADH-BUD14This study
DKY137FAY145 lte1βˆ†::kanMX6 bud14βˆ†::HIS3MX6 pRS416-ADH-BUD14-F379AThis study
DKY145ESM356 kar9βˆ†::HIS3MX6 bud14βˆ†::klTRP1 pRS316This study
DKY147ESM356 kar9βˆ†::HIS3MX6 bud14βˆ†::klTRP1 pMK125This study
DKY149ESM356 kar9βˆ†::HIS3MX6 bud14βˆ†::klTRP1 pMK131This study
DKY167ESM356 KIN4-GFP-HIS3MX6 Spc42-eqFP-natNT2 bud14βˆ†::klTRP1 lte1βˆ†::hphNT1This study
DKY179YPH499 cdc5-10 pRS316-CDC5 bud14Ξ”::klTRP3This study
DKY188ESM356 KIN4-GFP-HIS3MX6 Spc42-eqFP-natNT2 lte1βˆ†::URA3This study
ESM2156YPH499 GFP-Tub1-URA3 dyn1βˆ†::klTRP1 kin4βˆ†::hphNT1Pereira and Schiebel, 2005
ESM2282YPH499 cdc15-1 pRS316-CDC15Caydasi et al., 2017
ESM2283YPH499 dbf2-2 pRS316-DBF2Caydasi et al., 2017
ESM2285YPH499 mob1-67 pRS316-MOB1Caydasi et al., 2017
ESM2326ESM356 KIN4-6HA-hphNT1Caydasi et al., 2010b
ESM356MATa ura3-52 leu2βˆ†one his3βˆ†200 trp1βˆ†63Pereira and Schiebel, 2001
FAY145MATa ura3-52 his3βˆ†200 leu2βˆ†one lte1βˆ†::kanMX6 pRS316-LTE1Bertazzi et al., 2011
GPY1033ESM356-1 KIN4-GFP-his3MX6 Spc42-eqFP-natNT2Pereira and Schiebel, 2005
GPY1054ESM356 Mob1-GFP-kanMX6 Spc42-eqFP-hphNT1This study
GPY491YPH499 cdc14-2 pRS316- CDC14Caydasi et al., 2017
GYBY005ESM356 Glc7-TAP-kanMX6 (CBP-Tev-4ProtA-kanMX6)This study
HKY099ESM356 BFA1-GFP-kanMX6 Spc42-eqFP-hphNT1 kar9βˆ†::klTRP1 pRS316-KAR9 mCherry-Tub1-LEU2This study
HKY100ESM356 BFA1-GFP-kanMX6 Spc42-eqFP-hphNT1 kar9βˆ†::klTRP1 pRS316-KAR9 kin4βˆ†::HIS3MX6 mCherry-Tub1-LEU2This study
HKY101ESM356 BFA1-GFP-kanMX6 Spc42-eqFP-hphNT1 kar9βˆ†::klTRP1 pRS316-KAR9 bud14βˆ†::HIS3MX6 mCherry-Tub1-LEU2This study
HKY114ESM356 Bfa1-GFP-kanMX6 Spc42-eqFP-hphNT1 mCherry-Tub1-LEU2 pRS416This study
HKY115ESM356 Bfa1-GFP-kanMX6 Spc42-eqFP-hphNT1 bud14βˆ†::klTRP1 mCherry-Tub1-LEU2 pRS416This study
HKY116ESM356 Bfa1-GFP-kanMX6 Spc42-eqFP-hphNT1 bud14βˆ†::klTRP1 mCherry-Tub1-LEU2 pRS416-BUD14This study
HKY133FAY145 lte1βˆ†::kanMX6 pRS316-LTE1 kel1βˆ†::hphNT1This study
HKY134FAY145 lte1βˆ†::kanMX6 pRS316-LTE1 kel2βˆ†::hphNT1This study
HKY135FAY145 lte1βˆ†::kanMX6 pRS316-LTE1 spo12βˆ†::natNT2 kel1βˆ†::hphNT1This study
HKY136FAY145 lte1βˆ†::kanMX6 pRS316-LTE1 spo12βˆ†::natNT2 kel2βˆ†::hphNT1This study
HKY139ESM356 kar9βˆ†::HIS3MX6 pRS316-KAR9 kel1βˆ†::hphNT1This study
HKY140ESM356 kar9βˆ†::HIS3MX6 pRS316-KAR9 kel2βˆ†::hphNT1This study
HKY155ESM356 BUD14-TAP-kanMX6This study
HKY164ESM356 Bud14-TAP-kanMX6 Glc7-9myc-HIS3MX6This study
HKY171ESM356 Mob1-GFP-kanMX6 Spc42-eqFP-hphNT1 mCherry-Tub1-LEU2This study
HKY172ESM356 Mob1-GFP-kanMX6 Spc42-eqFP-hphNT1 mCherry-Tub1-LEU2 bud14βˆ†::HIS3MX6This study
HKY173ESM356 Mob1-GFP-kanMX6 Spc42-eqFP-hphNT1 mCherry-Tub1-LEU2 kin4βˆ†::natNT2This study
HKY174ESM356 Mob1-GFP-kanMX6 Spc42-eqFP-hphNT1 mCherry-Tub1-LEU2 bfa1βˆ†::natNT2This study
HKY175YPH499 kar9βˆ†::klTRP1 pRS316-KAR9 GFP-Tub1-ADE2 bud14βˆ†::HIS3MX6 bfa1βˆ†::natNT2This study
HKY177YPH499 bfa1βˆ†::kITRP1This study
HKY180YPH499 mob1-67 pRS316-MOB1 bfa1Ξ”::klTRP3This study
HKY182YPH499 cdc15-1 pRS316-CDC15 bfa1Ξ”::klTRP3This study
HKY183YPH499 cdc5-10 pRS316-CDC5 bfa1Ξ”::klTRP3This study
HKY184ESM356 cdc15::CDC15-as1(L99G)-URA3 Bfa1-3HA- kITRP1 bud14βˆ†::HIS3MX6This study
HKY185ESM356 pRS416This study
HKY186ESM356 pMK60 (Bud14-GFP)This study
HKY187ESM356 pHK002 (Bud14-F379A-GFP)This study
HKY188ESM356 pDK003 (Bud14-βˆ†SH3-GFP)This study
HKY189ESM356 pDK001 (Bud14-5A-GFP)This study
HKY190ESM356 BFA1-GFP-kanMX6 Spc42-eqFP-hphNT1 bud14βˆ†::klTRP1 mCherry-TUB1-Leu2 pMK131This study
IKY075SGY37-VIII,3 kin4βˆ†::klTRP1This study
DKY133SGY37-VIII,3 bub2βˆ†::hphT1This study
IKY192ESM356 kar9βˆ†::HIS3MX6 bud14βˆ†::klTRP1 pRS416-endogenous BUD14-promoter- GFP-BUD14-5AThis study
IKY193ESM356 kar9βˆ†::HIS3MX6 bud14βˆ†::klTRP1 pRS416-endogenous BUD14-promoter- GFP-BUD14-βˆ†SH3This study
JOY79YPH499 cdc5-10 pRS316-CDC5Caydasi et al., 2017
PAY701W303 MATa ade2-1 his3-11,15 leu2-3,112 ura3-1 can1-100 ssd1-d2 glc7::LEU2 trp1-1::glc7-12::TRP1Andrews and Stark, 2000
PAY704W303 MATa ade2-1 his3-11,15 leu2-3,112 ura3-1 can1-100 ssd1-d2 glc7::LEU2 trp1-1::GLC7::TRP1Andrews and Stark, 2000
SEY032YPH499 kar9Ξ”::klTRP3 pRS316-KAR9 GFP-Tub1-ADE2 bud14βˆ†::HIS3MX6This study
SEY206ESM356 cdc15::CDC15-as1(L99G)-URA3 Bfa1-3HA- kITRP1This study
SEY212ESM356 cdc15::CDC15-as1(L99G)-URA3 Bfa1-3HA- kITRP1 Bud14-9myc-HIS3MX6This study
SEY218ESM356 cdc15::CDC15-as1(L99G)-URA3 Bfa1-3HA- kITRP1 bud14βˆ†::HIS3MX6 leu2::LEU2- pGal1-Bud14-9myc-hphNT1This study
SEY219ESM356 cdc15::CDC15-as1(L99G)-URA3 Bfa1-3HA- kITRP1 bud14βˆ†::HIS3MX6 leu2::LEU2- pGal1-Bud14- F379A-9myc-hphNT1This study
SGY37-VIII,3MATa leu2 his3 trp1 ADE2 ura3-52::URA3-lexA-op-LacZGeissler et al., 1996
SHM562YPH499 GFP-Tub1-URA3 dyn1βˆ†::klTRP1Maekawa et al., 2007
YDA101ESM356 kar9βˆ†::HIS3MX6 bud14βˆ†::klTRP1Caydasi and Pereira, 2017
YPH499MATa ura3-52 lys2-801 ade2-101 trp1βˆ†63 his3βˆ†200 leu2βˆ†1Sikorski and Hieter, 1989
Table 2
Plasmids used in this study.
Plasmid nameDescriptionReference
pRS316URA3-dependent CEN-based yeast-E. coli shuffle plasmid AmpRSikorski and Hieter, 1989
pRS416URA3-dependent CEN-based yeast-E. coli shuffle plasmid AmpRGeissler et al., 1996
pAFS125GFP-TUB1-URA3-containing integration plasmidStraight et al., 1997
pSM1027GFP-TUB1-ADE2-containing integration plasmidCaydasi et al., 2010b
pAK010mCherry-TUB1-kΔ±TRP1-containing integration plasmidKhmelinskii et al., 2007
pAK011mCherry-TUB1-URA3-containing integration plasmidKhmelinskii et al., 2007
pBK067mCherry-TUB1-LEU2-containing integration plasmidCaydasi et al., 2014
pWS103-1pRS304-Gal1-UPL-TEM1Shou et al., 1999
pMM5p423-Gal1-lexA-MycGeissler et al., 1996
pMM6p425-Gal1-Gal4-HA.Geissler et al., 1996
pCDV471pEG202 ADH1-LexA-DBD-GLC7Lenssen et al., 2005
pEG2022 ΞΌm ADH1-LexA-DBD-HIS3Pereira et al., 1999
pCL2-1pMM6-BUB2HΓΆfken and Schiebel, 2004
pCL4a-3pMM6-BFA1HΓΆfken and Schiebel, 2004
pHA132-2pMM6-KIN4 (1–750 aa)Gift from G. Pereira
pHA69-1pMM5-BUD14Gift from G. Pereira
pHA70-1pMM6-BUD14Gift from G. Pereira
pTH221pMM6-KELHΓΆfken and Schiebel, 2004
IKY006pMM5-BUD14-5AThis Study
IKY007pMM5-BUD14-F379AThis Study
IKY008pMM5-BUD14-βˆ†SH3This Study
IKY010pMM6-BUD14-5AThis Study
IKY011pMM6-BUD14-F379AThis Study
IKY012pMM6-BUD14-βˆ†SH3This Study
pMK125pRS416 ADH-BUD14Knaus et al., 2005
pMK131pRS416 ADH-BUD14-F379AKnaus et al., 2005
pMK60pRS416-endogenous BUD14-promoter-GFP-BUD14Knaus et al., 2005
pDKY001pRS416-endogenous BUD14-promoter-GFP-BUD145A (135A 137A 138A 139A 140A)This Study
pDKY003pRS416-endogenous BUD14-promoter-GFP-BUD14βˆ†SH3(βˆ†aa 262–318)This Study
pHK002pRS416-endogenous BUD14-promoter-GFP-Bud14-F379AThis study
pGW399-1pRS316-KAR9Caydasi et al., 2010b
pSM805pRS305-pGal1KΓΆnig et al., 2010
pHK003pSM805 pGal1-Bud14-Leu2This study
pHK004pSM805 pGal1-Bud14-F379A-Leu2This study
pIK007pMM5-Bud14-F379AThis study
pSM903-4pRS316-LTE1HΓΆfken and Schiebel, 2002
pSM908pRS316-DBF2Caydasi et al., 2017
pSM926pRS316-MOB1Caydasi et al., 2017
pUG120pRS316-CDC14Gruneberg et al., 2000
pBS9pRS316-CDC15Caydasi et al., 2017
pCL33pRS316-CDC5HΓΆfken and Schiebel, 2004

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  1. Dilara Kocakaplan
  2. HΓΌseyin KarabΓΌrk
  3. Cansu Dilege
  4. Idil KirdΓΆk
  5. Seyma Nur Bektas
  6. Ayse Koca Caydasi
(2021)
Protein phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae
eLife 10:e72833.
https://doi.org/10.7554/eLife.72833