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. 2021 Oct 11;10:e72833. doi: 10.7554/eLife.72833

Figure 7. Glc7-Bud14 promotes Bfa1 dephosphorylation in anaphase.

(A) Bfa1-3HA Gal1-UPL-Tem1-containing lte1∆ and lte1∆ bud14∆ 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 lte1∆bud14∆ 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 37°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 bud14∆ 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 kin4∆ cells in the presence or absence of 1.5 µM 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 7—source data 1. Labeled uncropped blot images for Figure 7A and B.
Figure 7—source data 2. Raw scans of the x-ray films for Figure 7—source data 1b, (A) anti-HA blot, (B) anti-Clb2 blot, (C) anti-tubulin blot, and (D) anti-HA blot.

Figure 7.

Figure 7—figure supplement 1. Analysis of Bfa1 mobility in lte1∆ and lte1∆ kin4∆ cells.

Figure 7—figure supplement 1.

(A) Bfa1-3HA Gal1-UPL-Tem1-containing lte1∆ and lte1∆kin4∆ 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 7—figure supplement 1—source data 1. Labeled uncropped blot images for Figure 7—figure supplement 1.
Figure 7—figure supplement 1—source data 2. Raw scans of the blot images for (A) anti-HA blot.
Figure 7—figure supplement 2. Kin4 overexpression toxicity in bud14∆ cells and phosphatase assay using Bud14-TAP.

Figure 7—figure supplement 2.

(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 kin4∆ 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 7—figure supplement 2—source data 1. Labeled uncropped blot images for Figure 7—figure supplement 2B.
Figure 7—figure supplement 2—source data 2. Raw scans of the blot images for (A) anti-HA blot and (B) anti-TAP and anti-Myc blots.