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. 2013 Sep 10;8(9):e73194.
doi: 10.1371/journal.pone.0073194. eCollection 2013.

The FEAR protein Slk19 restricts Cdc14 phosphatase to the nucleus until the end of anaphase, regulating its participation in mitotic exit in Saccharomyces cerevisiae

Ann Marie E Faust  1 , Catherine C L Wong, John R Yates 3rd, David G Drubin, Georjana Barnes
Affiliations

The FEAR protein Slk19 restricts Cdc14 phosphatase to the nucleus until the end of anaphase, regulating its participation in mitotic exit in Saccharomyces cerevisiae

Ann Marie E Faust et al. PLoS One. .

Abstract

In Saccharomyces cerevisiae mitosis, the protein Slk19 plays an important role in the initial release of Cdc14 phosphatase from the nucleolus to the nucleus in early anaphase, an event that is critical for proper anaphase progression. A role for Slk19 in later mitotic stages of Cdc14 regulation, however, has not been demonstrated. While investigating the role of Slk19 post-translational modification on Cdc14 regulation, we found that a triple point mutant of SLK19, slk19(3R) (three lysine-to-arginine mutations), strongly affects Cdc14 localization during late anaphase and mitotic exit. Using fluorescence live-cell microscopy, we found that, similar to slk19Δ cells, slk19(3R) cells exhibit no defect in spindle stability and only a mild defect in spindle elongation dynamics. Unlike slk19Δcells, however, slk19(3R) cells exhibit no defect in Cdc14 release from the nucleolus to the nucleus. Instead, slk19(3R) cells are defective in the timing of Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase. This mutant has a novel phenotype: slk19(3R) causes premature Cdc14 movement to the cytoplasm prior to, rather than concomitant with, spindle disassembly. One consequence of this premature Cdc14 movement is the inappropriate activation of the mitotic exit network, made evident by the fact that slk19(3R) partially rescues a mutant of the mitotic exit network kinase Cdc15. In conclusion, in addition to its role in regulating Cdc14 release from the nucleolus to the nucleus, we found that Slk19 is also important for regulating Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SLK19 point mutants and protein characteristics.
Slk19 proteins encoded by specific combination slk19 point mutants have smaller apparent sizes than wild type Slk19. (A) Diagram of the locations of the SLK19 point mutations generated in this study. (B) Western blot of Slk19-3xHA proteins from whole-cell extracts, run on an 8.0% polyacrylamide gel. Blot probed with α-HA and α-Pgk1 (loading control) antibodies. Lanes, from left: Slk19 WT; Slk19 (K412R); Slk19 (K440R); Slk19 (K524R); Slk19 (K545R); Slk19 (K640R); Slk19 (K412R, K440R) (2R); Slk19 (K412R, K440R, K524R) (3R); Slk19 (K412R, K440R, K524R, K545R) (4R); Slk19 (K412R, K440R, K524R, K545R, K640R) (5R). (C) Western blot of wild type Slk19-3xHA, Slk193R-3xHA and Slk195R-3xHA proteins from whole-cell extracts, run on a 4.5% polyacrylamide gel to enhance resolution. Blot probed with α-HA and α-Sac6 (loading control) antibodies.
Figure 2
Figure 2. Slk193R has an altered localization pattern during mitosis.
Live cell microscopy time course montages of the endogenous fusion proteins (A) Slk19-GFP and (B) Slk193R-GFP during anaphase in representative cells. Microtubules are visualized with mCherry-Tub1. For each time course, the “0 min” time point was set as the frame immediately prior to the anaphase spindle entering the bud.
Figure 3
Figure 3. slk193R cells lose the transition between the fast and slow phases of anaphase
. Anaphase spindle length traces of (A) SLK19 and (B) slk193R are represented by individual colored lines. Each line represents a single spindle (SLK19, n = 13; slk193R, n = 15). (C) Average spindle elongation traces of SLK19 (blue) and slk193R (red). Each time point represents a minimum of three spindle measurements. Time points with fewer than three spindle measurements were omitted from the average traces. The anaphase pause period is indicated with an arrow. The “0” time point was set at 2 minutes before the beginning of the fast phase of anaphase B.
Figure 4
Figure 4. slk193R does not cause a defect in Cdc14 nucleolar release during anaphase.
Live cell microscopy images of representative cells with Cdc14-GFP and mCherry-Tub1 with SLK19, slk193R and slk19Δ genotypes. In SLK19 and slk193R cells, Cdc14 is released into the nucleus in early-to-mid anaphase (spindle lengths in SLK19: 5.5 and 4.9 μM; spindle lengths in slk193R: 5.7 and 5.2 μM), while in slk19Δcells, Cdc14 is still sequestered in the nucleolus in late anaphase (spindle lengths in slk19Δ: 7.9 and 6.6 μM).
Figure 5
Figure 5. slk193R causes premature movement of Cdc14 from the nucleus to the cytoplasm at the end of anaphase.
Live cell microscopy time course montages of representative Cdc14-GFP and mCherry-Tub1 in (A) SLK19 and (B) slk193R cells. The appearance of Cdc14-GFP at the bud neck is indicated with arrows, and spindle disassembly is indicated with arrowheads. For the slk193R condition in (B), the “0 min” time point was set as the image frame immediately prior to the appearance of Cdc14-GFP at the bud neck. As spindle disassembly occurred during the “10 min” time frame in slk193R, a corresponding 10-min time period was depicted in the SLK19 condition in (A).
Figure 6
Figure 6. slk193R partially rescues the spindle disassembly arrest phenotype of cdc15-2 cells.
Graph represents the delay in spindle disassembly of SLK19 cdc15-2 (n = 10), slk193R cdc15-2 (n = 24) and slk19Δ cdc15-2 (n = 23) cells at 25°C and SLK19 cdc15-2 (n = 19), slk193R cdc15-2 (n = 23) and slk19Δ cdc15-2 (n = 18) cells at 37°C. SLK19 and slk19Δ are unable to rescue the cdc15-2 disassembly defect at the restrictive temperature (p<0.01 for both), but slk193R rescues the defect to levels that are not statistically significantly different from those at the permissive temperature.
Figure 7
Figure 7. slk193R does not rescue the temperature sensitivity phenotype of cdc15-2 cells.
The indicated strains were spotted in serial dilutions from 0.5 OD600 units/mL to 0.5×10−3 OD600 units/mL onto YPD plates and grown at 25°C or 37°C for 3 days. Strains with cdc15-2 and either SLK19, slk193R or slk19Δ formed colonies at 25°C but did not form colonies at 37NC. A strain with CDC15 and slk193R formed colonies at both temperatures.
Figure 8
Figure 8. Model of the proposed activities of Slk19 and Slk193R during anaphase with respect to the regulation of Cdc14 localization.
In early anaphase, the FEAR network is activated to release Cdc14 from the nucleolus to the nucleus, and both Slk19 and Slk193R are proficient at promoting the release of Cdc14 from the nucleolus (diagram, left side). In late anaphase, wild type Slk19 retains Cdc14 within the nucleus until after spindle disassembly, while Slk193R cannot retain Cdc14 within the nucleus, resulting in the premature release of Cdc14 to the bud neck in the cytoplasm prior to spindle disassembly (diagram, right side). MEN proteins, which are involved in the release of Cdc14 from the nucleus to the bud neck during mitotic exit, might directly oppose Slk19 in late anaphase and might be more effective at promoting the release of Cdc14 to the bud neck in the presence of the mutant Slk193R protein. Alternatively, Slk19 might retain Cdc14 in the nucleus by a mechanism that not indirect opposition of the MEN, and mutant Slk193R protein is unable to retain Cdc14 within the nucleus until the end of anaphase.
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