BPs near the C terminus of Cdc14 comprise an NLS. (A) Diagram of Cdc14 showing the relative positions of the type II phosphatase domain and putative NES and NLS. BP1–4 refer to four pairs of basic amino acids. (B) The C-terminal domain of Cdc14 promotes nuclear localization. Cdc14 containing the indicated number of amino acids (full-length protein is 551 residues) to GFP and expressed from the GAL1,10 promoter. Fusion protein expression was induced for 75 min with 3% galactose, and cells were imaged by fluorescence microscopy. The table shows the number of cells for each genotype in which the GFP signal was present in both the cytoplasm and nucleus (C + N) or restricted to the nucleus (N). The statistical significance of the deviation from wild-type behavior (calculated using a χ² test) is shown in the third column. (C) The C-terminal region of Cdc14 contains NLS activity. (left) The last 100 amino acids of Cdc14 were fused to the N terminus of GFP and transiently expressed from the GAL1,10 promoter. NLS-GFP accumulated in the nucleus of unbudded and small-budded cells. (middle) Arg 527, 528, 533, and 534 of Cdc14 were substituted for Ala (BP1,2A). (right) GFP alone was present in both the cytoplasm and nucleus. Arrows indicate the colocalization of Cdc14-GFP (green) with nucleolar Nop1-DsRed (red; γ adjusted). Bars, 2 µm.

Cytoplasmic localization of NLS-GFP during late mitosis is dependent on MEN. (A) Localization of NLS-GFP (green) in temperature-sensitive dbf2-2 and wild-type (WT) W303 grown at 37°C (nonpermissive). mCherry-Tub1 was imaged to identify interphase (short spindle) and late anaphase (long spindle) cells. 73% of wild-type (n = 75) cells with spindles >5 µm exhibited pancellular localization of the NLS-GFP reporter (carrot) compared with 16% of dbf2-2 (n = 50) cells, indicating that Dbf2 activity is required for inactivation of Cdc14’s C-terminal NLS during late anaphase. Only 4% of wild-type (n = 50) cells and no dbf2-2 (n = 50) cells with spindles <5 µm exhibited pancellular GFP staining. The graph shows the percentage of cells with released Cdc14 (i.e., both cytoplasmic and nuclear fluorescence) as a function of spindle length (<5 or >5 µm). (B) Wild-type and temperature-sensitive dbf2-2 yeast strains were grown at 37°C for 45 min, resuspended in 2.5% LMP agarose with 1× synthetic complete medium, and placed on slides. Slides were held at 37°C by a temperature-controlled stage for ≥30 min before imaging the cells every 6 min. Representative time-lapse series are shown for wild-type (n = 7) and dbf2-2 (n = 5) cells. The NLS-GFP reporter was released from the nucleus for approximately two time points (red) in all of the wild-type cells but exhibited persistent nuclear localization in all five dbf2-2 cells. Bars, 2 µm.

Cdc14 is phosphorylated by Dbf2–Mob1. (A) Phosphorylation of Cdc14 by Dbf2–Mob1. Purified GST-Cdc14, GST-Cdc14(PS1–3A), or histone H1 were treated with wild-type (wt) or kinase-dead Dbf2–Mob1 (dbf2) complex. Dbf2–Mob1 was activated in vitro by protein kinase Cdc15. Phosphorylated Cdc14 was detected by SDS-PAGE and autoradiography. PS1–3A refers to a hextuple mutant in which the residues in PA pairs 1–3 (PS1–3; B) were changed to Ala. (B) The C terminus of Cdc14 contains three potential regions of Dbf2–Mob1 phosphorylation. The sites that were altered for this study, PS1–3, are indicated in black font. RXXS refers to the preferred site for phosphorylation by Dbf2 (Mah et al., 2005). (C) Phosphorylation of Cdc14’s NLS region by Dbf2–Mob1 depends on PS1–3. The last 100 amino acids of the C terminus of Cdc14 were fused to GST. Purified GST-NLS was treated with either wild-type Dbf2–Mob1 or kinase-dead Dbf2–Mob1. Dbf2–Mob1 complexes were activated with protein kinase Cdc15. Phosphorylation of wild type, a BP mutant, BP4A (Fig. 1 A), and a six-site Ser/Thr to Ala mutant, PS1–3A, were assayed. Asterisks indicate predicted trypsin cleavage sites.

Phosphorylation sites within Cdc14’s NLS are required for its cell cycle regulation. (A) Mutation of Dbf2–Mob1 phosphorylation sites in NLS changes the nuclear/cytoplasmic distribution of NLS-GFP (green) reporter fusions. Wild-type (WT), PS1,2A, PS1,3A, PS1–3A, and PS1,2E NLS-GFP reporters are shown. Red fluorescent mCherry-Tub1 (red; γ adjusted) was used to identify interphase (short spindle) and late anaphase (long spindle) cells. The indicated NLS constructs were transiently expressed (3% galactose for 90 min) from the GAL1,10 promoter. Live cells were imaged. Wild-type NLS-GFP was concentrated in the nucleus of interphase cells (short spindle; arrows) and dispersed into the cytoplasm of late anaphase cells (long spindle; carrot). NLS-GFP alleles PS1,2A, PS1,3A, and PS1–3A do not exhibit cytoplasmic release of the reporter. In the table, the first column shows genotypes, the second column shows the number of cells for which the spindle was >5 µm long and the GFP signal was dispersed into the cytoplasm or concentrated in the nucleus, and the third column shows the statistical significance of the deviation from wild type (χ² test). In contrast to the nonphosphorylatable mutants, PS1,2E, a putative phosphomimetic mutant, is defective for nuclear accumulation. The residues that comprise PS1–3 are specified in Fig. 3 B. (B) The PS mutant NLS-GFP is not released from the nucleus during late mitosis. Time-lapse microscopy was performed as described in Fig. 2 B. Representative time-lapse series are shown for wild-type and PS1,2A reporters. The wild-type NLS-GFP fusion was dispersed throughout the cell for approximately two time points (indicated by red numbers; n = 5). In contrast, mutant PS1,2A reporters did not redistribute to the cytoplasm for the duration of the time course (n = 5). Bars, 2 µm.

Basic residues and phosphorylation sites within the C-terminal NLS are required for proper cell cycle–dependent control of Cdc14-GFP localization. (A) Localization of full-length Cdc14-GFP fusions (green) with either wild-type (WT) or mutated NLSs. Wild-type, PS1,2A, BP1,2A, and PS1,2E mutants expressed for 16 h from the GAL1,10 promoter with the addition of galactose to 0.015% are shown. Nop1-DsRed (red; γ adjusted) marks the nucleolus and relative position of the nucleus. Late anaphase cells with segregated nucleoli are shown. (B) Cdc14-GFP reporters were expressed from the GAL1,10 as described in A. Red fluorescent mCherry-Tub1 (red; γ adjusted) was used to select cells in late anaphase (spindles >5 µm). The intensity of Cdc14-GFP fluorescence inside the nucleus (red circle nearest the spindle pole) and inside the cytoplasm (red circle adjacent to the cell wall) was determined. (left) Representative images for wild-type and PS1,2A reporters are shown. (middle) Red squares define the regions of interest shown to the right. Red circles define the areas for which mean pixel intensity was calculated. (right) The ratio of fluorescence intensities (nuclear/cytoplasm) was calculated for multiple cells and plotted for each reporter. The mean ratio ± SD was calculated for wild type (1.8 ± 0.3 SD, n = 16) and PS1,2A (3.3 ± 0.8 SD, n = 14). The difference between the mean ratios (1.5 ± 0.2 SEM) was determined to be significant by an unpaired t test (P < 0.0001). (C) Defective dispersion of a full-length Cdc14-GFP PS mutant during late mitosis. Full-length Cdc14-GFP constructs were transiently expressed from the GAL1,10 promoter by treating cells with 3% galactose for 75 min. Wild-type and PS1,2A Cdc14-GFP reporters were observed through a single cell division by time-lapse fluorescence microscopy as described in Fig. 2 B. Representative time series of wild type (top; n = 5) and PS1,2A (bottom; n = 5) are shown. Wild-type Cdc14-GFP accumulated in the nucleolus and was released during late anaphase. The time point when Cdc14-GFP was maximally released is indicated in red. Note that GFP fluorescence is difficult to see at this time point because of its dispersion throughout the cell. In contrast, mutant Cdc14 (PS1,2A)-GFP accumulated in the nucleolus and nucleus before division and was released from the nucleolus but retained in the nucleus during anaphase (arrows). Bars, 2 µm.

Phosphorylation sites within NLS are required for efficient release from a late mitotic block. (A) Mutation of NLS phosphorylation sites impaired mitotic exit after reversal of cell cycle arrest. CDC14 and cdc14-(PS1,2A) cultures were placed at 37°C for 2 h to achieve a late mitotic cdc15-2 arrest and returned to permissive temperature (25°C). Clb2p and Cdc28 levels were followed by immunoblot analysis. (B) Cell aliquots from A were stained with DAPI and evaluated by fluorescence microscopy. Combined DIC and DAPI fluorescence images are shown. (C, left) Clb2 protein detected in A was quantified and normalized to Cdc28 levels. Relative units of fluorescence intensity were plotted for each time point. (right) The frequency of large-budded cells (n > 200) with segregated nuclei (B) was plotted for each time point. WT, wild type. Bars, 2 µm.

Bypass of MEN by inactivation of Cdc14’s C-terminal NLS compromises the checkpoint that monitors spindle position. (A) Inactivation of Cdc14’s C-terminal NLS bypasses the essential function of the MEN kinase Cdc15. Cdc15Δ yeast carrying plasmid-borne copies of the indicated CDC14 alleles and kept alive by a CDC15 URA3 plasmid were serially diluted and plated on 5-fluoroorotic acid to select for transformants that were able to grow without the CDC15 URA3 plasmid. (B) Growth of cdc15Δ and CDC15⁺ cells harboring the endogenous CDC14 gene and plasmid (pRS315)-borne alleles. Growth was measured by threefold serial dilution of yeast cells that were spotted onto synthetic media lacking leucine and incubated at 30°C. Mutant PS1,2E and BP1,2A alleles bypass cdc15Δ but grow more slowly than wild-type (WT) cells, suggesting that NLS mutations do not fully restore CDC15’s essential functions. (C) Model for MEN regulation of Cdc14. (i) MEN activity promotes release of Cdc14 from nucleolar Net1, and some of this Cdc14 makes its way to the cytoplasm. MEN also prevents reuptake of this cytoplasmic Cdc14 into the nucleus by inactivating the C-terminal NLS, allowing Cdc14 to accumulate in the cytoplasm. (ii) In a MEN mutant (e.g., cdc15Δ), release of Cdc14 from the nucleolus is inhibited. Those few molecules that are released and translocated to the cytoplasm fail to accumulate there because the C-terminal NLS remains active. Consequently, both the nuclear and cytoplasmic pools of Cdc14 are very low. (iii) The same as in ii is shown except that the C-terminal NLS is mutated. Consequently, the small number of Cdc14 molecules that leak through to the cytoplasm can accumulate there, and thereby enable suppression of cdc15Δ. (D) Constitutive inactivation of Cdc14’s C-terminal NLS compromises growth of dyn1Δ cells. Threefold serial dilutions of strains carrying the indicated CDC14 NLS mutant alleles on a low copy centromeric plasmid in either CDC14 dyn1Δ or CDC14 DYN1 backgrounds are shown. (E, left) DIC and DAPI fluorescence images of dyn1Δ cells harboring the indicated CDC14 alleles on a low copy centromeric plasmid. Yeast cultures were grown at 25°C. Cells with multiple nuclei are indicated by arrows. (right) Graph indicating the frequency with which dyn1Δ cells accumulated multiple nuclei in the presence of various CDC14 alleles (error bars indicate SD of three replicate experiments; n > 350 for each strain). VC, vector control. Bars, 2 µm.