The degradation of Cdc5p is dependent on Cdc23p function. Wild-type and cdc23-1 cells were transformed with a 2μm-based plasmid carrying GAL1-HA-CDC5 (PCH740). For both strains, an overnight culture was grown selectively at 23°C in SC lacking histidine. This was inoculated into YEP plus raffinose, grown to early log phase at 23°C and arrested in G₁ with α-factor. When >90% of the cells exhibited a schmoo-like morphology, as determined by microscopy, the temperature of the culture was shifted to 37°C (the restrictive temperature for cdc23-1). FACS analysis on these blocked samples also indicated that >90% of the cells had a 1n content of DNA and were in G₁ (data not shown). After 30 min at the restrictive temperature, galactose was added for 15 min to induce expression of HA-CDC5 from the GAL promoter. Subsequently, at time 0, glucose was added to turn off expression of HA-CDC5 from the GAL promoter while maintaining the G₁ arrest at the restrictive temperature. Samples of equal density were taken at the designated time points and assayed by immunoblotting for HA-Cdc5p and actin. Actin detection was used as an internal loading control.

Nuclear staining of Cdc5p in asynchronous Cdc5-ProA SPC42-GFP (YCH301) cells. Four views of each cell are shown: Nomarski optics (Nom), staining of DNA (DAPI), Cdc5p-ProA staining (Texas red), and Spc42-GFP using direct fluorescence. On the basis of these four characteristics, cells were placed into the following four groups. I (single G₁ cells), II (budded cells with a single nucleus and duplicated SPBs; IIa without and IIb with Cdc5p staining), III (cells in anaphase with nuclei in the mother and daughter neck and separate SPBs present in both mother and daughter), IV (cells which have undergone anaphase, with separate nuclei and polar positioning of SPBs; IVa with and IVb without Cdc5p staining). For each class of cells at given points in the cell cycle, the number and fraction of cells with (IIb, III, and IVa) or without (I, IIa, and IVb) Cdc5p staining are indicated. All images were taken with a 100× objective and printed at the same magnification. Bar = 5 μm.

Cdc5p is present in cells synchronized in telophase as detected by immunofluorescence. CDC5-ProA cdc15-1 cells (YCH307) were synchronized in telophase by growth at the restrictive temperature and processed for immunofluorescence. CDC5 and SPC42 are not tagged in cdc15-2* cells (YCH238). Four views of each cell are shown: Nomarski optics (NOM), staining of DNA (DAPI), Cdc5p-ProA staining (Texas red), and Spc42-GFP (direct fluorescence). All images were taken with a 100× objective and printed at the same magnification. Bar = 5 μm. ND, not done.

Cdc5p levels and Cdc5p-associated kinase activity fluctuate during the cell cycle. CDC5-ProA bar1 (YCH199) cells were synchronized in G₁ by addition of α-factor and released into fresh medium lacking α-factor at 23°C. Samples for FACS analysis and extract preparation were taken at the time intervals shown. Cdc5-ProA (a) and (b) actin were detected by immunoblotting with anti-IgG and antiactin antibodies, respectively. (c) Cdc5-ProA was immunoprecipitated (IP) from 400 μg of extract using IgG-Sepharose beads, (d) Kinase activity in the immunocomplexes was measured as described in Materials and Methods with casein as the substrate. (e) A bar graph of the [³²P]casein levels is shown. Levels were quantitated with a Molecular Dynamics PhosphorImager. (f) FACS analysis of DNA content.

Cdc5p-associated kinase activity is phosphorylation dependent. Cdc5-ProA cells (YCH199) were synchronized in metaphase by treatment with nocodazole. Cdc5-ProA was immunoprrecipitated from extract by using IgG-Sepharose, washed into kinase buffer, and divided equally among three samples. (a) CIP (100 U) was either not added (−), added (+), or added to the sample as a control after the CIP was boiled for 10 min to remove phosphatase activity (+*). All three tubes were placed at 37°C for 30 min. The samples were spun down, CIP was washed out using kinase buffer, [γ-³²P]ATP and cold ATP were added, and a kinase assay was performed at 30°C. The samples were loaded onto a SDS–9% polyacrylamide gel, and immunoblot analysis was performed using anti-IgG (α IgG) to detect Cdc5-ProA. After color development of the immunoblot (left panel), the filter was exposed to film (right panel). (b) Kinase assays were performed as described above for panel a, except that the phosphatase reactions were carried out in the absence (−) or presence (+) of CIP and in the absence (−) or presence (+) (200 mM) of the CIP inhibitor β-glycerophosphate (β-glyc.).

Decrease in Cdc5p levels as cells exit mitosis. CDC5-ProA cdc 15-2 (YCH236) cells were synchronized in telophase by growth at 37°C for 3 h prior to release into fresh medium at 23°C. Samples for immunoblot analysis of Cdc5-ProA and actin, FACS analysis of DNA content, and detection of anaphase spindles by immunofluorescence staining of tubulin were taken at the times (in minutes) indicated. The percentage of cells with anaphase spindles is shown on the left side of the FACS analysis profile. Spindle morphology in cells was determined by indirect immunofluorescence staining with an antitubulin antibody. Actin detection was used as an internal loading control. Cells released from a cdc15-2 block are delayed in cytokinesis which results in a 2n and 4n shift in DNA content after replication.

Detection of Cdc5p by immunofluorescence in mitotic cells. Cells derived from asynchronous cultures were fixed with methanol and formaldehyde and processed for indirect immunofluorescence. (Left) Population of asynchronous Cdc5-ProA cells (YCH194) stained with anti-IgG to detect Cdc5-ProA. (Right) 4′,6-Diamidino-2-phenylindole (DAPI) to detect DNA using Nomarski optics (NOM-DAPI). Bar = 10 μm.

Cdc5p is present in cells blocked in S and M phase as detected by immunofluorescence. Wild-type and cdc mutant cells were synchronized at different stages of the cell cycle using either chemicals or by growth of the cdc mutant at the restrictive temperature of 37°C and processed for immunofluorescence. Four views of each cell are shown: Nomarski optics (NOM), staining of DNA (DAPI), Cdc5p-ProA staining (Texas red), and Spc42-GFP (direct fluorescence). The cells were synchronized at the different stages as follows: late G₁, with α-factor (YCH301) and cdc4-1 (YCH303); S phase, with HU (YCH301); Metaphase, cdc13-1 (YCH309), nocodazole (Na) (YCH301),and cdc23-1 (YCH305). Samples of the arrested cells used in this study were also used for FACS, immunoblot, and kinase assay analyses shown in Fig. 7. All images were taken with a 100× objective and printed at the same magnification. Bar = 5 μm.

Cdc5p is detected in late G₁; S- and M-phase synchronized cells, but Cdc5p-associated kinase activity is restricted to cells arrested in M Phase. Wild-type and cdc mutant cells were arrested in specific stages of the cell cycle using either chemicals or by growth at the restrictive temperature of 37°C, respectively. The cells were synchronized at the various stages as follows: late G₁, with α-factor (YCH301) and cdc4-1 (YCH303); G₁/S, cells were initially blocked in G₁ with α-factor (YCH199) and then released into fresh medium containing HU; S phase, with HU (YCH301); metaphase (Meta), with cdc13-1 (YCH309), nocodazole (NZ) (YCH301), and cdc23-1 (YCH305); and telophase (Tel.), with cdc15-2 (YCH307) and cdc5-1 (YCH214). Extracts from asynchronous (Async) cells were derived from cultures of Cdc5-ProA (YCH199) and strain W303a expressing an untagged Cdc5p. Samples for FACS analysis and extract preparation were taken when >95% of the cells in the culture were appropriately arrested, as detected by light microscopy. Cdc5-ProA (a) and actin (b) in the crude extract were detected by immunoblotting with anti-IgG and antiactin antibodies, respectively. (c) Cdc5-ProA was routinely immunoprecipitated from 400 μg of extract with IgG-Sepharose beads and detected as above by immunoblotting. (d) Kinase activity in these immunocomplexes was measured as described in Materials and Methods with casein as the substrate, except in the case of the 4× HU lane, where 1.6 mg of extract was used. (e) A bar graph of the [³²P]-casein levels is shown. Levels were quantitated with a Molecular Dynamics PhosphorImager. (f) Cdc5-ProA kinase activity was determined for cdc5-1-proA (YCH214) cultures grown either asynchronously (Async) (23°C), in the presence of nocodazole (NZ) (3 h, 23°C) or in the presence of nocodazole (3 h, 23°C) followed by a 1-h shift to 37°C (NZ→37). Cdc5-ProA kinase activity was also determined for a wild-type strain expressing Cdc5-ProA (YCH199) arrested in the presence of nocodazole and loaded on the same gel. (g) FACS analysis of DNA content.

Modification of Cdc5p in cdc13-1 cells grown at the nonpermissive temperature is dependent on the DNA damage checkpoint genes, MEC1, RAD53/MEC2, and RAD9. (a) cdc13-1 (YCH216) cells were incubated at 37°C, the nonpermissive temperature for 8 h. Samples for budding index determination and extract preparation were taken at the time intervals shown. (b) cdc13-1 rad53-21 (YCH318), cdc13-1 mec1-1 (YCH317), cdc13-1 rad9::URA3 (YCH316) (rad9-n) and cdc13-1 (YCH216) [+]) cells were incubated at 37°C, the nonpermissive temperature for 3 h. cdc13-1 is the only temperature-sensitive mutation in these strains. Samples for extract preparation were taken at 3 h. (c) cdc13-1 rad 53-21 (YCH318), cdc13-1 mec1-1 (YCH317), and cdc13-1 rad9::URA3 (YCH316) (rad9-n) cells were incubated at 37°C, the nonpermissive temperature for 7 h. Samples for extract preparation were taken at 0 and 7 h. Cdc5-ProA and actin were detected by immunoblotting with anti-IgG and antiactin antibodies, respectively.