Article Figures & Data
Figures
- FIG. 1.
Steady-state levels of Cdc6p through the cell cycle. (A) Yeast cells with a copy of MET-CDC6 and another copy of myc-tagged CDC6 expressed under its own promoter (YSB75) and containing a chromosomal copy of Pds1-HA (YSB441) or Clb2-HA (YSB407) were grown in synthetic medium and arrested with α-factor. Cells were released in rich medium, and samples were taken at 20 min after release and, from then on, every 5 min to be processed for immunoblotting. α-Factor was readded 60 min after release to prevent entry into a new cell cycle. For a comparison of levels of Cdc6 expressed from its own promoter with those reached by GAL overexpression, we measured the levels of Cdc6 F in cells arrested with α-factor in YPRaf medium, treated with galactose for 15 min, and released in YPRaf-Gal. Note that four times less protein was loaded in gels from cells expressing GAL-myc-Cdc6 F than in endogenous myc-Cdc6. (B) Advancement of Pds1 and Clb2 degradation by reduction in Cdc6 levels. Yeast cells with a copy of MET-CDC6 and another copy of myc-tagged CDC6 expressed under its own promoter (YSB75) or an empty vector (YSB431) and containing a chromosomal copy of Pds1-HA (YSB441 and YSB442), Clb2-HA (YSB407 and YSB408), or Clb5-HA (YSB410 and YSB411) were grown in synthetic medium without methionine and arrested with α-factor. Cells were released in synthetic medium without methionine, and 30 min after release, methionine was added. At the indicated times, samples were taken to be processed for immunoblotting and flow cytometry. α-Factor was readded 70 min after release to prevent reentry into another cell cycle. Notice that for panel A, cells were released in rich yeast extract-peptone-dextrose medium and, in panel B, they were released in synthetic medium. Therefore the peak of accumulation of Cdc6p is slightly delayed in panel B.
- FIG. 2.
(A) Schematic diagram of Cdc6 protein. Vertical lines represent CDK sites A to F, and the amino acid numbers at the top of the diagram are given for consensus threonines and serines. (B). Cell cycle delay of cells expressing myc-tagged wild-type and mutant Cdc6p. Cells growing logarithmically in YPRaf medium were transferred to 2% galactose-containing medium, and samples were taken for flow cytometry analysis at the indicated times. The strains used were YSB57, empty vector; YSB58, GAL-CDC6; YSB117, GAL-CDC6 A-C; YSB116, GAL-Cdc6 F; YSB60, GAL-CDC6 A-C+F; YSB61, GAL-CDC6 A-F; YSB62, GAL-CDC6 ΔN8-48; and YSB196, GAL-CDC6 ΔN8-48+F. (C) The degradation of cell cycle-regulated Pds1p and Clb2p is delayed in strains overexpressing Cdc6p. W303 strains expressing either the indicated myc-tagged mutant Cdc6 proteins from the GAL1,10 promoter or an empty vector and expressing either HA-tagged Pds1p (upper panels) or HA-tagged Clb2p (lower panels) were grown in YPRaf to early log phase and arrested with α-factor. After arrest, Cdc6p expression was induced by the addition of galactose. Fifteen minutes later, cells were washed and released from the arrest into YPRaf-Gal. Samples were taken at the time of release and every 10 min thereafter during a 130-min time course to be processed for immunoblotting to detect Pds1-HA and Clb2-HA. The Pds1-HA strains were YSB169, empty vector; YSB170, GAL-CDC6; YSB171, GAL-Cdc6 F; YSB306, GAL-CDC6 A-C+F; YSB307, GAL-CDC6 A-F; and YSB308, GAL-CDC6 ΔN8-48. The Clb2-HA strains were YSB271, empty vector; YSB272, GAL-CDC6; YSB273, GAL-Cdc6 F; YSB274, GAL-CDC6 A-C+F; YSB275, GAL-CDC6 A-F; and YSB304, GAL-CDC6 ΔN8-48. wt, wild type. (D) Benomyl hypersensitivity of Cdc6 phosphorylation site mutants. W303 strains expressing either the indicated myc-tagged mutant Cdc6 proteins from the GAL1,10 promoter or an empty vector, described in the legend for panel B, were grown in glucose-containing selective medium to stationary phase, serially diluted 10-fold, spotted in raffinose (as a control) and raffinose plus galactose plates containing 5 μg/ml benomyl, and incubated at room temperature for 1 week. −, absence of; +, presence of.
- FIG. 3.
(A) Lethality of Cdc6 phosphorylation site mutants in a mih1Δ background. W303 or isogenic mih1Δ (W303 mih1Δ) strains expressing either the indicated myc-tagged mutant Cdc6 proteins from the GAL1,10 promoter or an empty vector were grown in glucose-containing selective medium to stationary phase, serially diluted 10-fold, spotted in either raffinose (as a control) or raffinose plus galactose selective plates, and incubated at 30°C for 2 days. The mih1Δ strains were YSB198, empty vector; YSB199, GAL-CDC6; YSB157, GAL-CDC6 E+F; YSB201, GAL-CDC6 A-C+F; YSB202, GAL-CDC6 A-F; and YSB203, GAL-CDC6 ΔN8-48. (B) Interaction between Cdc6p and Cdc28p. Strains (YSB161, empty vector; YSB162, GAL-CDC6; YSB163, GAL-Cdc6 F; YSB164, GAL-CDC6 A-C; YSB166, GAL-CDC6 A-C+F; and YSB167, GAL-CDC6 A-F) containing HA-tagged Cdc28p under the control of its natural promoter and expressing either the indicated myc-tagged mutant Cdc6 proteins from the GAL1,10 promoter or an empty vector were grown in YPR to mid-log phase, and galactose was added to induce the expression of Cdc6p. After 2 h, total yeast extracts were prepared and subjected to immunoprecipitation (IP). The top panel corresponds to HA immunoprecipitates blotted with the 9E10 anti-myc antibody. The bottom panel corresponds to myc immunoprecipitates blotted with the 12CA5 anti-HA antibody. (C) Yeast cells with a copy of MET-CDC6 and another copy of myc-tagged wild-type CDC6 (YSB75) or CDC6 A-C (YSB133) expressed under its own promoter and containing a chromosomal copy of Pds1-HA or Clb2-HA were grown in synthetic medium without methionine and arrested with α-factor (the Pds1-HA strains were YSB441 and YSB478, and the Clb2-HA strains were YSB407 and YSB472). The cells were further treated as described in the legend for Fig. 1B. (D) Effect of wild-type and mutant Cdc6p overexpression on Cdc28 kinase activity. W303 strains (YSB359, empty vector; YSB360, GAL-CDC6; YSB361, GAL-CDC6 F; and YSB364, GAL-CDC6 A-C+F) containing HA-tagged p86/91 and expressing the indicated forms of myc-tagged Cdc6p under the GAL1,10 promoter were grown to early log phase in YPRaf and arrested with nocodazole (Noc). After nocodazole arrest, galactose was added for 2.5 h and samples were taken to be processed for Western blotting to detect p86/91. wt, wild type.
- FIG. 4.
Suppression of mitotic delay. (A) W303 and W303 cdc55Δ cells expressing the indicated myc-tagged mutant Cdc6 proteins from the GAL1,10 promoter and growing logarithmically in either YPRaf or YPSuc were transferred to 2% galactose-containing medium, and samples were taken for flow cytometry analysis at the indicated times. Flow cytometry profiles of normally cycling cultures (asynchronous; no galactose added) were also obtained. The cdc55Δ strains were YSB335, empty vector; YSB336, GAL-CDC6; YSB337, GAL-CDC6 F; and YSB340, GAL-CDC6 A-C+F. (B) cdc16-264 and cdc16-264 cdc55Δ strains expressing either the indicated myc-tagged mutant Cdc6 proteins from the GAL1,10 promoter or an empty vector were grown in glucose-containing selective medium to stationary phase, serially diluted 10-fold, spotted in either glucose (as a control) or raffinose plus galactose selective plates, and incubated at 30°C for 2 days. The cdc16-264 strains were YSB172, empty vector; YSB173, GAL-CDC6; YSB176, GAL-CDC6 E+F; YSB177, GAL-CDC6 A-C+F; YSB178 GAL-CDC6 A-F; and YSB323 GAL-CDC6 ΔN8-48. The cdc16-264 cdc55Δ strains were YSB416, empty vector; YSB417, GAL-CDC6; YSB418, GAL-CDC6 E+F; YSB419, GAL-CDC6 A-C+F; YSB420, GAL-CDC6 A-F; and YSB421, GAL-CDC6 ΔN8-48. (C) Wild-type (YSB169), cdc55Δ (strains described in the legend for panel A), and swe1Δ strains (YSB248) expressing the indicated myc-tagged mutant Cdc6 proteins from the GAL1,10 promoter and expressing HA-tagged Pds1p were grown to early log phase in YPSuc (cdc55Δ) or YPRaf (swe1Δ) and arrested with α-factor. After arrest, Cdc6p expression was induced by the addition of galactose. Fifteen minutes later, cells were washed and released from the arrest into YPSuc-Gal or YPRaf-Gal. Samples were taken at the time of release and every 10 min thereafter during a 130-min time course to be processed for immunoblotting.
- FIG. 5.
(A) Interaction between Cdc6p and Cdc55p. W303 strains expressing HA-tagged Cdc55p (YSB399) and either the indicated myc-tagged mutant Cdc6p from the GAL1,10 promoter or an empty vector were grown to mid-log phase in YPRaf, galactose was added to induce the expression of Cdc6p for 3 h, and total extracts were prepared. The top and middle panels represent a fraction (1/500) of the total input material in the immunoprecipitation (IP) blotted with the anti-myc-Cdc6 (9E10) and anti-Cdc55-HA (12CA5) antibodies, respectively. The bottom panel represents one-third of the Cdc6-myc immunoprecipitates blotted with the anti-HA antibody 12CA5. The Cdc55-HA strains used were YSB289, empty vector; YSB290, GAL-CDC6; and YSB291, GAL-CDC6 F. wt, wild type. (B) Cell cycle-specific interaction between Cdc6, Cdc55, and Tpd3. Yeast cells with a copy of MET-CDC6 and another copy of myc-tagged CDC6 expressed under its own promoter and containing a chromosomal copy of Cdc55-HA (YSB393) was grown in synthetic medium and arrested with α-factor and released in rich medium. Samples were taken, and total cell extracts were prepared from arrested cells and from cells at the indicated times after release. Cdc6-myc immunoprecipitates prepared with the 9E10 antibody were blotted and probed with 9E10, 12CA5, and α-Tpd3 for Cdc6-myc, Cdc55-HA, and Tpd3, respectively. (C and D) Cdc55-PP2A activity during the cell cycle in wild-type cells (C) or in cells depleted of Cdc6 in mitosis (D). Yeast cells with a copy of MET-CDC6 and another copy of myc-tagged CDC6 expressed under its own promoter (YSB393) or an empty vector (YSB534) and containing a chromosomal copy of Cdc55-HA were grown in synthetic medium without methionine and arrested with α-factor. They were further treated as described in the legend for Fig. 1B. Extracts, immunoprecipitations, and phosphatase assays were carried out at the indicated time points. Histograms represent the mean values and standard errors of three experiments. Immunoprecipitated HA-Cdc55 from both yeast strains that was used to normalize the phosphatase levels is shown below the graphs. −, absence of; +, presence of.
- FIG. 6.
(A) Structure/function analysis of the effect of various Cd6p deletion mutations on the extent of interaction with Cdc55p. W303 strains expressing HA-tagged Cdc55p (YSB399) and carrying either an empty vector or vectors expressing the indicated myc-tagged Cdc6p C-terminal deletion mutant from the GAL1,10 promoter were grown to mid-log phase in YPRaf, galactose was added to induce the expression of Cdc6p for 3 h, and total extracts were prepared and subjected to immunoprecipitation. The top panel corresponds to myc immunoprecipitates blotted with the 9E10 anti-myc antibody. The bottom panel corresponds to myc immunoprecipitates blotted with the 12CA5 anti-HA antibody. The Cdc55-HA strains were YSB289, empty vector; YSB290, GAL-CDC6; YSB423, GAL-CDC6 Δ401-513; YSB424, GAL-CDC6 Δ428-513; YSB425, GAL-CDC6 Δ494-513; YSB427, GAL-CDC6 Δ401-513 A-C; YSB428, GAL-CDC6 Δ428-513 A-C; and YSB429, GAL-CDC6 Δ494-513 A-C. (B) Pds1-HA degradation kinetics in cells expressing endogenous levels of either wild-type (wt) Cdc6p or different Cdc6 deletion mutants, each of which is indicated to the left of each blot. As a control of nonfunctional Cdc6p, the effect of the expression of Cdc6 K114E is also included. Yeast cells with a copy of MET-CDC6 (K4055) and a plasmid-borne copy of the indicated myc-tagged CDC6 mutant expressed under the control of its own promoter or an empty vector and also containing a chromosomal copy of Pds1-HA were subjected to the same experimental conditions as those described in the legend for Fig. 1B. The strains used were YSB441, CDC6; YSB442, empty vector; YSB488, CDC6 Δ401-513; YSB490, Δ494-513; YSB482, Δ401-513 A-C; YSB483 Δ494-513 A-C; and YSB479, CDC6 K114E.
