The Ubp15 deubiquitinase promotes timely entry into S phase in Saccharomyces cerevisiae

Abstract

The anaphase-promoting complex in partnership with its activator, Cdh1, is an E3 ubiquitin ligase responsible for targeting cell cycle proteins during G1 phase. In the budding yeast Saccharomyces cerevisiae, Cdh1 associates with the deubiquitinating enzyme Ubp15, but the significance of this interaction is unclear. To better understand the physiological role(s) of Ubp15, we examined cell cycle phenotypes of cells lacking Ubp15. We found that ubp15∆ cells exhibited delayed progression from G1 into S phase and increased sensitivity to the DNA synthesis inhibitor hydroxyurea. Both phenotypes of ubp15∆ cells were rescued by additional copies of the S-phase cyclin gene CLB5. Clb5 is an unstable protein targeted for proteasome-mediated degradation by several pathways. We found that during G1 phase, the APC(Cdh1)-mediated degradation of Clb5 was accelerated in ubp15∆ cells. Ubp15 interacted with Clb5 independent of Cdh1 and deubiquitinated Clb5 in a reconstituted system. Thus deubiquitination by Ubp15 counteracts APC activity toward cyclin Clb5 to allow Clb5 accumulation and a timely entry into S phase.

FIGURE 1:

UBP15 activity is required for normal progression through S phase. (A) Wild-type and ubp15∆ cells were synchronized in G1 phase in the presence of the mating pheromone α-factor and released from the arrest. The progression of the synchronized cells through the next cell cycle was monitored by FACS analysis. (B) The ratios of the numbers of cells with 2N and 1N DNA contents as determined by FACS analysis were calculated and plotted for each time point. The plots represent dynamical changes in 2N:1N content for wild-type and ubp15∆ strains. (C) The percentage of small-budded cells was determined in wild-type, ubp15∆, and ubp15∆ 2xCLB5 cells at the indicated times after release from a G1 phase arrest as in A.

FIGURE 2:

Ubp15 stabilizes Clb5. (A, B) Wild-type (A) and ubp15∆ (B) cells were synchronized and released from G1 arrest as in Figure 1A. The abundance of endogenous Clb5-TAP and Clb6-Myc was determined by immunoblotting with anti-TAP and anti-Myc antibodies, respectively. The blots were stripped and reprobed with anti-PSTAIR antibodies to detect Cdc28 as a loading control. (C) The expression of GAL1p-CLB5 (lanes 1–10) and CLB5-mdb (lanes 11–15) was induced in α-factor–arrested wild-type and ubp15∆ cells. The levels of Clb5-TAP were determined by immunoblotting as in A at the indicated times after addition of 2% dextrose and 500 μg/ml cycloheximide. (D) The Clb5 levels in C were quantitated using ImageJ software (National Institutes of Health, Bethesda, MD), normalized, and plotted.

FIGURE 3:

APC^Cdh1 mediates Clb5 degradation during G1 phase. (A) Strains carrying the temperature-sensitive cdc28-13 allele and the indicated additional mutations were arrested in G1 phase by shift to the nonpermissive temperature. The expression of GAL1p-CLB5 was induced and Clb5 levels analyzed after addition of dextrose and cycloheximide as in Figure 2C. The levels of Clb5 protein in the indicated yeast strains was measured, normalized, and plotted as in Figure 2D. (B) APC-dependent ubiquitination of Clb5-N150 in the absence (lane 1) and presence (lane 2) of Cdh1. HA-Clb5 and its ubiquitin conjugates were detected by immunoblotting with 12CA5 antibodies to the HA tag. (C) ³⁵S-labeled Clb5 was ubiquitinated by APC^Cdh1 in the presence of methyl-ubiquitin (lanes 2–4). Ubiquitination reactions also contained Ubp15 (lane 3) or catalytically inactive Ubp15-C214 (lane 4), as indicated. The reaction products were visualized by autoradiography.

FIGURE 4:

An extra copy of CLB5 restores near wild-type Clb5 expression in ubp15∆ cells. (A) Expression of Clb5 in asynchronous cultures of ubp15∆ cells carrying 1xCLB5-TAP (lane 1), 2xCLB5-TAP (lane 2), and 3xCLB5-TAP (lane 3). Cell extracts were immunoblotted with PAP antibodies to detect the TAP-tagged Clb5 proteins. (B) Wild-type and ubp15∆ cells carrying one or two copies of CLB5-TAP were released from G1 arrest and probed for the presence of Clb5 at the indicated times.

FIGURE 5:

An extra copy of CLB5 restores a normal cell cycle to ubp15∆ cells. (A) Wild-type and ubp15∆ strains carrying one or two copies of CLB5-TAP were released from G1 arrest, and their progression through the cell cycle was monitored by FACS analysis. The asynchronous sample of ubp15∆ cells is the same as in Figure 1A. (B) Plots of the ratios of the numbers of cells with 2N and 1N DNA contents in A. (C) Serial dilutions of the indicated strains were spotted on YPD plates in the absence and presence of 100 mM hydroxyurea. Plates were incubated for 2 d at 30°C.

FIGURE 6:

Insertion of a KEN box transforms Clb5 into a better APC^Cdh1 substrate but does not phenocopy ubp15∆ cells. (A) Clb5, Clb5-K⁸⁷EN, and Clb5-mdb-K⁸⁷EN were expressed from a GAL1p promoter in α-factor–arrested cells. The levels of Clb5-TAP were determined by immunoblotting as in Figure 2C at the indicated times after addition of 2% dextrose and 500 μg/ml cycloheximide. (B) The Clb5 levels in A were quantitated using ImageJ software, normalized, and plotted. (C) Pattern of Clb5 and Clb5-K⁸⁷EN expression in cells synchronized in G1 and released. TAP-tagged proteins were detected by immunoblotting. (D) The indicated strains were synchronized in G1 and released from the arrest, and their progression through the cell cycle was monitored by FACS analysis. The ubp15∆ samples are the same as in Figure 1A, as is the asynchronous wild-type sample. (E) Wild-type, ubp15∆, clb5∆, and CLB5-K⁸⁷EN cells were serially diluted and plated on YPD in the absence or presence of 100 mM HU. The plates were incubated for 2 d at 30°C and photographed.

FIGURE 7:

The interaction of Ubp15 with Cdh1 is not necessary for deubiquitination of Clb5. (A) Cdh1-bead binding assay. Yeast extracts carrying the indicated UBP-TAP fusion proteins were incubated with empty (odd lanes) or Cdh1-bound beads (even lanes). Protein binding to the beads was detected using PAP antibodies. (B) Ubp15-bead binding assay. Yeast extracts from TAP-tagged strains were incubated with GST-Ubp15 beads, and bound proteins were visualized by immunoblotting with PAP antibodies. The bands shown in each row were from the same gel and had similar exposures. (C) Strains cdc28-13 cdh1∆ (lanes 1–5), cdc28-13 cdh1∆ GAL-CDC20 (lanes 6–10), and cdc28-13 cdh1∆ ubp15∆ GAL-CDC20 (lanes 11–15) were arrested in G1 phase by growth at the nonpermissive temperature (37°C) for 3 h. The expression of CDC20 and CLB5 was induced, and the stability of Clb5 was examined after addition of cycloheximide as in Figure 3A. The relative levels of Clb5 in each strains were quantitated and plotted as in Figure 2D.