Figure 1.
Identification of candidate APC/C substrates by protein expression profiling.
(A) Analysis of protein levels in G1 and M. Cells expressing the indicated TAP-tag fusion proteins were arrested in G1 or M phase and the corresponding cell extracts were immunoblotted with PAP antibodies to detect TAP-tagged proteins. (B) Protein degradation in G1 and M. Cells were arrested in either G1 or M phase and treated with 500 µg/ml cycloheximide for the indicated times. Cell extracts were prepared and immunoblotted to examine TAP-tag protein levels as in (A).
Figure 2.
APC/CCdh1-dependent degradation of Fir1, Mps1, and Ybr138C in G1.
(A) Wild-type and cdc23-1 cells were arrested in G1 with alpha factor and transferred to 37°C to inactivate Cdc23-1, a core subunit of the APC/C. cdc28-13 cdh1Δ cells were arrested in G1 by incubation at the non-permissive temperature (37°C). The expression of FIR1, MPS1, and YBR138C was induced from GAL1p followed by addition of 2% dextrose and 500 µg/ml cycloheximide to terminate protein synthesis. Samples were withdrawn at the indicated times and processed for immunoblotting with PAP antibodies. (B) Cells carrying an empty vector (lanes 1–4) or GALLp-cdh1-m11 (lanes 5–8) were grown in the presence of raffinose and induced with 2% galactose for the indicated times. Cdh1-m11 lacks sites of inhibitory phosphorylation and is constitutively active. Samples were processed for immunoblotting to examine endogenous levels of Fir1, Mps1, and Ybr138C.
Figure 3.
Degradation of Ybr138C and Fir1 and their interaction with Cdh1.
(A)Two hybrid assay to detect Cdh1-substrate interactions. The bait in this assay was either wild-type Cdh1-ΔN200 or Cdh1-ΔN200-D12, which contains mutations within Cdh1's WD40 domain previously demonstrated to disrupt Cdh1's interaction with the substrate D-Box [37], [41]. The prey consisted of the APC/C substrate Hsl1 or the APC/C pseudosubstrate inhibitor Acm1 [37]. (B) Yeast two-hybrid interaction between Cdh1 and Ybr138C. Cells expressing Cdh1 or Cdh1-D12 and Ybr138C, Ybr138C-ΔN200, or Ybr138C-N270 were tested for growth on selective medium, which indicates interaction of the respective proteins. Bottom: Schematic representation of Ybr138C indicating positions of potential D-boxes (DB). (C) Degradation of Ybr138C requires its N-terminal region but not putative D-boxes. Cells expressing wild-type YBR138C, YBR138C-3mdb, and YBR138C-ΔN80 from a GAL1 promoter were arrested in G1 and induced with galactose. Samples were withdrawn at the indicated times after cycloheximide addition and processed for blotting with PAP antibodies as in Fig. 2A. (D) cdc15-2 and cdc15-2 cdh1Δ cells carrying endogenous YBR138C-TAP were synchronized in mitosis by incubation at 37°C for 3 hours and released at 23°C at time zero. Samples were withdrawn at the indicated times and processed for immunoblotting. Cdc28 was used as a loading control. (E) Yeast two-hybrid interaction between Cdh1 and Fir1. As in (B) but cells carried Fir1 or Fir1-N200 instead of Ybr138C. Bottom: Schematic of Fir1 indicating its potential D-boxes (DB) and its SUMO-interacting motif (SIM).
Figure 4.
Cdh1 bead binding assay for TAP-tagged fusion proteins from whole cell extracts.
(A) Tos4 and Pdr3 interact with wild-type Cdh1 but not with Cdh1-D12 in the yeast two-hybrid system. (B) A Cdh1-bead binding assay. An extract from cells expressing Clb2-TAP was incubated with the indicated beads and binding was detected using PAP antibodies. WCE, whole cell extract. (C) Left panel: Extracts of cells expressing the indicated TAP-tag fusion proteins were incubated with Cdh1 beads or control beads and the levels of bound TAP-tagged proteins were detected by blotting with PAP antibodies (lanes, 1–10). Right panel: 2% of the input material (whole cell extract, WCE) for the binding assays is shown (lanes 11–15).
Figure 5.
Tos4 and Pdr3 are potential APC/CCdh1 substrates.
(A) Tos4 and Pdr3 are partially stabilized in cdh1Δ cells. Asynchronously growing WT or cdh1Δ cells expressing Tos4-HA or Pdr3-HA were treated with cycloheximide for the indicated times. HA-fusion protein levels from the cell extracts were determined by immunoblotting with anti-HA antibodies. A non-specific cross-reactive protein (*) served as a loading control. (B) Overexpressed Pdr3-HA but not Tos4-HA was partially stabilized in an APC/C mutant strain. Cells were arrested in G1 and induced to express the indicated HA-tagged protein by galactose addition. Cells were shifted to 37°C to inactivate the APC/C in the cdc23-1 mutant cells, and then treated with cycloheximide for the indicated times prior to extract preparation and protein detection by immunoblotting with anti-HA antibodies. (C) The carboxyl-terminal region (C222) of Tos4 contains multiple motifs that mediate interaction with Cdh1 in the yeast two-hybrid system. Top: Schematic of Tos4 constructs showing the locations of the putative KEN box and DB motifs. Tos4-mut. 5 contains mutations of the four C-terminal D-box and one KEN box motifs. (D) Tos4-C222 was efficiently ubiquitinated by APC/CCdh1 in vitro. The N267 and C222 forms of Tos4 are shown in (C), as are the sites of the various DB mutations. Locations of the putative Tos4 degrons are as follows: DB1, 232; DB2, 237; KEN, 365; DB3, 414; DB4, 418; DB5, 458; DB6, 469.
Figure 6.
Stabilization of Ybr138C or Mps1 reduces cell fitness.
(A) Co-cultures of wild-type and 2xYBR138C-TAP cells. Cells carrying two copies of the YBR138C gene were genetically marked (with TRP1) and grown together with wild-type (W303) cells (marked with LEU2) in complete medium for four days with dilution of the co-cultures once per day. The cultures were tested daily for the presence of TRP1 and LEU2 auxotrophs by plating on selective media. The markers were swapped, the experiment repeated, and the averaged results of two trials were plotted. The fraction of 2xYBR138C-TAP cells on day 0 was arbitrarily set to 100%. (B) Cell cycle degradation of Mps1 and Mps1-3mdb. MET-CDC20 cells carrying endogenously expressed TAP-tagged MPS1 or mps1-3mdb were synchronized in mitosis by incubation with 5 mM methionine for 2.5 hours to deplete Cdc20. Cells were released from the arrest into methionine-free medium. Samples were taken at the indicated times after release and processed for blotting to detect TAP-tagged proteins. (C) Stabilization of Mps1 by mutation of its D-boxes. The stabilities of Mps1 and Mps1-3mdb in G1 were assessed as in Figure 2A. (D) Elevated expression of Mps1-3mdb. Cells carrying endogenously expressed Mps1 or Mps1-3mdb were arrested in G1 and M phase. The levels of Mps1-TAP were visualized by immunoblotting. Lanes 1 and 4 show the levels of these proteins in cell extracts prepared from asynchronous cell cultures. (E) Expression of Mps1-3mdb reduces cell fitness in the presence of mild spindle disruption. mps1-3mdb cells were genetically marked and grown together with wild-type MPS1 cells. Cells were grown in complete medium in the absence (open squares) or presence (closed circles) of 6 µg/ml nocodazole for three days with dilution of the co-cultures once per day. Samples of the cultures were plated daily to determine the relative numbers of MPS1 and mps1-3mdb cells present. The markers were swapped, the experiment repeated, and the averaged results of three independent trials are shown. (F) Expression of Mps1-3mdb delays mitotic exit in the presence of mild spindle disruption. Asynchronous cells expressing endogenous MPS1 (open squares) or mps1-3mdb (closed circles) were treated with 100 ng/ml alpha factor in the absence or presence of a semi-permissive concentration of nocodazole (6 µg/ml) at time zero. G1-arrested cells were counted at 30-minute intervals. The averaged results of two trials showing the percentage of cells passing through mitosis into G1 was calculated and plotted. In (A), (E) and (F), error bars denote standard errors.