Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1

Abstract

Ubiquitin-mediated proteolysis controls the abundance of many cell cycle regulatory proteins. Recent work in Saccharomyces cerevisiae suggests that a complex consisting of Cdc53, Skp1, and a third component known as an F-box protein (termed SCF) in combination with Cdc34 specifically targets regulatory proteins for degradation, and that substrate specificity is likely to be mediated by the F-box subunit. A screen for genetic interactions with a cdc34 mutation yielded MET30, which encodes an F-box protein. MET30 is an essential gene required for cell cycle progression and met30 mutations interact genetically with mutations in SCF components. Furthermore, physical interactions between Met30, Cdc53, Cdc34, and Skp1 in vivo provide evidence for an SCFMet30 complex. We demonstrate the involvement of Met30 in the degradation of the Cdk-inhibitory kinase Swe1. Swe1 is stabilized in met30 mutants and GST-Met30 pull-down experiments reveal that Met30 specifically binds Swe1 in vivo. Furthermore, extracts prepared from cdc34 or met30 mutants are defective in polyubiquitination of Swe1. Taken together, these data suggest that SCF-mediated proteolysis may contribute to the regulation of entry into mitosis. Our data, in combination with previously published results, also provide evidence for distinct SCF complexes in vivo and support the idea that their F-box subunits mediate SCF substrate specificity.

Figure 1

Genetic interactions between MET30, CDC34, and SCF components. (A) Herterozygous disruption of MET30 enhances the temperature sensitivity of diploid cdc34-3 mutants. Cells were grown at 23°C, sequentially replica plated to four new plates, and incubated at the temperatures indicated. (B) High-copy suppression. met30-6, cdc53-1, and cdc4-3 mutants were transformed with 2μ-based plasmids harboring no insert (vector), MET30 (2μ-MET30), or CDC34 (2μ-CDC34). Transformants were grown at 23°C and replicas were incubated at 34°C (met30-6 and cdc53-1) or 30°C (cdc4-3). To analyze genetic interactions between MET30 and SKP1, met30-6 mutants expressing SKP1 under control of the GAL1 promoter were grown on glucose plates at 23°C, replica plated to galactose plates to induce expression of SKP1 and incubated at the restrictive temperature of 34°C.

Figure 2

Physical interaction of Met30 with Skp1, Cdc34, and Cdc53. (A) Met30 forms a complex with Skp1 in vivo. Cells harboring an endogenously expressed (HA)3-tagged MET30 and expressing GST–SKP1 or GST under the control of the CUP1 promotor were grown in the presence of 100 μm copper to an OD₆₀₀ ≅ 0.5. Protein extracts were incubated with glutathione beads. After several washes with binding buffer the beads were washed with binding buffer, including 0.8 m of sodium chloride (wash). Bound proteins were eluted with glutathione and subjected to Western blot analysis with an antibody directed against the HA epitope. Probing the blot with antibodies directed against GST revealed a two- to threefold excess of GST compared with GST–Skp1 (data not shown). (B) Met30 is in a complex with Cdc34 and Cdc53 in vivo. Cells expressing a RGS6H epitope-tagged MET30 under control of the GAL1 promotor and harboring either endogenously expressed (HA)3-tagged CDC34 or CDC53 were grown in galactose to an OD₆₀₀ ≅ 0.5. Immunocomplexes were purified with 12CA5 antibodies (directed against the HA epitope) coupled to protein A, separated by SDS-PAGE, and analyzed by immunoblotting with antibodies directed against the HA or the RGS6H epitopes to detect Cdc53 and Cdc34 or Met30, respectively. Cells expressing Met30(RGS6H) but no HA-tagged protein were used as a control (no HA tag).

Figure 3

Hyperactivation of the Swe1 pathway in met30 and cdc34 mutants. Cells were grown at 23°C in YEPD and shifted to 37°C for 5 hr, followed by fixation in 70% ethanol and visualization by DIC microscopy. Strains were as follows (percent of elongated cells or buds in the population are indicated in parentheses): (A) wild type (0%), (B) mih1::LEU2 (0%), (C) met30-6 (23%), (D) met30-6 mih1::LEU2 (50%), (E) met30-6 mih1::LEU2 swe1::URA3 (10%—these were smaller and less straight than the elongated buds in C and D), (F) cdc34-3 sic1::URA3 (10%), (G) cdc34-3 sic1::URA3 mih1::LEU2 (65%). Two hundred cells were counted in each sample. Bar, 10 μm. The graph illustrates the percentage of cells with elongated buds.

Figure 4

Swe1 is stabilized in met30 but not cdc4 mutants. (A) Wild-type (top), cdc4-3 (middle), or met30-6 (bottom) cells containing the GAL1–SWE1–myc12 construct were shifted to 37°C for 2 hr. Galactose was then added to 2% and the cells were incubated for a further 10 min to induce Swe1–myc12 expression. Cells were then labeled 10 min at 37°C followed by termination of Swe1 synthesis and labeling during a 4-hr time course. The immunoprecipitated Swe1 was analyzed by SDS-PAGE and PhosphorImaging. (Left lane) Immunoprecipitates from cells lacking the Swe1–myc construct. The band running below Swe1–myc12 (asterisk) is a background band. (B) A similar experiment was conducted with synchronized cells. Wild-type and met30-6 cells were arrested by addition of 200 mm hydroxyurea (HU) for 3 hr at 23°C prior to the temperature shift to 37°C. Cell cycle arrest was monitored visually (>80% large budded cells). Pulse-chase analysis was performed as described in A but in the continuous presence of 200 mm HU. (C) The ³⁵S-labeled Swe1–myc12 signal was quantitated by PhosphorImager and ImageQuant v1.2 software, normalized to the background band and expressed as percent of signal at t = 0.

Figure 5

Met30 interacts physically with Swe1. Cells were grown in YEP raffinose to an OD₆₀₀ ≅ 0.3 and expression of Swe1(myc)12, Clb2 and GST–Met30 was induced by addition of galactose for 3 hr. As a control, a strain expressing GST instead of GST–Met30 was processed in parallel. Protein complexes were purified on glutathione beads and parallel samples were separated on two SDS–polyacrylamide gels. After immunoblotting, one of the blots was probed with a polyclonal antibody directed against GST to detect GST–Met30 and GST, respectively. The other blot was analyzed with the 9E10 antibody against the myc epitope on Swe1. (Asterisk) A protein crossreacting with the anti-GST antibody.

Figure 6

Swe1 in vitro ubiquitination. (A) Swe1 is ubiquitinated in whole cell extracts. ³⁵S-labeled Swe1 was incubated with whole yeast cell extracts at 23°C (lanes 2,3) or on ice (lane 1) in the presence of an ATP-regenerating system and ubiquitin (lanes 1,2) or mutant R48-ubiquitin (lane 3), respectively. The reaction mix was separated by SDS-PAGE and analyzed with a PhosphorImager. (B) Swe1 ubiquitination is defective in cdc34 and met30 mutants. In vitro ubiquitination activity of extracts prepared from cdc34-3 or met30-6 mutants was compared with wild-type extracts. (Lane 5) Complementation experiment in which both mutant extracts were mixed (ratio, 2:1 = cdc34-3:met30-6). Extracts were preincubated at 23°C for 5 min to allow complex formation. ³⁵S-labeled Swe1, ATP-regenerating system and ubiquitin were then added and reactions incubated at 23°C (lanes 2– 6) or on ice (lane 1). No extract was added to the reaction shown in lane 6. Reaction products were analyzed as described in A.

Figure 7

Met30 is closely related to Drosophila Slimb. (A) Dendrogram illustrating that Met30 is more closely related to Slimb and a human homolog h-βTrCP (Margottin et al. 1998) than Cdc4. The dendrogram was generated with GeneWorks 2.5 software. (B) Met30 and Slimb sequences were aligned with Clustalw. Identical amino acids are shown as white letters on black background, and related residues as black letters on gray background.

Figure 7

Met30 is closely related to Drosophila Slimb. (A) Dendrogram illustrating that Met30 is more closely related to Slimb and a human homolog h-βTrCP (Margottin et al. 1998) than Cdc4. The dendrogram was generated with GeneWorks 2.5 software. (B) Met30 and Slimb sequences were aligned with Clustalw. Identical amino acids are shown as white letters on black background, and related residues as black letters on gray background.