Overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p inhibits ubiquitin ligase activities of their SCF complexes

Abstract

Ubiquitin ligases direct the transfer of ubiquitin onto substrate proteins and thus target the substrate for proteasome-dependent degradation. SCF complexes are a family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. Distinct SCF complexes, defined by a particular F-box protein, target different substrate proteins for degradation. Although a few have been identified to be involved in important biological pathways, such as the cell division cycle and coordinating cellular responses to changes in environmental conditions, the role of the overwhelming majority of F-box proteins is not clear. Creating inhibitors that will block the in vivo activities of specific SCF ubiquitin ligases may provide identification of substrates of these uncharacterized F-box proteins. Using Saccharomyces cerevisiae as a model system, we demonstrate that overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p results in specific inhibition of their SCF complexes. Analyses of mutant amino-terminal alleles demonstrate that the interaction of these polypeptides with their full-length counterparts is an important step in the inhibitory process. These results suggest a common means to inhibit specific SCF complexes in vivo.

FIG. 1.

Overproduction of GST-Cdc4(1-278)p inhibits SCF^Cdc4p complex activity. (A) Schematic diagram of Cdc4p, illustrating the positions of the F box and WD-40 repeats, the GST-Cdc4p fusions made in this study, and a summary of their ability to permit growth when overproduced in Y382 cells. (B and C) Comparison of the terminal phenotypes of cells containing the cdc4-3 temperature-sensitive mutation and cells overproducing GST-Cdc4(1-278)p. Panel B is an overlay of differential interference contrast and fluorescent images of the indicated cells stained with propidium iodide, and panel C shows results of flow cytometry analysis. In panels B and C, the left panels show cdc4-3-containing cells and the right panels show Y382 cells overproducing GST-Cdc4(1-278)p. For both panel B and panel C, cells were grown to mid-logarithmic phase. cdc4-3-containing cells (strain A.2.7.A3p) were then shifted from 23 to 37°C for either 3 h (B) or the indicated length of time (C). Y382 cells containing pGST-Cdc4(1-278) were grown to mid-logarithmic phase and shifted to medium containing galactose for 8 h (B) or for the indicated time (C). (D) The terminal morphology of GST-Cdc4(1-278)p overproduction is dependent on SIC1. Shown are an overlay of differential interference contrast and fluorescent images of sic1Δ cells overproducing GST-Cdc4(1-278)p. PMY1 cells (sic1Δ) transformed with pGST-CDC4(1-278) were plated onto solid medium containing galactose and incubated overnight. (E) GST-Cdc4(1-278)p overproduction results in the accumulation of Sic1p. Lysates prepared from cells producing either HA-Sic1p or untagged Sic1p and the indicated GST fusion were subjected to Western immunoblot analysis with antibodies raised against GST (upper panel) or HA (middle panel). The lower panel is the signal obtained from a band cross-reacting to anti-HA antibodies and is used as a loading control. (F) GST-Cdc4(1-278)p overproduction does not result in Cln2p accumulation. Shown are results of Western immunoblot analysis of lysate prepared from cdc34-1-containing cells (strain G101) and Y382 cells, producing the indicated GST fusion, to detect HA-Cln2p expressed from the GAL1 promoter. G101 cells were grown to early logarithmic phase at 23°C, shifted to 37°C for 3 h, and incubated in the presence of galactose for a further hour. Y382 cells were grown to mid-logarithmic phase and incubated in the presence of galactose for 8 h. The position of HA-Cln2p is as indicated by a bracket, and the asterisk indicates a cross-reacting band. (G) The toxicity of GST-Cdc4(1-278)p overproduction is suppressed by elevated levels of Cdc4p. Tenfold serial dilutions of saturated Y382 cultures overproducing the indicated proteins were plated onto medium containing galactose and incubated at 30°C for 4 days.

FIG. 2.

Overproduction of GST-Met30(1-225)p inhibits SCF^Met30p complex activity. (A) Schematic diagram of Met30p, illustrating the positions of the F box and WD-40 repeats, the GST-Met30p fusions made in this study, and a summary of their ability to permit growth when overproduced in Y382 cells. (B to D) Suppression of GST-Met30(1-225)p- and GST-Met30(150-225)p-mediated toxicity. Tenfold serial dilutions of saturated cultures overproducing the indicated proteins were plated onto medium containing galactose and incubated at 30°C for 4 days. Strain Y382 is used in panels B and C, and strains KS410 (MET4) and KS255 (met4Δ) are used in panel D. (E) GST-Met30(1-225)p overproduction results in decreased abundance of Met4p modification. Shown are results of Western immunoblot analysis with anti-MYC antibodies of lysate prepared from cells containing MET4-(18myc) that were grown to mid-logarithmic phase and incubated in the presence of galactose for 5 h to induce the production of the indicated GST fusion. Relative exposure times are indicated. The bottom panel, used as a loading control, is a cross-reacting band. The arrow indicates unmodified Met4p; slower-migrating species are modified. (F) GST-Met30(1-225)p overproduction causes a G₁ arrest. Shown are results of flow cytometry analysis of Met30(1-225)p-overproducing cells incubated in galactose-containing medium for the indicated times.

FIG. 3.

GFP-Cdc4(1-278)p and GFP-Met30(1-225)p accumulate in the nucleus. Y382 cells producing the indicated GFP fusion protein from the GAL1 promoter were grown in sucrose to mid-logarithmic phase and incubated in the presence of galactose for a further 3 to 4 h. Nuclei were visualized by adding DAPI to the culture before observation as described in Materials and Methods. DIC, differential interference contrast.

FIG. 4.

GST-Cdc4(1-278)p is present in a high-molecular-weight complex. Shown are results of Western immunoblot analysis of the indicated GST fusions with anti-GST antibodies obtained after fractionation of whole-cell lysate on a Sephacryl S-300 HR column. The lysate was prepared from Y382 cells grown to mid-logarithmic phase in sucrose-containing medium and then shifted to galactose-containing medium. The top two panels, for GST-Cdc4(1-278)p and GST, show results for samples that expressed these proteins separately. The lower two panels, for GST-Cdc4(1-278)p and GST-Cdc4p, show results for samples that coexpressed these proteins. Size markers are shown in kilodaltons.

FIG. 5.

Cdc4(1-278)p physically associates with Cdc4p. (A) Coimmunoprecipitation of GST-Cdc4p and GST-Cdc4(1-278)p with FLAG-Cdc4p. Lysates from Y382 cells expressing the indicated GST-Cdc4p fusions with either FLAG-Cdc4p or untagged Cdc4p were either subjected to Western immunoblot analysis with anti-GST antibodies (upper panel) or incubated in the presence of anti-FLAG resin. Proteins immobilized on the resin were subjected to Western immunoblot analysis with antibodies raised against FLAG and GST (middle and lower panels). (B) Two-hybrid analysis of Cdc4p constructs. Patches of PJ69-4A cells transformed with the indicated constructs were replica plated onto medium lacking adenine and incubated for 4 days at 30°C.

FIG. 6.

Analysis of Cdc4(1-278)p mutants. (A) Growth assay of Y382 cells overproducing wild-type and mutant GST-Cdc4(1-278)p alleles. Tenfold serial dilutions of saturated cultures overproducing the indicated proteins were plated onto medium containing galactose and incubated at 30°C for 4 days. (B) Morphology of Y382 cells overproducing wild-type and mutant GST-Cdc4(1-278)p alleles, as indicated. (C) Western immunoblot analysis of lysate prepared from cells producing the indicated protein: anti-GST signal (upper panel) and a cross-reacting band (lower panel). (D) Two-hybrid analysis was performed on PJ69-4A cells containing pGAD-CDC4 and the indicated pGBD construct, and the cells were plated onto medium lacking adenine and incubated at 30°C for 4 days.

FIG. 7.

Analysis of Met30(1-225)p mutants. (A) Growth assay of Y382 cells overproducing wild-type and mutant GST-Met30(1-225)p alleles. Tenfold serial dilutions of saturated cultures overproducing the indicated proteins were plated onto medium containing galactose and incubated at 30°C for 4 days. (B) Western immunoblot analysis of lysate prepared from cells producing the indicated GST-Met30(1-225)p allele. (C) Coimmunoprecipitation analysis of GST-Met30(1-225)p alleles with HA-Met30p. Lysates prepared from Y382 cells producing the indicated GST-Met30(1-225)p allele with or without HA-Met30p, as indicated, were subjected to Western immunoblot analysis with anti-GST antibodies (upper panel) or incubated in the presence of anti-HA resin. Proteins immobilized on the resin were subjected to Western immunoblot analysis with antibodies raised against HA (middle panel) and GST (lower panel).