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. 2000 Nov;20(21):7845-52.
doi: 10.1128/MCB.20.21.7845-7852.2000.

The abundance of Met30p limits SCF(Met30p) complex activity and is regulated by methionine availability

D B Smothers  1 , L Kozubowski, C Dixon, M G Goebl, N Mathias
Affiliations

The abundance of Met30p limits SCF(Met30p) complex activity and is regulated by methionine availability

D B Smothers et al. Mol Cell Biol. 2000 Nov.

Abstract

Ubiquitin-mediated degradation plays a crucial role in many fundamental biological pathways, including the mediation of cellular responses to changes in environmental conditions. A family of ubiquitin ligase complexes, called SCF complexes, found throughout eukaryotes, is involved in a variety of biological pathways. In Saccharomyces cerevisiae, an SCF complex contains a common set of components, namely, Cdc53p, Skp1p, and Hrt1p. Substrate specificity is defined by a variable component called an F-box protein. The F- box is a approximately 40-amino-acid motif that allows the F-box protein to bind Skp1p. Each SCF complex recognizes different substrates according to which F-box protein is associated with the complex. In yeasts, three SCF complexes have been demonstrated to associate with the ubiquitin-conjugating enzyme Cdc34p and have ubiquitin ligase activity. F-box proteins are not abundant and are unstable. As part of the SCF(Met30p) complex, the F-box protein Met30p represses methionine biosynthetic gene expression when availability of L-methionine is high. Here we demonstrate that in vivo SCF(Met30p) complex activity can be regulated by the abundance of Met30p. Furthermore, we provide evidence that Met30p abundance is regulated by the availability of L-methionine. We propose that the cellular responses mediated by an SCF complex are directly regulated by environmental conditions through the control of F-box protein stability.

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Figures

FIG. 1
FIG. 1
Characterization of the GST-Met30p fusion. (a) Low-level production of GST-Met30p is sufficient for complementation of a met30-6 temperature-sensitive allele. PY283 cells containing the met30-6 temperature-sensitive mutation were transformed with either pEG(KG) or pGSTMET30-3, which allowed the production of the indicated protein. Patches were made on SD medium, and cells were incubated at the indicated temperature for 3 days. (b) NMYmet30ΔMET25-lacZ cells containing pGSTMET30-3 or a wild-type MET30 congenic strain were grown in medium in the presence (+) of a repressing concentration of l-methionine or in the absence (−) of l-methioine. The reported values represent averages of two independent assays and were expressed as nanomoles of substrate transformed per minute per milligram of protein. The individual measurements deviated from the average values shown here by 20% or less. (c) Induction time course of GST-Met30p and GST. We performed an anti-GST Western immunoblot analysis of lysate prepared from cells, grown to mid-logarithmic growth phase, containing either pEG(KG) or pGST-MET30, which produced the indicated protein that had been induced for 0, 1, 2, and 3 h by galactose. A cross-reacting band is used as a loading control. (d) Met30p is an unstable protein. We performed time course experiments to measure the stability of GST-Met30p fusion (see Materials and Methods for details). A cross-reacting band is used as a loading control.
FIG. 2
FIG. 2
Effect of Met30p overproduction. (a) Methionine prototrophic Y382 cells were transformed with either pEG(KG) or pGSTMET30-3, which allowed the production of the indicated protein. Patches were made on the indicated medium, and cells were incubated for 3 to 4 days. (b) Flow cytometry analysis of Y382 cells, transformed with pGSTMET30-3, grown in the absence of methionine, before production of GST-Met30p (upper panel) and after production of GST-Met30p (lower panel). (c) C114 cells, MET25-lacZ, transformed with a plasmid that allowed the production of the indicated protein, were grown in medium containing (+) a repressing concentration of l-methionine or in the absence (−) of l-methionine. The reported values represent averages of three independent assays and were expressed as nanomoles of substrate transformed per minute per milligram of protein. The individual measurements deviated from the average values shown here by 20% or less.
FIG. 3
FIG. 3
GST-Met30p steady-state abundance and stability are dependent on methionine availability. (a) Anti-GST and anti-Cdc34p immunoblot analysis of soluble protein extracts from Y382 cells containing either pEG(KG) or pGSTMET30-3, whose expression was induced for 3 h by the addition of galactose. Cdc34p, whose abundance is unaffected by methionine (Fig. 4), is used as a loading control, and its position is indicated (*). (b) A fivefold-longer exposure time of panel a. (c) Quantitation of GST and GST-Met30p immunoblot signals of which panel a is an example. Values represent averages derived from five independent experiments. (d) Time course experiments to measure the stability of GST-Met30p fusions after promoter shutoff in medium containing or lacking methionine. Y382 cells, containing pGSTMET30-3, were grown either in the presence or in the absence of 2 mM methionine, and GST-Met30p synthesis was induced by the addition of galactose for 3 h. After the addition of dextrose and cycloheximide, samples were taken at the indicated time points. (e) Quantification of data in panel d by Storm Phosphorimager analysis. The amount of GST-Met30p protein detected at the indicated time points is represented as a percentage of GST-Met30p protein detected at time point zero.
FIG. 4
FIG. 4
Met30p is the only component of SCFMet30p whose steady-state abundance is methionine dependent. Y382 cells transformed with the appropriate epitope-tagged plasmid were grown either in the presence (+) or in the absence (−) of 2 mM methionine. GST-Met30p and GST-Skp1p were detected using anti-GST antibodies (Sigma), Cdc34p was detected by anti-Cdc34p antibodies, and HA-Cdc53p was detected using anti-HA antibodies (Roche Molecular Biochemicals).
FIG. 5
FIG. 5
Met30p steady-state abundance is dependent on Skp1p activity. skp1-3-containing cells, transformed with pGSTMET30-3, were grown either in the presence (+) or in the absence (−) or 2 mM methionine and induced to produce the GST fusion protein for 3 h by the addition of galactose at the indicated temperature.
FIG. 6
FIG. 6
Identification of a methionine-responsive element in the Met30p family of proteins. (a and c) Anti-GST Western immunoblot analysis of soluble protein extracted from Y382 cells transformed with either pEG(KG), pGSTMET30-3, pGSTMET30(176–275), or pGSTMET30(Δ185–277) producing the indicated GST-Met30p fusion. Cells were grown either in the presence (+) or in the absence (−) of 2 mM methionine and induced to produce the GST fusion protein for 3 h by the addition of galactose. Either Cdc34p (∗) or a cross-reacting band was used as a loading control. (b) Quantification of the degradation rate of GST-Met30(176–275)p in the presence or absence of 2 mM methionine by Storm Phosphorimager analysis. Y382 cells transformed with pGSTMET30(176–275) were grown in the presence or absence of methionine and induced to produce GST-Met(176–275)p by the addition of galactose for 3 h. The amount of GST-Met30(176–275)p detected at the indicated time points is represented as a percentage of GST-Met30(176–275)p protein detected at time point zero.
FIG. 7
FIG. 7
Sequence alignment of the region that regulates Met30p in a methionine-dependent manner in the Met30p family of proteins. Residues 177 to 252 in Met30p from S. cerevisiae are shown aligned with the same region of SCONB from Emericella nidulans, open reading frame YDJ5 from S. pombe, and scon2 from N. crassa. Identical residues in at least three species are highlighted. The location of the F box is indicated.
FIG. 8
FIG. 8
Proposed model for the regulation of SCFMet30p complex activity. Met30p abundance is regulated by methionine availability. In the presence of methionine, Met30p stability increases to permit SCFMet30p complex formation and subsequent repression of methionine biosynthetic gene expression by mediating Met4p degradation. In the absence of methionine, Met30p abundance is decreased, lowering the activity of SCFMet30p and, thus, derepressing methionine biosynthetic gene expression (expr) by allowing the accumulation of Met4p.
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