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

Protein degradation is an essential regulatory mechanism used by the cell in many fundamental processes, including response to changing environmental conditions. In eukaryotes, a major proteolytic mechanism is the ubiquitin (Ub)-proteasome pathway (10, 12). Ub is a member of a family of conserved polypeptides that are covalently attached to protein substrates. Reiteration of Ub modification creates a poly-Ub chain on the substrate that is then targeted for degradation by the proteasome, a large multiprotein complex protease. The transfer of Ub to a protein substrate is a multistep process requiring at least three proteins. Free Ub is activated at the expense of ATP by a Ub-activating enzyme, or E1. Activated Ub is then transferred to a Ub-conjugating enzyme, or E2. Ub-ligases, or E3s, facilitate the transfer of Ub from E2 to protein substrate. Some E3s act as intermediary Ub carriers in the transfer of Ub from E2 to substrate (35), while other E3s act as adapters tethering the E2 to its substrate (6). A multiprotein complex called the SCF (named after the original components Skp1p, Cdc53p, and F-box protein) complex is represented in all eukaryotic taxa and has recently emerged as a major family of Ub ligases (6, 28, 42). In budding yeast, SCF complexes are comprised of a common set of components, namely, Cdc53p (or cullin), Skp1p, and Hrt1p (or Rbx1p or Roc1p) (for reviews, see references 5, 6, 28, and 42). Each SCF complex also contains an F-box protein, which is responsible for substrate recognition (8, 37). The F box, a ∼40-amino-acid motif, is believed to link the F-box protein with the common SCF components by binding Skp1p. Although several F-box proteins exist in the cell, only one F-box protein is present within any one SCF complex (29). Thus, a family of complexes that are distinguished by the F-box protein exists. The Ub-conjugating enzyme Cdc34p is associated with several SCF complexes and is necessary for SCF-dependent ubiquitination (8, 17, 23, 29, 36, 37, 43). Three yeast F-box proteins, Cdc4p, Grr1p, and Met30p, are present within SCF complexes, referred to as SCFCdc4p, SCFGrr1p, and SCFMet30p, respectively, and have roles in Cdc34p-mediated protein degradation events (6, 42). An emerging feature of these three SCF Ub ligases is their dual role in mediating cell cycle progression and metabolism. SCFCdc4p is required for entry into S phase by degrading the cyclin-dependent kinase-inhibitory protein Sic1p. Additionally, SCFCdc4p controls the biosynthesis of amino acids and purines (11, 13) by regulating the abundance of the transcription factor Gcn4p (25). Grr1p regulates G1 cyclin abundance (2) in addition to playing roles in heavy metal resistance, amino acid transport, cell growth, and glucose repression (4, 9, 14, 15, 20). SCFMet30p regulates methionine biosynthetic gene expression in addition to playing a positive role in cell cycle progression (16, 29, 30, 40). A well-described role for SCFMet30p is its regulation of methionine biosynthesis (29, 32). The MET genes encode enzymes and transporters necessary for the uptake of inorganic sulfur and its assimilation into methionine and cysteine (39). A negative feedback loop regulates MET gene expression: cells supplied with l-methionine repress MET gene expression, whereas depression occurs in media lacking l-methionine. The transcription factor Met4p positively regulates methionine biosynthesis. Recently, Met4p was proposed to be a substrate for the SCFMet30p Ub ligase complex (32). How SCFMet30p activity is regulated by l-methionine is unknown. One means of regulating the activity of some SCF complexes is at the level of substrate recognition. There are well-characterized examples of substrates being phosphorylated prior to their recognition by an SCF complex (42). However, Met4p modification has not been attributed to phosphorylation, and thus, SCFMet30p complex activity may be regulated by an alternate mechanism. Here we show that increased Met30p abundance positively regulates SCFMet30p activity. Additionally, we report that Met30p abundance is regulated by the availability of l-methionine. Therefore, SCFMet30p complex activity is, at least partially, regulated at the level of Met30p abundance. Finally, the F-box region of Met30p plays a critical role in regulating Met30p stability in a methionine-dependent manner, suggesting that this sequence may have crucial regulatory roles in addition to linking the F-box protein with the SCF complex.