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Mol Genet Genomics. 2005 Jun;273(5):361-70. Epub 2005 May 10.

Identification of residues in the WD-40 repeat motif of the F-box protein Met30p required for interaction with its substrate Met4p.

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  • 1Department of Biochemistry and Molecular Biology, Louisiana State University Health Science Center, 1501 Kings Highway, Shreveport, LA, 71130-3932, USA.

Abstract

The SCF family of ubiquitin-ligases consists of a common core machinery, namelySkp1p, Cdc53p, Hrt1p, and a variable component, the F-box protein that is responsible for substrate recognition. The F-box motif, which consists of approximately 40 amino acids, connects the F-box protein to the core ubiquitin-ligase machinery. Distinct SCF complexes, defined by distinct F-box proteins, target different substrate proteins for proteasome-dependent degradation. As part of the SCF(Met30p) complex, the F-box protein Met30p selects the substrate Met4p, a transcriptional activator for MET biosynthetic genes that mediate sulfur uptake and biosynthesis of sulfur containing compounds. When cells are grown in the absence of methionine, Met4p evades degradation by the SCF(Met30p) complex and activates the MET biosynthetic pathway. However, overproduction of Met30p represses MET gene expression and induces methionine auxotrophy in an otherwise methionine prototrophic strain. Here we demonstrate that overproduction of the C-terminal portion of Met30p, which is composed almost entirely of seven WD-40 repeat motifs, is necessary and sufficient to induce methionine auxotrophy and complement the temperature sensitive (ts) met30-6 mutation. Furthermore, we show that this region of Met30p is important for binding Met4p and that mutations that disrupt this interaction prevent both the induction of methionine auxotrophy and complementation of the met30-6 mutation. These assays have been exploited to identify residues that are important for the interaction of Met30p with its substrate. Since the C-terminal domain of Met30p lacks the F-box and cannot support the ubiquitination of Met4p, our results indicate that the recruitment of Met4p to the SCF(Met30p) complex itself results in inactivation of Met4p, independently of its ubiquitination.

PMID:
15883825
[PubMed - indexed for MEDLINE]
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