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

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.

Identifying the Hotspots on the Top Faces of WD40-Repeat Proteins from Their Primary Sequences by β-Bulges and DHSW Tetrads

“…A total of six R 1 , seven R 1 -2, four D-1, two X and one R 1 -3 mutations were found in Met30( S. cerevisiae ) [36], MDV1( S. cerevisiae ) [37], Tup11( S. pombe ) [38], COP1( Arabidopsis ) [39] and SPA1( Arabidopsis ) [59].…”

“…To obtain the loop d–a and loop b–c, the predicted WD40 blades are aligned with those in the templates. Then, the residues in two loops are selectively studied if the corresponding residues in the templates are already proved to be indispensable for protein-protein [36] , [37] , [38] , [39] .…”

Lessons from Fungal F-Box Proteins

“…The way Met4 is regulated by Met30 has been under discussion for several years (23,24,83,140,141). A picture in which Met30 regulates Met4 in multiple ways has emerged (30).…”

Adaptive evolution in two large families of ubiquitin-ligase adapters in nematodes and plants

“…The F-box domain binds to Cullin1 via Skp1-related (Skr) proteins (Bai et al 1996;Zheng et al 2002); diverse regions outside the F-box domain bind to specific substrate proteins (Winston et al 1999;Hsiung et al 2001;Brunson et al 2005;Nayak et al 2005). In these adapter proteins, the F-box is near the N terminus, and the remainder of the protein falls into several families, including kelch repeat, WD-40 repeat, LRR, FTH, FBA, FBA1, and FBA2 domain-containing families (http://www.sanger.ac.uk/Software/Pfam/2005) (Jiang and Struhl 1998;Ilyin et al 1999;Winston et al 1999;Clifford et al 2000;Andrade et al 2001;Gagne et al 2002).…”

“…The BTB domain binds directly to Cullin3, and other domains in the BTB protein confer substrate specificity. Like Fbox proteins, BTB-containing adapters have a variety of substrate-binding domains, including MATH, WD-40 repeat, Znfinger repeat, and kelch repeat domains (Winston et al 1999;Pintard et al 2003;Prag and Adams 2003;Xu et al 2003;van den Heuvel 2004;Brunson et al 2005;Figueroa et al 2005;Stogios et al 2005). In C. elegans, most of these adapters have a BTB domain near their C terminus and an N-terminal MATH domain, which is responsible for substrate binding (Pintard et al 2003;Xu et al 2003).…”