Identification of residues in the WD-40 repeat motif of the F-box protein Met30p required for interaction with its substrate Met4p.
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.
Brunson
Lee Ellen
LE
Department of Biochemistry and Molecular Biology, Louisiana State University Health Science Center, 1501 Kings Highway, Shreveport, LA, 71130-3932, USA.
Dixon
Cheryl
C
LeFebvre
Aaron
A
Sun
Lisa
L
Mathias
Neal
N
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D015153:Blotting, Western; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D044783:F-Box Proteins; D005816:Genetic Complementation Test; D005982:Glutathione Transferase / Q000378:metabolism; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry*; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D015534:Trans-Activators / Q000737:chemistry*; D015533:Transcriptional Activation; D025801:Ubiquitin / Q000737:chemistry; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry*
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D044783:F-Box Proteins; C470285:MET30 protein, S cerevisiae; C073675:MET4 protein, S cerevisiae; D012097:Repressor Proteins; D044786:S-Phase Kinase-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D025801:Ubiquitin; D004247:DNA; D008715:Methionine; D043743:Ubiquitin-Protein Ligase Complexes; D005982:Glutathione Transferase; D046988:Proteasome Endopeptidase Complex