The amino-terminal portion of the F-box protein Met30p mediates its nuclear import and assimilation into an SCF complex.
SCF complexes are a conserved family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. The F-box motif links the F-box protein to the core components via its interaction with Skp1p. In yeast, the SCFMet30p complex contains the Met30p F-box protein and regulates Met4p, a transcription factor that mediates sulfur fixation and methionine biosynthesis. Although a nuclear protein, Met30p lacks a definable nuclear localization sequence. Here we show that the entire amino-terminal half of Met30p is required for its proper nuclear localization. Mutations in the F-box, but not mutations in Skp1p, affect Met30p nuclear localization, indicating that the F-box motif plays an important role in Met30p trafficking independent of its interaction with Skp1p binding. Met30p mutants that poorly localize to the nucleus display increased nuclear to cytoplasmic exchange, indicating that the amino terminus mediates nuclear retention in addition to nuclear import. The Met30p F-box motif, residues 180-225, is necessary and sufficient to bind Skp1p; however, mutations upstream of the Met30p F-box inhibit Skp1p binding. We propose that additional factors bind the amino-terminal region of Met30p and mediate its nuclear localization and assimilation into an SCF complex.
Brunson
Lee Ellen
LE
Department of Biochemistry and Molecular Biology, Louisiana State University Health Science Center, Shreveport, Louisiana 71130-3932, USA.
Dixon
Cheryl
C
Kozubowski
Lukasz
L
Mathias
Neal
N
D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D050976:Basic-Leucine Zipper Transcription Factors; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D044783:F-Box Proteins; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins / Q000737:chemistry; D044843:SKP Cullin F-Box Protein Ligases / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000378:metabolism
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D044783:F-Box Proteins; D008164:Luminescent Proteins; C470285:MET30 protein, S cerevisiae; C073675:MET4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D044786:S-Phase Kinase-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D004247:DNA; D043743:Ubiquitin-Protein Ligase Complexes; D044843:SKP Cullin F-Box Protein Ligases; D005982:Glutathione Transferase