Cloning and characterization of scon-3+, a new member of the Neurospora crassa sulfur regulatory system.
The sulfur regulatory system of Neurospora crassa consists of a group of sulfur-regulated structural genes (e.g., arylsulfatase) that are under coordinate control of the CYS3 positive regulator and sulfur controller (SCON) negative regulators. Here we report on the cloning of scon-3(+), which encodes a polypeptide of 171 amino acids and is a Skp1 family homolog. Repeat-induced point mutation of scon-3(+) resulted in a phenotype of constitutive expression of arylsulfatase, a phenotype consistent with other sulfur controller mutants. Northern analysis indicated that, unlike other members of the sulfur regulatory system, expression of scon-3(+) is not under the direct control of the CYS3 transcriptional activator. In particular, scon-3(+) mRNA was detectable under sulfur repressing or derepressing conditions in a Deltacys-3 mutant. In yeast, Skp1p and an F-box protein binding partner are core constituents of a class of E3 ubiquitin ligases known as SCF complexes. The N. crassa negative regulator SCON2 contains an F-box motif essential for the operation of the sulfur regulatory system and suggests a role for an SCF complex in the N. crassa sulfur regulatory system. A crucial set of experiments, by using a yeast two-hybrid approach with confirming coimmunoprecipitation assays, demonstrated that SCON3 interacts with SCON2 in a manner dependent upon the F-box motif of SCON2. The protein-protein interaction detected between SCON2 and SCON3 represents the initial demonstration in a filamentous fungus of functional interaction between putative core components of a SCF complex.
Sizemore
Steven T
ST
Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio 45435, USA.
Paietta
John V
JV
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001192:Arylsulfatases / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics*; D008025:Ligases; D008969:Molecular Sequence Data; D009154:Mutation; D009492:Neurospora crassa / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013455:Sulfur / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases
D005656:Fungal Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D013455:Sulfur; D044767:Ubiquitin-Protein Ligases; D001192:Arylsulfatases; D008025:Ligases