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Cloning and Characterization of scon-3 + , a New Member of the Neurospora crassa Sulfur Regulatory System

2002; American Society for Microbiology; Volume: 1; Issue: 6 Linguagem: Inglês

10.1128/ec.1.6.875-883.2002

1535-9778

Steven T. Sizemore, John V. Paietta,

Enzyme Catalysis and Immobilization

ABSTRACT 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 Δ cys-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.