An additional role for the F-box motif: gene regulation within the Neurospora crassa sulfur control network.
The F-box represents a protein motif originally identified as a conserved amino-terminal domain within the Neurospora crassa negative regulator sulfur controller-2. Recently, F-boxes have been found within a number of cell cycle regulatory proteins, where they mediate ubiquitin-driven proteolytic events required for major cell cycle transitions. F-box function, however, is not restricted solely to cell cycle pathways. Here we present evidence expanding F-box function to encompass gene regulatory processes independent of the cell cycle through in vivo analysis of an F-box acting within the N. crassa sulfur regulatory network. The Neurospora sulfur circuit features a set of regulatory genes acting to modulate gene expression based on environmental sulfur conditions. These sulfur regulatory genes include cys-3+, which encodes a basic region-leucine zipper transcriptional activator, as well as the negative regulatory gene scon-2+. Through site-directed mutagenesis of the SCON2 F-box, we have generated a sulfur auxotrophic phenotype previously unobserved in any scon-2 mutant. Using Northern analysis, we have traced this auxotrophy to a complete shutdown of cys-3+ gene expression. We have further analyzed F-box function by constructing a series of chimeric SCON2 proteins containing swapped F-box domains from the yeast transcriptional inhibitor Met30p and the Candida albicans cell cycle regulator Cdc4p. The ability of these chimeric proteins to restore partial wild-type sulfur regulation in vivo emphasizes the universal nature of this motif and confirms the functional importance of the F-box within noncell cycle regulatory pathways.
Kumar
A
A
Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435, USA.
Paietta
J V
JV
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013455:Sulfur / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D005656:Fungal Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C093148:SCON-2 protein, Neurospora crassa; D013455:Sulfur