Synthesis and Differential Turnover of the CYS3 Regulatory Protein of Neurospora crassa Are Subject to Sulfur Control

ABSTRACT The transcription factor CYS3 of Neurospora crassa is a positive regulator of the sulfur regulatory circuit which contains many structural genes involved in sulfur metabolism. Expression and degradation of the CYS3 protein are precisely regulated in a sulfur-dependent manner. cys-3 expression was found to be fully repressed by high concentrations of methionine or inorganic sulfate present in the culture medium and to be derepressed when these favored sulfur sources were limited. cys-3 transcripts could be readily detected within 2 h after derepression, whereas the CYS3 protein was not found until after 4 h. CYS3 is stable, with a half-life greater than 4 h under low-sulfur conditions when it is required for cell growth. However, it is degraded relatively quickly when methionine or inorganic sulfate becomes available. Upon sulfur repression, cys-3 transcripts disappeared within 30 min with an estimated half-life of 5 min whereas CYS3 protein almost entirely disappeared in 1 h with a half-life of approximately 10 min. These results suggest that a selective elimination of CYS3 is a highly regulated process. Site-directed mutagenesis showed that Lys-105 of CYS3 is important for its instability. The change of this single residue from lysine to glutamine resulted in a prolonged half life of CYS3 and impaired responsiveness of CYS3 degradation to sulfur level changes.

Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa

The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. The N. crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant. Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis. The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions. In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.

Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa.

The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. The N. crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant. Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis. The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions. In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.