Regulation of laccase synthesis in induced Neurospora crassa cultures.

Rapidly growing cultures of N. crassa do not produce laccase. Exposure of this fungus to different inducing agents leads to a de novo biosynthesis of extracellular laccase in vegetative cultures. In this study the induction of laccase after addition of cycloheximide and D-phenylalanine is reported. De novo synthesis of laccase mRNA was followed over 96 h after induction. A fast appearance of the message, as well as its presence over a rather long period, indicates a regulation on a transcriptional and maybe on a post-transcriptional level. In contrast to the kinetics of mRNA production, Western analysis with a polyclonal anti-laccase antibody showed a remarkably delayed appearance of the intracellular, as well as of the extracellular, protein product after induction with cycloheximide. Furthermore, activity measurements at different times after induction of both crude extracts and media of the vegetative cultures showed that in extracted mycelia the activity occurs at least 20 h after the protein is immunologically detectable. Laccase activity in the medium starts to increase only 30 h after translation. These data, together with the published structure of the laccase gene, indicate a regulation on the transcriptional, post-transcriptional and on a post-translational level. In cultures induced with D-phenylalanine a rather fast appearance of laccase-specific mRNA also indicates a transcriptional regulation. Compared to cycloheximide-induced laccase biosynthesis no delayed appearance of laccase protein levels of laccase activity is observed after induction with D-phenylalanine.

ATP-induced protyrosinase synthesis and carboxyl-terminal processing in Neurospora crassa.

The effects of 3'-5' cyclic AMP and ATP upon tyrosinase induction in Neurospora crassa were examined. Northern analysis of total cellular RNA revealed rapid de novo synthesis of protyrosinase after addition of these substances to stationary-phase mycelia. The maturation of protyrosinase in crude extracts of mycelia was followed by Western analysis. Polyclonal rabbit antiserum directed against the denatured carboxyl-terminal extension of protyrosinase does recognize the proform and several intermediate forms of different molecular weight but not mature tyrosinase. Disruption of ATP-induced mycelia in sodium phosphate buffer (pH 6.0) demonstrate processing at the carboxyl-terminal end of protyrosinase. The activity assays revealed that protyrosinase is an inactive precursor and that at least two active forms of slightly different molecular weight are present in crude extracts. Maturation of protyrosinase thus involves specific and sequential proteolytic cleavage at the carboxyl-terminus. These results suggest the presence of a tyrosinase activator in Neurospora crassa mycelia, which is kept apart from protyrosinase in the intact mycelium.