Genetic analysis of signal transduction through light-induced protein phosphorylation in Neurospora crassa perithecia

We have previously demonstrated that blue light induces the phosphorylation of a 15-kDa protein in crude membrane fractions of Neurospora crassa mycelia. Here we report the isolation and characterization of a mutant (?psp; phosphorylation of small proteins) that is completely defective for phosphorylation of that protein, as assayed in both crude membrane and soluble fractions. This mutation defines a unique locus that maps to linkage group VR between al-3 and his-6. To elucidate the photobiological significance of the phosphorylation of the protein, we analyzed known photobiological phenomena and discovered that the positioning of beaks on the perithecia, defined as perithecial polarity, was light-dependent in the wild type. In the psp mutant, beaks were phototropic as in the wild type, but their position was random. In a wc-1 mutant, however, beaks were positioned at random and were not phototropic. Thus light-induced perithecial polarity and phototropism of perithecial beaks are controlled differently. A psp; wc-1 double mutant showed the same phenotype as that of wc-1 with respect to these two photomorphogenetic characters. These results indicate that the wc-1 gene is epistatic to psp in the light-signal transduction pathway that controls both phototropism and perithecial polarity.     

Coordinated Regulation of Radioadaptive Response by Protein Kinase C and p38 Mitogen-Activated Protein Kinase

Eukaryotic cells are known to have an inducible or adaptive response that enhances radioresistance after a low priming dose of radiation. This radioadaptive response seems to present a novel cellular defense mechanism. However, its molecular processing and signaling mechanisms are largely unknown. Here, we studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the expression of radioadaptive response in cultured mouse cells. Protein immunoblot analysis using isoform-specific antibodies showed an immediate activation of PKC-alpha upon X-irradiation as indicated by a translocation from cytosol to membrane. A low priming dose caused a prolonged translocation, while a nonadaptive high dose dramatically downregulated the total PKC level. Low-dose X-rays also activated the p38 MAPK. The activation of p38 MAPK and resistance to chromosome aberration formation were blocked by SB203580, an inhibitor of p38 MAPK, and Calphostin C, an inhibitor of PKC. Furthermore, it was demonstrated that p38 MAPK was physically associated with delta1 isoform of phospholipase C (PLC-delta1), which hydrolyzed phosphatidylinositol bisphosphate into diacylglycerol, an activator of PKC, and that SB203580 also blocked the activation of PKC-alpha. These results indicate the presence of a novel mechanism for coordinated regulation of adaptive response to low-dose X-rays by a nexus of PKC-alpha/p38 MAPK/PLC-delta1 circuitry feedback signaling pathway with its breakage operated by downregulation of labile PKC-alpha at high doses or excess stimuli.     

A regulatory protein for orthophosphate-regulated cyclic phosphodiesterase in Neurospora crassa

A regulatory protein for orthophosphate-regulated cyclic phosphodiesterase (cPDase) was detected in mycelial extracts of Neurospora crassa. The protein, designated neucrassin, was precipitated by ammonium sulfate between 60 to 100% saturation, and fractionated by gel filtration through a TSK-gel column. The molecular weight was estimated to be 65,000. Neucrassin inhibited the hydrolyzing activity of cPDase for cyclic 3',5'-AMP in the presence of MnCl2 in a noncompetitive manner, whereas it stimulated the activity for cyclic 2',3'-AMP over a wide ranges of pH's, between 2.8 to 6.8.     

Avoiding entry into intracellular protein degradation pathways by signal mutations increases protein secretion in Pichia pastoris

In our previous study, we serendipitously discovered that protein secretion in the methylotrophic yeast Pichia pastoris is enhanced by a mutation (V50A) in the mating factor alpha (MFα) prepro-leader signal derived from Saccharomyces cerevisiae. In the present study, we investigated 20 single-amino-acid substitutions, including V50A, located within the MFα signal peptide, indicating that V50A and several single mutations alone provided significant increase in production of the secreted proteins. In addition to hydrophobicity index analysis, both an unfolded protein response (UPR) biosensor analysis and a microscopic observation showed a clear difference on the levels of UPR induction and mis-sorting of secretory protein into vacuoles among the wild-type and mutated MFα signal peptides. This work demonstrates the importance of avoiding entry of secretory proteins into the intracellular protein degradation pathways, an observation that is expected to contribute to the engineering of strains with increased production of recombinant secreted proteins.