A Point Mutation in Nucleoside Diphosphate Kinase Results in a Deficient Light Response for Perithecial Polarity in Neurospora crassa

Abstract

In Neurospora crassa, the phosphorylation of nucleoside diphosphate kinase (NDK)-1 is rapidly enhanced after blue light irradiation. We have investigated the function of NDK-1 in the blue light signal transduction pathway. A mutant called psp (phosphorylation of small protein) shows undetectable phosphorylation of NDK-1 and is defective in light-responsive regulation of perithecial polarity. Sequencing analysis of ndk-1 cDNA by reverse transcription-polymerase chain reaction revealed that proline 72 of ndk-1 was replaced with histidine in psp. The mutation ndk-1P72H resulted in accumulation of normal levels of mRNA and of about 25% of NDK-1P72H protein compared with that of wild type as determined by Western blot analysis. The ectopic expression of cDNA and introduction of genomic DNA of wild type ndk-1 in psp (ndk-1P72H) suppressed the reduction in accumulation and phosphorylation of NDK-1 and the light-insensitive phenotype. These findings demonstrated that the phenotype of psp was caused by the ndk-1 P72H mutation. Biochemical analysis using recombinant NDK-1 and NDK-1P72H indicated that the P72H substitution in NDK-1 was responsible for the decrease in phosphotransfer activities, 5% of autophosphorylation activity, and 2% of Vmax for protein kinase activity phosphorylating myelin basic protein, compared with those of wild type NDK-1, respectively.