A point mutation in nucleoside diphosphate kinase results in a deficient light response for perithecial polarity in Neurospora crassa.
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-1(P72H) resulted in accumulation of normal levels of mRNA and of about 25% of NDK-1(P72H) 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-1(P72H)) 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-1(P72H) indicated that the P72H substitution in NDK-1 was responsible for the decrease in phosphotransfer activities, 5% of autophosphorylation activity, and 2% of V(max) for protein kinase activity phosphorylating myelin basic protein, compared with those of wild type NDK-1, respectively.
Ogura
Y
Y
Kihara Institute for Biological Research and Graduate School of Integrated Science, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan.
Yoshida
Y
Y
Yabe
N
N
Hasunuma
K
K
D019943:Amino Acid Substitution; D016764:Cell Polarity / Q000502:physiology* / Q000528:radiation effects; D003624:Darkness; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine; D008027:Light; D009492:Neurospora crassa / Q000166:cytology* / Q000201:enzymology* / Q000528:radiation effects; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011392:Proline; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006639:Histidine; D011392:Proline; D005982:Glutathione Transferase; D009701:Nucleoside-Diphosphate Kinase