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

• Published in: The Journal of biological chemistry Vol. 276; no. 24; pp. 21228 - 21234
• Main Authors: Ogura, Yasunobu, Yoshida, Yusuke, Yabe, Naoto, Hasunuma, Kohji
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.06.2001; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; blue light; Cell Polarity; Cell Polarity - physiology; Cell Polarity - radiation effects; chemistry; cytology; Darkness; enzyme activity; enzymology; fungal anatomy; gene expression; genetics; Glutathione Transferase; Glutathione Transferase - metabolism; Histidine; irradiation; Light; metabolism; mutation; ndk-1 gene; Neurospora crassa; Neurospora crassa - cytology; Neurospora crassa - enzymology; Neurospora crassa - radiation effects; Nucleoside-Diphosphate Kinase; Nucleoside-Diphosphate Kinase - chemistry; Nucleoside-Diphosphate Kinase - genetics; Nucleoside-Diphosphate Kinase - metabolism; phenotype; Phosphorylation; phosphotransferases (kinases); physiology; Point Mutation; Proline; radiation effects; Recombinant Fusion Proteins; Recombinant Fusion Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Messenger - genetics; Transcription, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M011381200

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 ofsmall protein) shows undetectable phosphorylation of NDK-1 and is defective in light-responsive regulation of perithecial polarity. Sequencing analysis ofndk-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-1P72Hprotein 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 ofpsp was caused by the ndk-1P72Hmutation. 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 Vmaxfor protein kinase activity phosphorylating myelin basic protein, compared with those of wild type NDK-1, respectively.