Two high-mobility group domains of MHG1 are necessary to maintain mtDNA in Neurospora crassa.

The mhg1 (NCU02695/ada-23) gene encodes the mitochondrial high-mobility group box (HMG-box or HMGB) protein in Neurospora crassa. The mhg1 KO strain (mhg1KO) has mitochondrial DNA (mtDNA) instability and a short lifespan; however, the function of MHG1 remains unclear. To investigate the role of this protein in the maintenance of mtDNA, domain deleted MHG1 proteins were expressed in the mhg1KO strain, and their effects were analyzed. We identified two putative HMG-domains, HMGBI and HMGBII. Although deletion of the HMG-box did not abolish MHG1's mitochondrial localization, the mhg1KO phenotype of a severe growth defect and a high sensitivity to mutagens could not be restored by introduction of HMG-box deleted mhg1 gene into the KO strain. It was indicated that recombinant full-length MHG1, i.e., mitochondrial targeting sequence (MTS) containing protein, did not exhibit explicit DNA binding, whereas the MHG1 protein truncated for the MTS sequence did in vitro by an electrophoretic mobility shift assay. Furthermore, recombinant MHG1 protein lacking MTS and HMG-domains, either HMGBI or HMGBII, had DNA affinity and an altered band shift pattern compared with MTS-truncated MHG1 protein. These results suggest that cleavage of MTS and appropriate DNA binding via HMG-domains are indispensable for maintaining mtDNA in N. crassa.

Phenotypic analysis of newly isolated short-lifespan Neurospora crassa mutant deficient in a high mobility group box protein.

To elucidate genetic mechanisms affecting the lifespan of the filamentous fungus Neurospora crassa, we attempted to identify a gene of which a defect causes a short-lifespan. By screening a Neurospora knockout library, provided by the Fungal Genetics Stock Center at Kansas State University, several KO strains with a short-lifespan were isolated. FGSC#11693 is one of these, which shows similar phenotypes to known Neurospora short-lifespan mutants as follows: 1) hyphal growth ceases after about 2weeks of cultivation, despite that of the wild-type continuing for over 2years, 2) viability of conidia is lower than that of the wild-type, and 3) high sensitivity to mutagens such as methyl methanesulfonate, ultraviolet radiation, and hydroxyl urea is exhibited. The NCU number of the knocked-out gene in the KO strain is NCU02695, and recovery from the short-lifespan and mutagen sensitivity was achieved by the introduction of this gene from the wild-type. The putative amino acid sequence of the knocked-out gene contains two high mobility group box domains and a mitochondrial localization signal is found at the N-terminal of this sequence. Upon analyzing the subcellular localization of the gene product fused with GFP, GFP signals were detected in mitochondria. From these observations, the gene and KO strain were named mitochondrial high mobility group box protein 1 (MHG1) and mhg1KO strain, respectively. The amount of mtDNA relative to the nuclear amount was lower in the mhg1KO strain than in the wild-type. mtDNA aberration was also observed in the mhg1KO strain. These results suggest that the MHG1 protein plays an important role in the maintenance of mitochondrial DNA, and mitochondrial abnormality caused by mtDNA aberration is responsible for the short-lifespan of the mhg1KO strain.