Mitochondrial DNA (mtDNA) is generally packaged into the mitochondrial nucleoid (mt-nucleoid) by a high-mobility group (HMG) protein. Glom is an mtDNA-packaging HMG protein in Physarum polycephalum. Here we identified a new mtDNA-packaging protein, Glom2, which had a region homologous with yeast Mgm101. Glom2 could bind to an entire mtDNA and worked synergistically with Glom for condensation of mtDNA in vitro. Down-regulation of Glom2 enhanced the alteration of mt-nucleoid morphology and the loss of mtDNA induced by down-regulation of Glom, and impaired mRNA accumulation of some mtDNA-encoded genes. These data suggest that Glom2 may organize the mt-nucleoid coordinately with Glom.

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Summary Mitochondrial DNA (mtDNA) is highly organized into a compact structure, the mitochondrial nucleoid (mt-nucleoid). To facilitate our understanding of the regulation of mtDNA genetic activity within the mt-nucleoid structure, we have identified a novel mt-nucleoid protein Pmn34 (Physarum polycephalum mitochondrial nucleoid protein 34), having a molecular weight of 34 kDa, from pure mt-nucleoids isolated from the true slime mold, Physarum polycephalum. The Pmn34 protein is composed of 326 amino acids with mitochondrial transit peptides and its primary sequence contains a conserved 3Ј to 5Ј exonuclease motif of the "DEDD" superfamily. DNA mobility shift assays demonstrated that recombinant Pmn34 binds weakly to both mtDNA and lDNA with no apparent sequence specificity. Furthermore, immunoblotting and immunostaining analyses revealed that Pmn34 localizes specifically in the peripheral region of mt-nucleoids. These results indicate that Pmn34 functions in the peripheral region of mt-nucleoids, suggesting that the mt-nucleoid is compartmentalized into functional domains.

Under various environmental stresses, the true slime mold Physarum polycephalum converts into dormant forms, such as microcysts, sclerotia, and spores, which can survive in adverse environments for a considerable period of time. In drought-induced sclerotia, actin is threonine phosphorylated, which blocks its ability to polymerize into filaments. It is known that fragmin and actin-fragmin kinase (AFK) mediate this phosphorylation event. In this work, we demonstrate that high levels of actin threonine phosphorylation are also found in other dormant cells, including microcysts and spores. As the threonine phosphorylation of actin in microcysts and sclerotia were induced by drought stress but not by other stresses, we suggest that drought stress is essential for actin phosphorylation in both cell types. Although characteristic filamentous actin structures (dot- or rod-like structures) were observed in microcysts, sclerotia, and spores, actin phosphorylation was not required for the formation of these structures. Prior to the formation of both microcysts and sclerotia, AFK mRNA expression was activated transiently, whereas fragmin mRNA levels decreased. Our results suggest that drought stress and AFK might be involved in the threonine phosphorylation of actin.