Semi-in vitro detection of Mg2+-dependent DNase that specifically digest mitochondrial nucleoids in the zygote of Physarum polycephalum

The maternal/uniparental inheritance of mitochondria is controlled by the selective elimination of paternal/uniparental mitochondria and digestion of their mitochondrial DNA (mtDNA). In isogamy, the selective digestion of mtDNA in uniparental mitochondria is initiated after mating and is completed prior to the elimination of mitochondria, but the molecular mechanism of the digestion of uniparental mtDNA remains unknown. In this study, we developed a semi-in vitro assay for DNase, wherein the digestion of mitochondrial nucleoids (mt-nucleoids) was microscopically observed using isolated mitochondria from Physarum polycephalum and the DNase involved in uniparental inheritance was characterized. When myxamoebae of AI35 and DP246 are crossed, mtDNA and mt-nucleoid from only the DP246 parent are digested. The digestion of mt-nucleoids was observed in zygotes 3 h after plating for mating. During the digestion of mt-nucleoids, mitochondrial membrane integrity was maintained. In the semi-in vitro assay, the digestion of mt-nucleoids was only observed in the presence of Mg2+ at pH 7.5–9.0. Moreover, such Mg2+-dependent DNase activity was specifically detected in mitochondria isolated from zygotes 3 h after plating for mating. Therefore, Mg2+-dependent DNase is potentially involved in uniparental inheritance. Our findings provide insights into the DNase involved in uniparental inheritance and its regulatory mechanism.


Supplementary materials and methods
Expression cloning of PpEndoG-like genes: The cDNAs coding for PpEndoG-like genes were obtained by reverse transcription of mRNA isolated from AI35 using the RNeasy ® Plant Mini Kit (Qiagen, Hilden, Germany). The cDNAs encoding PpEndoG-like genes without the coding regions for the respective N-terminal mitochondrial targeting sequences were amplified by PCR using KOD Neo Plus and Primer sets 1 and 2. The primer sequences used were as follows: 1. 5'-CCGAGCTCCTACCCAGCGAAGAAAATCTCCATT -3' 2. 5'-CCCAAGCTTTTACTTTTGGTCGCTTTTGGCATCTTT -3' The cloned cDNA of the predicted mature protein of PpEndoG-like (98-407 a.a.) was inserted into the TOPO vector and transformed into Escherichia coli DH5α. A portion of the cDNA of PpEndoG-like was cut from the TOPO vector and inserted into the expression vector pET28a.
PpEndoG-like expression and purification: E. coli BL21 (DE3) bacterial cells were transformed with pET28a-PpEndoG-like. The expression of rPpEndoG-like was induced by incubation for 4 h with 1 mM IPTG at 37 °C. The bacterial cells were collected by centrifugation at 3,000 ×g at 4 °C for 15 min, and the supernatant was removed. The collected bacterial cells were suspended in sonication buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1.25 mM EDTA (pH 8.0), 250 μg/mL EDTA-free protease inhibitor cocktail) in the presence of 5 μg/μL DNase I and 0.25 mg/ml lysozyme, and then broken using an Ultrasonic Liquid Processor XL-2000 SERIES (Misonix, Farmingdale, NY, USA). The broken cells were collected by centrifugation at 9,100 ×g at 4 °C for 10 min, and the supernatant was removed. The broken cells were resuspended in sonication wash buffer (0.5 % Triton-X, 1 mM EDTA (pH 8.0), 250 μg/mL EDTA-free protease inhibitor cocktail) were sonicated and collected by centrifugation at 9,100 ×g at 4 °C for 10 min. This procedure was performed four times. The broken cells were suspended in dissolution buffer (6 M guanidinium chloride, 100 mM NaH2PO4, 7.6 mM Tris-HCl, pH 8.0) and shaken for 1 h at 24 ℃, and the supernatant was collected by centrifugation at 9,100 ×g at 4 °C for 30 min. The recombinant protein of PpEndoG-like was purified by Ni 2+ -NTAaffinity chromatography using a HisTrap FF Crude column (GE Healthcare, Danderyd, Sweden).

Refolding of recombinant PpEndoG-like:
The rPpEndoG-like was dialyzed into Dialysis Buffer (0.5 M NaCl, 50 mM phosphate buffer (pH 7.0)) at 4 °C overnight, and soluble rPpEndoG-like was collected as supernatant by centrifugation at 20,400 ×g at 4 °C for 1 min.

Preparation of antibody:
The refolded rPpEndoG-like was used to raise polyclonal rabbit antisera. The antibodies for PpEndoG-like were purified from serum using AminoLink Coupling Resin (Thermo Fisher Scientific, Dreieich, Germany).
Western blotting: Isolated mitochondria were separated using SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes (Immobilon ® -P Transfer Membrane, 0.45 μm pore size, Merck Millipore, Darmstadt, Germany) for approximately 60 min at 100 V in a blotting buffer (2.5 mM Tris, 19.2 mM glycine, 0.02 % SDS, 20 % methanol). Membranes were blocked with 5 % non-fat milk in TBST (20 mM Tris, 150 mM NaCl, containing 0.05 % Tween-20, pH 7.4) at 4 °C overnight. Membranes were incubated with anti-PpEndoG-like or anti-ANT antibodies, which were diluted 1:500 in TBST with 1 % non-fat milk, at room temperature for 2 h. After washing the membranes with TBST with 1 % non-fat milk three times, the membranes were incubated with anti-rabbit HRP, diluted 1:30 000 in TBST with 1 % non-fat milk at room temperature for 2 h. After washing the membranes with TBST three times and TBS (20 mM Tris, 150 mM NaCl, pH 7.4) twice, membranes were exposed to Immobilon Western chemiluminescent HRP substrate (Merck Millipore) for 1 min at room temperature and visualized using an ImageQuant LAS 4000 mini (GE Healthcare).