Journal: Nucleic Acids Research
Article Title: Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication
Figure Lengend Snippet: Twinkle is membrane associated. ( A ) Isolated mitochondria of HEK293E cells were subjected to either KCl or sodium carbonate extraction (Na 2 CO 3 ) as described in the main text. Endogenous Twinkle was detected using a monoclonal antibody and blots were re-probed with antibodies for TFAM and mtSSB. Results show that endogenous Twinkle fractionates mostly to the pellet fraction using both methods illustrating its strong membrane association, similar to overexpressed Twinkle ( Supplementary Figure S6C ), whereas TFAM and mtSSB mostly became soluble, in particular, in combination with 0.5 M KCl and sonication. Please note that to detect Twinkle with confidence, more protein was used for these western blots sometimes resulting in overloading of TFAM. Sonication in combination with DNAseI (D) or DNaseI/RNase A (R, RNase)/Benzonase ( B ) released more TFAM and mtSSB than sonication alone, showing that a proportion of TFAM and mtSSB can be found in the insoluble fractions of the various experiments by means of their interaction with mtDNA (rightmost blot panel A). (B) Na 2 CO 3 fractionation shows that overexpressed Twinkle–Myc is almost exclusively in the pellet (p) fraction again indicative of tight membrane association, whereas TFAM is partially soluble (s = supernatant) (left panel). Treatment of the Na 2 CO 3 pellet fraction by DNAseI and subsequent re-extraction using Na 2 CO 3 further released a proportion of TFAM (middle panel). Finally, Na 2 CO 3 in combination with Triton-X100 (TX100) treatment solubilized both Twinkle–Myc and endogenous TFAM (right panel). ( C ) Similar to results shown in panel A, sonication in combination with DNAseI ( D ) or DNaseI/RNase A (R, RNase)/Benzonase (B) released more TFAM than sonication alone or combined with RNase treatment, showing that a proportion of TFAM can be found in the insoluble fractions of the various experiments by means of its interaction with mtDNA, whereas in this case, overexpressed Twinkle–Myc remains in the pellet fraction with all treatments. (D) Digitonin-based isolation of a mitochondrial membrane fraction again showed the retention of Twinkle and proportions of nucleoid-associated proteins such as TFAM, mtSSB and POLG1 (see main text and Supplementary Figure S6D ). This membrane fraction (marked as ‘Digitonin pellet’) was here subjected to flotation by layering a gradient of iodixanol on top of this fraction that was first re-solubilized with 1% TX100 (see ‘Materials and Methods’ for details). The gradient was then subjected to ultracentrifugation. In parallel, a mitochondrial fraction that was directly solubilized by 1% TX100 (marked as ‘TX100’) was subjected to the same procedure. Collected fractions were isolated as indicated and subjected to western blot analysis as well as dot blot analysis to detect mtDNA. The results show that mtDNA and nucleoid-associated proteins in the digitonin-lysis membrane fraction moved up the gradient to a single low-density iodixanol concentration, showing that they likely form a single complex. In contrast, COXII and a marker for the large ribosomal subunit MRPL49 have remained at relatively high-density fractions. In the total mitochondrial TX100 lysate, Twinkle and other nucleoid proteins appear more dispersed as does mtDNA.
Article Snippet: Treatment of isolated mitochondria by sonication and nucleases Mitochondria were resuspended in enzyme-buffer (50 mM Tris–HCl, pH 7.5, 50 mM NaCl, 3 mM CaCl2 , 2 mM MgCl2 ), sonicated on ice at 40% power for three times 20 s before addition of the enzymes as indicated DNase I (Fermentas) 10U, RNAse A (Fermentas) 20 µg, Micrococcal nuclease (Fermentas) 50 U and Benzonase nuclease (Sigma) 50U, and incubated at +37°C for 30 min. Where appropriate, lysates were further subjected to carbonate extraction as described above.
Techniques: Isolation, Sonication, Western Blot, Fractionation, Dot Blot, Lysis, Concentration Assay, Marker