Developing a genetic approach to investigate the mechanism of mitochondrial competence for DNA import

Abstract

Mitochondrial gene products are essential for the viability of eukaryote obligate aerobes. Consequently, mutations of the mitochondrial genome cause severe diseases in man and generate traits widely used in plant breeding. Pathogenic mutations can often be identified but direct genetic rescue remains impossible because mitochondrial transformation is still to be achieved in higher eukaryotes. Along this line, it has been shown that isolated plant and mammalian mitochondria are naturally competent for importing linear DNA. However, it has proven difficult to understand how such large polyanions cross the mitochondrial membranes. The genetic tractability of Saccharomyces cerevisae could be a powerful tool to unravel this molecular mechanism. Here we show that isolated S. cerevisiae mitochondria can import linear DNA in a process sharing similar characteristics to plant and mammalian mitochondria. Based on biochemical data, translocation through the outer membrane is believed to be mediated by voltage-dependent anion channel (VDAC) isoforms in higher eukaryotes. Both confirming this hypothesis and validating the yeast model, we illustrate that mitochondria from S. cerevisiae strains deleted for the VDAC-1 or VDAC-2 gene are severely compromised in DNA import. The prospect is now open to screen further mutant yeast strains to identify the elusive inner membrane DNA transporter.

Fig. 1

DNA can be imported into yeast mitochondria. (A) Testing different media. Labeled DNA representing a 0.5 kb fragment of CIRV orf1 (0.5 kb CIRV1, see Materials and methods) was incubated for 40 min at 30 °C with isolated yeast (S. cerevisiae) mitochondria (parental strain M3, see Materials and methods) in media containing different combinations of buffer (Tris–HCl or potassium phosphate 40 mM, pH as indicated) and sugar (0.5 M sucrose or 0.6 M mannitol as indicated) prior to DNase-I digestion. Mitochondrial nucleic acids were subsequently extracted, fractionated by agarose gel electrophoresis and transferred onto a nylon membrane which was autoradiographed. (B) Time course and temperature dependence of DNA uptake. Labeled linear maize (Zea mays) 2.3 kb mitochondrial plasmid (2.3 kb ZmPL, see Materials and methods) was incubated for different times at 30 °C (lanes 10 to 13) or at different temperatures for 40 min (lanes 14 to 16) with yeast mitochondria in the Tris–HCl/mannitol medium. Uptake was analysed as in A. (C) DNA uptake is enhanced by glutamate/malate and the DNA reaches the matrix. Lanes 17 and 18: labeled maize 2.3 kb plasmid (2.3 kb ZmPL) was incubated with yeast mitochondria in the absence or presence of glutamate (10 mM) and malate (1 mM) in the Tris–HCl/mannitol medium for 40 min at 30 °C. Lanes 19 and 20: following incorporation of labeled maize 2.3 kb plasmid for 40 min, yeast mitochondria were mock-treated (Mt) or submitted to osmotic shock (Mpl) before DNase-I treatment. Intermembrane space and matrix marker enzymes were assayed with the same samples to assess efficiency of mitoplasting. Uptake was analysed as in A. Migration of the incorporated substrates is indicated (0.5 kb CIRV1 and 2.3 kb ZmPL).

Fig. 2

DNA import into plant and mammalian mitochondria is inhibited by VDAC effectors. (A) DIDS and heparin inhibit DNA uptake into plant mitochondria. Labeled maize 2.3 kb plasmid (2.3 kb ZmPL) was incubated for 45 min at 25 °C with isolated cauliflower (Brassica oleracea) mitochondria in the absence or presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, lanes 1 to 3) or heparin (lanes 4 and 5) in standard plant DNA import conditions prior to DNase-I digestion. As the DIDS stock solution was prepared in DMSO, the corresponding control assay (lane 1) was run in the presence of 0.5% v/v DMSO, which was representative for the amount of solvent introduced by the highest DIDS concentration used (500 μM). Uptake was analysed as in Fig. 1A. Lanes 6 and 7: cauliflower mitochondria were preincubated for 30 min in import conditions in the absence or presence of heparin, pelleted, resuspended in import medium and used for a standard import assay with labeled maize 2.3 kb plasmid. Uptake was analysed as in Fig. 1A. (B) Heparin inhibits DNA uptake into mammalian mitochondria. Rat (Rattus norvegicus) mitochondria were preincubated for 30 min in standard mammalian DNA import conditions in the absence or presence of heparin; the organelles were then pelleted, resuspended in import medium and used for a standard import assay (80 min at 30 °C) with a 1 kb labeled DNA fragment corresponding to the non-coding region of the rat mitochondrial genome flanked by the tRNA^Phe and tRNA^Pro genes (1.0 kb RnFncrP, see Materials and methods). Uptake was analysed as in Fig. 1A. Migration of the incorporated substrates is indicated (2.3 kb ZmPL and 1.0 kb RnFncrP).

Fig. 3

DNA import into yeast mitochondria is inhibited by VDAC effectors. Labeled maize 2.3 kb plasmid (2.3 kb ZmPL) was incubated for 40 min at 30 °C with isolated yeast (parental strain M3) mitochondria in the absence or presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, lanes 1 to 3), heparin (lanes 4 to 6) or Ruthenium Red (RuR, lanes 7 to 9) in standard DNA import conditions prior to DNase-I digestion. As the DIDS stock solution was prepared in DMSO, the corresponding control assay (lane 1) was run in the presence of 0.2% v/v DMSO, which was representative for the amount of solvent introduced by the highest DIDS concentration used (200 μM). Uptake was analysed as in Fig. 1A. Migration of the incorporated substrate is indicated (2.3 kb ZmPL).

Fig. 4

DNA import into mitochondria from yeast individual VDAC mutants is impaired. (A) DNA import assays. Labeled maize 2.3 kb plasmid (2.3 kb ZmPL) was incubated for 40 min at 30 °C in standard DNA import conditions with mitochondria isolated from the parental yeast strain M3, from the M22-2 Δpor1 strain or from the M3-2 Δpor2 strain. Following DNase-I digestion, uptake was analysed as in Fig. 1A. Migration of the incorporated substrate is indicated (2.3 kb ZmPL). (B) Western blot analysis of VDAC-1. Mitochondrial membrane proteins from the parental yeast strain M3, the M22-2 Δpor1 strain and the M3-2 Δpor2 strain were fractionated by SDS-PAGE and blotted. The blot was probed with an antiserum specific for yeast VDAC-1 and revealed by chemiluminescence with a peroxidase-conjugated secondary antibody. Migration of VDAC-1 is indicated.

Fig. 5

Upon native analysis of mitochondrial import samples the DNA is recovered in complexes likely to contain the VDAC. (A) BN-PAGE analysis. A standard DNA import assay was run with labeled maize 2.3 kb plasmid (2.3 kb ZmPL⁎) and mitochondria isolated from the parental yeast strain M3. Following DNase-I treatment, the mitochondrial sample was lysed and the membrane fraction was submitted to electrophoresis on a Blue Native polyacrylamide gel (see Materials and methods) so as to fractionate the protein complexes (lane 1); an aliquote of the labeled DNA probe was run in parallel on the same gel (lane 2). The radioactivity was revealed upon exposure of the gel to an imaging plate. Migration of the DNA probe alone is indicated (Free DNA⁎). (B) Mass spectrometry analysis. The region corresponding to the high molecular weight complex(es) associated with labeled DNA (Complex⁎) was excised from the Blue Native gel and its protein content was analysed by nano-LC-MSMS mass spectrometry. The proteins identified are listed, with the accession number, the number of matching peptides and the percent of coverage given in brackets in the mentioned order.

Fig. 6

Mitochondria from the yeast double VDAC mutant retain competence for DNA uptake. (A) Mitochondrial membrane protein pattern analysis. Mitochondrial membrane proteins from the parental yeast strain M3, the M22-2 Δpor1 strain, the M3-2 Δpor2 strain and the M22-2-1 Δpor1–Δpor2 strain were fractionated by SDS-PAGE and stained with Coomassie Blue. ATPase-1 (accession P05030/Mascot score 112), ATP synthase subunit β (accession P00830/Mascot score 179), glyceraldehyde-3′-phosphate dehydrogenase-1 (GAPDH-1; accession P00360/Mascot score 305), prohibitin-1 (accession P40961/Mascot score 76) and the 26 kDa heat shock protein (HSP26; accession P15992/Mascot score 214) indicated on the right side of the panel were identified by MALDI-TOF mass spectrometry analysis after excision of the corresponding polyacrylamide slices from lane 4 of the gel and in-gel trypsin digestion. VDAC-1 indicated on the left side of the panel was identified by western blot analysis as in Fig. 4B following fractionation of the same samples on an identical gel. (B) DNA import assays. Labeled maize 2.3 kb plasmid (2.3 kb ZmPL) was incubated for 40 min at 30 °C in standard DNA import conditions with mitochondria isolated from the parental yeast strain M3 or from the M22-2-1 por1–Δpor2 strain. Following DNase-I digestion, uptake was analysed as in Fig. 1A. Migration of the incorporated substrate is indicated (2.3 kb ZmPL).