DNA import competence and mitochondrial genetics

Institute of Plant Molecular Biology, CNRS and University of Strasbourg (UdS) 12, General Zimmer Str., Strasbourg, France, 67084 Siberian Institute Plant Physiology and Biochemistry, Siberian Branch of the RAS 132, Lermontova Str., Irkutsk, Russian Federation, 664033 Institute for Cell and Molecular Biosciences, Medical School, Newcastle University Framlington Place, Newcastle upon Tyne, UK, NE2 4HH School of Pharmacy, MCPHS University, 179, Longwood Ave., Boston, USA, MA 02115

Introduction.Mitochondrial genome expression is essential for organelle functional efficiency and intercompartment cross-talk.Manipulation of mitochondrial genetics is thus of interest for a range of fundamental investigations and is appealing to treat neurodegenerative diseases caused by organelle DNA mutations.In plants, mitochondrial genetics underlies key breeding tools.Given the importance of these issues, transforming mitochondria has been a long standing goal that was unfortunately reached only in a couple of unicellular organisms.Contrasting with the failure to transform the organelles in whole cells, we established that isolated plant and mammalian mitochondria can functionally import double-stranded DNA through an active mechanism [1,2].The process is sensitive to a number of effectors and can accommodate large size linear DNA [3].Remarkably, the imported DNA functionally joins the organelle genetic system.Marker sequences under the control of a mitochondrial promoter are expressed in organello [1,2].Imported DNA carrying oxidative lesions is repaired [4,5].Constructs carrying fragments of mitochondrial DNA undergo homologous recombination with the resident DNA [6].On that basis, we aim to understand the mechanism(s) underlying mitochondrial competence for DNA uptake and to develop cell uptake followed by mitochondrial targeting of functional gene constructs.
Materials and methods.We developed DNA uptake experiments with mitochondria isolated from potato (Solanum tuberosum) or from Saccharomyces cerevisiae mutants defective for various nucleus-encoded mi-tochondrial proteins and carriers.We used 1-2.3 kb radiolabeled DNA fragments as substrates.Isolation of mitochondria and uptake assays were performed as described earlier [1,7].For mitochondrial targeting of DNA in mammalian cells, a liposomal formulation was prepared by a standard film hydration method [8].A 2.2 kb DNA construct was complexed with the liposomal carrier and incubated with a rat cell culture.qPCR and RT-qPCR analyses assessed the level of cell-internalized construct and putative transcription.
Results and discussion.The voltage-dependent anion channel (VDAC) seems to be involved in DNA translocation through the mitochondrial outer membrane [1,2,7].For the inner membrane, inhibition studies of the uptake using specific effectors pointed to an involvement of the adenine nucleotide translocator in plants [1], but the challenge of understanding which channel(s) can be recruited or hijacked by double-stranded DNA molecules remains mostly open.In the present studies, we used both biochemical approaches and S. cerevisiae genetic tools to identify the still elusive inner membrane proteins participating in mitochondrial DNA import.Strikingly, among the candidates from the inner membrane carrier family selected on the basis of biochemical data with plant organelles, only the two minor forms of the adenine nucleotide translocator turned out to be required for optimal DNA translocation into isolated yeast mitochondria (Figure).Conversely, we highlighted a putative contribution of proteins that control mitochondrial morphology in S. cerevisiae.
Building on the hypothesis that the competence for DNA uptake is also a property of the organelles in vivo, we attempted to use nanocarriers to target DNA to mitochondria in intact cells.We explored the use of a mitochondriotropic liposomal formulation to deliver a DNA construct encoding a recoded green fluorescent protein (gfp) gene controled by a rat mitochondrial promoter into the mitochondria in live rat cells.In comparison to free DNA and vehicle controls, incubation of the cells with liposome/DNA complexes led to significant incorporation of the construct and generation of gfp mRNA.
Conclusions.Taken together, our data imply that there are significant variations in the mitochondrial DNA import mechanism between different organisms and that even in a given organism multiple pathways might operate.Our first in vivo results suggest that mitochond-riotropic liposomes can deliver DNA into mitochondria of live mammalian cells, potentially opening novel prospects for mitochondrial transfection.
Funding.This work was supported by regular funding from the CNRS and the University of Strasbourg, as well as by the Investissements d'Avenir from the French Ministry for Research (grant number ANR-11-LABX-0057_MITOCROSS).