Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant.
We explore the possibility of re-engineering mitochondrial genes and expressing them from the nucleus as an approach to rescue defects arising from mitochondrial DNA mutations. We have used a patient cybrid cell line with a single point mutation in the overlap region of the ATP8 and ATP6 genes of the human mitochondrial genome. These cells are null for the ATP8 protein, have significantly lowered ATP6 protein levels and no Complex V function. Nuclear expression of only the ATP8 gene with the ATP5G1 mitochondrial targeting sequence appended restored viability on Krebs cycle substrates and ATP synthesis capabilities but, failed to restore ATP hydrolysis and was insensitive to various inhibitors of oxidative phosphorylation. Co-expressing both ATP8 and ATP6 genes under similar conditions resulted in stable protein expression leading to successful integration into Complex V of the oxidative phosphorylation machinery. Tests for ATP hydrolysis / synthesis, oxygen consumption, glycolytic metabolism and viability all indicate a significant functional rescue of the mutant phenotype (including re-assembly of Complex V) following stable co-expression of ATP8 and ATP6 Thus, we report the stable allotopic expression, import and function of two mitochondria encoded genes, ATP8 and ATP6, resulting in simultaneous rescue of the loss of both mitochondrial proteins.
SENS Research Foundation Research Center, Mountain View, CA 94041, USA amutha.boominathan@sens.org.
Amutha
A
Boominathan
SENS Research Foundation Research Center, Mountain View, CA 94041, USA.
Shon
S
Vanhoozer
Keck School of Medicine of USC, Los Angeles, CA 90033, USA.
Nathan
N
Basisty
SENS Research Foundation Research Center, Mountain View, CA 94041, USA.
Kathleen
K
Powers
Buck Institute for Research on Aging, Novato, CA 94945, USA.
Alexandra L
AL
Crampton
Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
Xiaobin
X
Wang
Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Natalie
N
Friedricks
Keck School of Medicine of USC, Los Angeles, CA 90033, USA.
Birgit
B
Schilling
Keck School of Medicine of USC, Los Angeles, CA 90033, USA.
Martin D
MD
Brand
SENS Research Foundation Research Center, Mountain View, CA 94041, USA matthew.oconnor@sens.org.
Matthew S
MS
O'Connor
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000255:Adenosine Triphosphate; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D015870:Gene Expression; D050259:Genes, Mitochondrial; D005816:Genetic Complementation Test; D006868:Hydrolysis; D008565:Membrane Proteins / Q000235:genetics*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases; D009154:Mutation
D002352:Carrier Proteins; D008565:Membrane Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein