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. We show that 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 activity, oxygen consumption, glycolytic metabolism, and viability all indicate a significant functional rescue of the mutant phenotype 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 the simultaneous rescue of the loss of both mitochondrial proteins.
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: SWATH ; data independent acquisition ; ATP6 ; mitochondria ; Complex 5
Principal Investigators: (in alphabetical order) |
Birgit Schilling |
Submitting User: | bschilling |
Boominathan A, Vanhoozer S, Basisty N, Powers K, Crampton AL, Wang X, Friedricks N, Schilling B, Brand MD, O'Connor MS.
Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant.
Nucleic Acids Res. Epub 2016 Sep 4.
Number of Files: | |
Total Size: | |
Spectra: | |
Subscribers: | |
Owner | Reanalyses | |
---|---|---|
Experimental Design | ||
Conditions:
|
||
Biological Replicates:
|
||
Technical Replicates:
|
||
Identification Results | ||
Proteins (Human, Remapped):
|
||
Proteins (Reported):
|
||
Peptides:
|
||
Variant Peptides:
|
||
PSMs:
|
||
Quantification Results | ||
Differential Proteins:
|
||
Quantified Proteins:
|
||
Browse Dataset Files | |
FTP Download Link (click to copy):
|