MiPsummer Programme pdf - Mitochondrial Physiology Society

62
Abstract # 32
The mechanism by which mitochondrial DNA 9205delTA mutation alters the structure and
function of ATP synthase and cytochrome c oxidase
K. Hejzlarová, V. Kaplanová, P. Ješina, Z. Drahota, H. Nsková, N. Kovářová and J. Houštěk
Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., Vídeská 1083, 14220
Prague 4, Czech Republic
Background Missense mutations in mtDNA MT-ATP6 gene frequently cause severe mitochondrial
disorders. A different type of MT-ATP6 mutation is represented by 9205delTA microdeletion which
cancels the STOP codon of MT-ATP6 gene and affects the cleavage site in MT-ATP8/MT-
ATP6/MT-CO3 polycistronic transcript, thereby interfering with the processing of mRNAs for
ATP6 subunit (F o -a) of the ATP synthase and Cox3 subunit of the cytochrome c oxidase (COX).
Objectives To gain more insight into the pathogenic mechanism, we investigated changes in the
structure (subunit levels and composition of the native enzymes) and function (respiration,
membrane potential and enzymatic activities) of ATP synthase and COX in control and 9205delTA
homoplasmic cybrids.
Methods All experiments were performed in control and 9205delTA homoplasmic cybrid cell lines
and some in + and 0 cells. Structural changes of ATP synthase and COX were analyzed using
SDS-PAGE, BNE- or hrCNE1-PAGE and WB or activity staining in gel. ADP-stimulated
respiration was measured by high-resolution respirometry (Oroboros, Oxygraph-2k), m was
measured with TPP + -selective electrode, ATP synthesis was determined in a luciferin-luciferase
reaction and ATP hydrolysis in ATP-regenerating system, COX activity was measured
spectrophotometrically.
Results We found that 9205delTA mutation diminishes the synthesis of F o -a protein and reduces
the content of Cox1, Cox2 and Cox3 proteins, alters the structure but not the content of ATP
synthase, decreases the content of COX, and prevents most of mitochondrial ATP production. The
ATP synthase complex was assembled without F o -a subunit but it was rather labile and unable to
synthesize ATP. The complex retained ATP hydrolytic activity that was unexpectedly oligomycinsensitive,
thus questioning the involvement of F o -a subunit in the inhibitor effect.
Conclusion The pathogenic mechanism of 9205delTA mutation is caused by combine defect of
both ATP synthase and COX enzymes, perhaps the COX deficiency is the primary and more critical
for the overall mitochondrial energy provision.
Acknowledgement This work was supported by the Grant Agency of the Czech Republic
(303/11/0970, 303/12/1363) and Ministry of Education, Youth and Sports of the Czech Republic
(AV0Z 50110509, RVO:67985823).