Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L.
Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.
Wittig
Ilka
I
Molecular Bioenergetics Group, Medical School, Goethe-University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. wittig@zbc.kgu.de
Meyer
Bjoern
B
Heide
Heinrich
H
Steger
Mirco
M
Bleier
Lea
L
Wumaier
Zibiernisha
Z
Karas
Michael
M
Schägger
Hermann
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics; D019281:Dimerization; D006801:Humans; D066298:In Vitro Techniques; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D017931:DNA Primers; D004272:DNA, Mitochondrial; D021122:Protein Subunits; D025261:Mitochondrial Proton-Translocating ATPases