Inhibition of Bak activation by VDAC2 is dependent on the Bak transmembrane anchor.
Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.
Lazarou
Michael
M
La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia.
Stojanovski
Diana
D
Frazier
Ann E
AE
Kotevski
Aneta
A
Dewson
Grant
G
Craigen
William J
WJ
Kluck
Ruth M
RM
Vaux
David L
DL
Ryan
Michael T
MT
D000818:Animals; D017209:Apoptosis; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D051336:Mitochondrial Membranes / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism; D050996:Voltage-Dependent Anion Channel 2 / Q000502:physiology*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000037:antagonists & inhibitors* / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000502:physiology*
C476525:FIS1 protein, human; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D050996:Voltage-Dependent Anion Channel 2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein