Comparative analysis of proapoptotic activity of cytochrome c mutants in living cells.
A non-traumatic electroporation procedure was developed to load exogenous cytochrome c into the cytoplasm and to study the apoptotic effect of cytochrome c, its K72-substitued mutants and "yeast --> horse" hybrid cytochrome c in living WEHI-3 cells. The minimum apoptosis-activating intracellular concentration of horse heart cytochrome c was estimated to be 2.7 +/- 0.5 microM (47 +/- 9 fg/cell). The equieffective concentrations of the K72A-, K72E- and K72L-substituted mutants of cytochrome c were five-, 15- and 70-fold higher. The "yeast --> horse" hybrid created by introducing S2D, K4E, A7K, T8K, and K11V substitutions (horse protein numbering) and deleting five N-terminal residues in yeast cytochrome c did not evoke apoptotic activity in mammalian cells. The apoptotic function of cytochrome c was abolished by the K72W substitution. The K72W-substituted cytochrome c possesses reduced affinity to the apoptotic protease activating factor-1 (Apaf-1) and forms an inactive complex. This mutant is competent as a respiratory-chain electron carrier and well suited for knock-in studies of cytochrome c-mediated apoptosis.
Sharonov
G V
GV
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia.
Feofanov
A V
AV
Bocharova
O V
OV
Astapova
M V
MV
Dedukhova
V I
VI
Chernyak
B V
BV
Dolgikh
D A
DA
Arseniev
A S
AS
Skulachev
V P
VP
Kirpichnikov
M P
MP
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D002460:Cell Line; D019069:Cell Respiration; D002470:Cell Survival; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism*; D018274:Electroporation; D006736:Horses; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008856:Microscopy, Fluorescence; D051336:Mitochondrial Membranes / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013997:Time Factors
D017304:Annexin A5; D050505:Mutant Proteins; D045304:Cytochromes c