| Sequence | Protein | Binding to mitochondria | Cytochrome c release | ||
|---|---|---|---|---|---|
| Yeast | Mammals | Yeast | Mammals | ||
| (1) Role of ART and Proline residues at positions 8 and 13 | |||||
| -P8RGGGP13- | full length Bax | +/− | +/− | +/− | +/− |
| BaxΨ/ΔART | +++ | +++ | +++ | +++ | |
| -G8RGGGG13- | ART mutant | +++ | n.d. | +++ | n.d. |
| -V8RGGGV13- | ART mutant | n.d. | +++ | n.d. | +++ |
| -V8RGGGP13- | ART mutant | n.d. | +/− | n.d. | +/− |
| -P8RGGGV13- | ART mutant | n.d. | +++ | n.d. | +++ |
| (2) Role of helix α1 | |||||
| -A24LLL27- | BaxΨ/ΔART | +++ | +++ | +++ | +++ |
| BaxΨ/ΔARTΔHα1 | n.d. | − | n.d. | − | |
| -R24LLL27- | BaxΨ/ΔARTHα1 mutant | + | − | + | − |
| -A24LGV27- | BaxΨ/ΔARTHα1 mutant | +++ | − | +++ | − |
| (3) Interaction between ART and BH2 (helix α7/8) | |||||
| -P8RGGGP13--I152QDQG156- | full length human Bax | +/− | +/− | +/− | +/− |
| -P8EGGGP13--I152QDQG156- | ART mutant | +++ | n.d. | +++ | n.d. |
| -P8RGGGP13--I152QKQG156- | BH2 mutant | +++ | n.d. | +++ | n.d. |
| -P8EGGGP13--I152QKQG156- | ART mutantBH2 mutant | − | n.d. | − | n.d. |
(1) The substitution of prolines 8 and 13, like the complete deletion of the 20 N-terminal residues, increase Bax binding and activity both in yeast and mammals; (2) substitutions in helix α1 decrease the binding of BaxΨ in mammals. The decrease is partly visible in yeast; (3) individual point mutations inverting charges in ART and in BH2 induce stimulation of Bax in yeast, but a double charge change revert to the wild-type behavior of Bax, suggesting the existence of an interaction between charged residues in these two domains, stabilizing the inactive conformation. Bold residues indicate mutations. −: inactive; n.d.: not determined; +/−: poorly active; +: active; +++: strongly active