| Abstract
| - Apoptosis regulators of the Bcl-2 family associate with intracellular membranes frommitochondria and the endoplasmic reticulum, where they perform their function. The activity of theseproteins is related to the release of apoptogenic factors, sequestered in the mitochondria, to the cytoplasm,probably through the formation of ion and/or protein transport channels. Most of these proteins containa C-terminal putative transmembrane (TM) fragment and a pair of hydrophobic α helices (α5−α6) similarto the membrane insertion fragments of the ion-channel domain of diphtheria toxin and colicins. Here,we report on the membrane-insertion properties of different segments from antiapoptotic Bcl-xL andproapoptotic Bax and Bid, that correspond to defined α helices in the structure of their soluble forms.According to prediction methods, there are only two putative TM fragments in Bcl-xL and Bax (theC-terminal α helix and α-helix 5) and one in activated tBid (α-helix 6). The rest of their sequence, includingthe second helix of the pore-forming domain, displays only weak hydrophobic peaks, which are belowthe prediction threshold. Subsequent analysis by glycosylation mapping of single α-helix segments in amodel chimeric system confirms the above predictions and allows finding an extra TM fragment made ofhelix α1 of Bax. Surprisingly, the amphipathic helices α6 of Bcl-xL and Bax and α7 of Bid do insert inmembranes only as part of the α5−α6 (Bcl-xL and Bax) or α6−α7 (Bid) hairpins but not when assayedindividually. This behavior suggests a synergistic insertion and folding of the two helices of the hairpinthat could be due to charge complementarity and additional stability provided by turn-inducing residuespresent at the interhelical region. Although these data come from chimeric systems, they show directpotentiality for acquiring a membrane inserted state. Thus, the above fragments should be considered forthe definition of plausible models of the active, membrane-bound species of Bcl-2 proteins.
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