The destabilization of lipid membranes induced by the C-terminal fragment of caspase 8-cleaved bid is inhibited by the N-terminal fragment.

Bid is a proapoptotic, BH3-domain-only member of the Bcl-2 family. In Fas-induced apoptosis, Bid is activated through cleavage by caspase 8 into a 15.5-kDa C-terminal fragment (t(c)Bid) and a 6.5 kDa N-terminal fragment (t(n)Bid). Following the cleavage, t(c)Bid translocates to the mitochondria and promotes the release of cytochrome c into the cytosol by a mechanism that is not understood. Here we report that recombinant t(c)Bid can act as a membrane destabilizing agent. t(c)Bid induces destabilization and breaking of planar lipid bilayers without appearance of ionic channels; its destabilizing activity is comparable with that of Bax and at least 30-fold higher than that of full-length Bid. Consistently, t(c)Bid, but not full-length Bid, permeabilizes liposomes at physiological pH. The destabilizing effect of t(c)Bid on liposomes and planar bilayers is independent of the BH3 domain. In contrast, mutations in the BH3 domain impair t(c)Bid ability to induce cytochrome c release from mitochondria. The permeabilizing effect of t(c)Bid on planar bilayers, liposomes, and mitochondria can be inhibited by t(n)Bid. In conclusion, our results suggest a dual role for Bid: BH3-independent membrane destabilization and BH3-dependent interaction with other proteins. Moreover, the dissociation of Bid after cleavage by caspase 8 represents an additional step at which apoptosis may be regulated.

Release of thioredoxin via the mechanosensitive channel MscL during osmotic downshock of Escherichia coli cells.

Escherichia coli cells possess several mechanosensitive ion channels but only MscL, the channel with the highest conductance, which is activated at the highest membrane tension, has been cloned. We investigated the putative involvement of MscL in the effluxes caused by osmotic downshock. Osmotic shock caused the release of potassium glutamate, trehalose, and glycine betaine from wild type cells and cells lacking MscL. There was no difference between the two strains, but the extreme rapidity of the efflux process, as shown herein for glycine betaine, suggests that it is channel-mediated. Osmotic downshock also induces the release of some cytosolic proteins from EDTA-treated cells. We investigated the release of thioredoxin. This protein was totally released from wild type cells but was retained by MscL- cells. Release was restored by expression of the gene coding for MscL. Thus MscL is not necessary for the excretion of osmoprotectants, but it does open in vivo during shock and catalyzes the efflux of thioredoxin and possibly other small cytosolic proteins. It follows that the other mechanosensitive channels, which are known to be activated at lower tension, must also open during osmotic shock. Their opening and that of MscL could account for the rapid release of osmolytes.

Gadolinium ion inhibits loss of metabolites induced by osmotic shock and large stretch-activated channels in bacteria.

Bacteria subjected to a hypotonic osmotic shock lose internal ions and also metabolites, without lysis of the cells. We show that the presence in the shock medium, at submillimolar concentrations, of the ion gadolinium, recently shown to block stretch-activated channels in Xenopus oocytes [Yang, X.-C. & Sachs, F. (1989) Science 243, 1068-1071], was sufficient to inhibit shock-induced release of metabolites such as lactose and ATP in Escherichia coli and ATP in Streptococcus faecalis. Moreover, gadolinium was observed, in patch-clamp experiments, to inhibit the giant stretch-activated channels of E. coli, S. faecalis. and Bacillus subtilis. Taken together, these data suggest that stretch-activated channels are localized in the cytoplasmic membrane of Gram-negative and Gram-positive bacteria, where they control the efflux of osmotic solutes, thus probably playing a major role in the response to hypotonic osmotic shock.