Reconstitution of caspase-mediated cell-death signalling in Schizosaccharomyces pombe

Two pro-apoptotic proteases, caspase-1 and caspase-3, have been expressed a s full-length proteins in the fission yeast Schizosaccharomyces pombe. Both proteins autoprocess to generate the corresponding active enzyme and both are lethal to the yeast cell. Lethality is due to catalytic activity since the expression of the inactive mutant forms of both caspases does not resul t in an obvious phenotype. Caspase-expressing yeast can be rescued by co-ex pression of the baculovirus protein p35, a known inhibitor of the caspase f amily. Go-expression of Bcl-2, another anti-apoptotic protein, does not pre vent the cell death induced by either caspase. However, Bcl-2 is itself cle aved by both caspase-1 and caspase-3 at two adjacent recognition sites, YEW D(31)A and DAGD(34)V respectively, immediately downstream from the N-termin al BH4 domain, a region of Bcl-2 which is essential for its anti-apoptotic activity; similar cleavage of Bcl-2 by caspases has been demonstrated in ma mmalian cells. Hence, key elements of the apoptotic pathway can be reliably reconstituted in fission yeast, opening the way to exploit yeast in order to study the control of apoptosis. Furthermore, the activity of caspase-3, although not caspase-1, can be demonstrated in vitro using chromogenic subs trates. This offers the possibility of using caspase-producing strains of y east to screen for chemical inhibitors either in vivo or in vitro.