There are a growing number of cell death (apoptosis) signaling proteins
found in the mitochondrial intermembrane space of mammalian cells.
Cytochrome C (CytC) is the best characterized among these apoptotic
proteins. Release of CytC leads to the cytosolic assembly of the
apoptosome (a caspase activating complex involving APAF-1 and caspase 9)
that induces the cell death cascade. It was shown that Saccharomyces
cerevisiae commits to a programmed cell death (PCD) with typical
apoptotic markers (1) and that a caspase related-protease is also
present (2). Moreover, CytC is translocated to the cytosol in S.
cerevisiae undergoing PCD induced by acetic acid (3) and as an
effect of BAX expression (4). It would be advantageous to be able to
screen CytC-protein interactions in order to elucidate the cell death
pathway triggered by CytC release in yeast. However, the localization of
mature CytC excludes the use of the standard transcription based yeast
two-hybrid system. For this reason, we are currently developing a two-
hybrid screening technique based on the dihydrofolate reductase (DHFR)
protein complementation assay (Remy I and Michnick SW 1999. PNAS
96:5394-9). The interacting proteins (bait and prey) are fused to each
of two rationally designed fragments of DHFR. Assisted by the
interaction between bait and prey, these fragments are able to fold
together and rescue a S. cerevisiae strain deficient in DHFR.
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