Download Identification and characterization of novel interaction partners of

12
th
E u r o c o n f e r e n c e on A p o p t o s i s
Poster Abstracts
Identification and characterization of novel interaction partners of
Drosophila apoptsis inhibitor DIAP1
Mirjana Gagic, Thomas Keßler and H.- Arno J. Müller;
Institut für Genetik, Heinrich-Heine Universität Düsseldorf, Germany
The Drosophila member of the IAP family, DIAP1 is necessary for cell survival and
thus for the early embryo. Zygotic null-mutants of DIAP1 (thread 109) exhibit massive
cell death during gastrulation. DIAP1 interacts both with caspases and with the
proapoptotic proteins Reaper Hid and Grim (RHG). Structurally DIAP1 has two BIRdomains, which mediate binding to caspases and also to RHG proteins. Additionally,
DIAP1 contains a C-terminal RING E3 ubiquitin ligase domain. Although DIAP1 is a
key anti-apoptotic protein in Drosophila, little is known about mechanisms that
regulate DIAP1 function.
To find additional proteins that might regulate DIAP1, we performed a yeast two
hybrid screen. From 29 potential interaction partners, two were chosen for further
analysis.
Rala is a Ras like small GTPase. Rala specifically interacts with the BIR1 domain of
DIAP1 in yeast. In GST pulldown experiments DIAP1 binds exclusively to a
constitutively activated form of Ral. Further experiments show that Ral inhibits
DIAP1 autoubiquitination. To characterize a relation between DIAP1 and Ral protein
levels in vivo, we investigate germline clones of Ral and perform overexpression
experiments with RalG20V as well as DIAP1 constructs. We hope to elucidate the
functional importance of the Ral/DIAP1 interaction for regulation of apoptosis in the
Drosophila embryo.
The other part of our research involves the B-subunit of the vacuolar H+-ATPase (vha
55). Vha55 is a component of a large complex localized to the lysosomes which
mediates (H+) transport to the lysosome and so regulates lysosomal pH. Inhibition of
the ATPase function with BafilomycinA in Drosophila S2-cells induced apoptosis in a
drICE dependent manner. We are currently testing whether induction of apoptosis is
a consequence of cathepsinD release from the lysosome to the cytosol. We are
interested whether DIAP1 plays a direct role in sustaining the function of the
ATPase.
P - 55