Khan et al.
Supplementary information
SUPPLEMENTARY METHODS
Affinity purification of recombinant dOmi from bacteria. Mature dOmi and its
mutants were induced by IPTG in 500 ml cultures in 2L flasks for 3.5 hours. Cell pellets
were collected and washed once in phosphate-buffered saline (10 mM phosphate, pH
7.4, containing 150 mM NaCl) and centrifuged, and the pellet was resuspended in 10 ml
of cell lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 1 mM
EDTA, 1 mM EGTA) supplemented with protease inhibitor cocktail tablets (complete,
EDTA free) from Roche. Cells were homogenized and the supernatant was subjected to
centrifugation at 12,500 x g.
The resultant supernatant was then incubated with
TALONTM-agarose for 1h at 4 °C. The bead-bound proteins were washed three times in
lysis buffer and then eluted with 100 mM EDTA at 4 °C for 1 h.
DEVD cleavage assay.
Drosophila caspases DCP1 and DRICE were expressed in
bacteria with C-terminal His6 tags using the pET-21a vector. Recombinant DCP1 and
DRICE were purified on TALONTM-agarose as described above. Purified DCP1 and
DRICE undergo spontaneous autoprocessing to yield the catalytically active form of the
enzyme. DCP1 and DRICE enzymatic assays were performed in a 100 µl volume in
buffer A (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM
EGTA, 1 mM dithiothreitol) at 25 °C. Pure GST-DIAP1 was incubated with pure DCP1
(20 nM) or DRICE (5 nM) with various amounts of mature wildtype and mutant dOmi
and 10 µM Asp-Glu-Val-Asp-7-Amino-4-trifluoromethyl coumarin (DEVD-AFC)
Khan et al.
substrate for 15 min. The amount of cleaved AFC was measured using 390 and 510 nm
as the excitation and emission wavelengths using a Thermo Electron Fluoroskan Ascent
spectrometer. Results were expressed as DEVD cleavage activity in relative fluorescence
units. The data represent the average of three independent experiments with error bars
showing standard deviation.
In vitro interaction assays. All interactions using in vitro translated 35S-labeled XIAP or
DIAP1 were performed by incubating 10 µl of TNT® (Promega) translation reaction at
4 °C for 1.5 h with equal amounts of proteins (200 ng) bound to 50 µl of TALONTMagarose (CLONTECH) beads in a 100-µl reaction. The beads in all the samples were
washed at least 4 times and then boiled in 30 µl of sample buffer. 15 µl of each sample
was loaded onto a 12.5% SDS polyacrylamide gel and separated by electrophoresis. The
gel was dried and exposed to x-ray film.
In vitro protease assay. Cleavage of 35S-labeled DIAP1 or DIAP2 by recombinant dOmi
was assayed as described previously 1. Bacterially expressed (His)6-tagged dOmi/HtrA2
was purified on Talon affinity resins and then incubated with
35
S-labeled DIAP1 or
DIAP2 for 1 h at room temperature. Cleavage products were analyzed by SDS-PAGE
and visualized by autoradiography.
The protease activity of endogenous Sf9-Omi or recombinant dOmi expressed in
Sf9 cells was assayed with the substrates
35
S-labeled -casein as described before 1.
Briefly, Sf9-Omi or dOmi was immunoprecipitated with an anti-dOmi polyclonal
antibody, and the antibody-Omi complexes were bound to protein G-sepharose and then
Khan et al.
washed several times. The protein G-sepharose-bound complexes were incubated with
S-labeled -casein for 1 h at 25° C. Cleavage products were analyzed by SDS-PAGE
35
and visualized by autoradiography.
Cleavage of DIAP1-C-GST by mature dOmi-L or dOmi-S was determined using
recombinant proteins expressed in bacteria. (His)6-tagged mature dOmi-L or dOmi-S
was purified on Talon affinity resin and then incubated with glutathione sepharose-bound
DIAP1-GST at 22°C for 1h. Cleavage products were analysed by SDS–PAGE and
visualized by coomassie staining. In some experiments, wildtype or mutant dOmi-L or
dOmi-S were bound to DIAP1-C-GST, DIAP1-BIR1-C-GST or DIAP1-BIR2-RING-CGST at 4°C for 1h. The DIAP1-C-GST-dOmi complexes were bound to glutathione
sepharose beads, washed and then incubated at room temperature for 1h. The reaction
products were analyzed by SDS–PAGE and visualized by coomassie staining.
Cytosolic and mitochondrial fractionation. Cells were homogenized in buffer A and
the homogenate was centrifuged at 800 x g to remove nuclei, cellular debris and unlysed
cells. The supernatant was centrifuged at 12,500 x g, and the resultant pellet (crude
mitochondrial fraction) and supernatant (cytosolic fraction) were collected and used for
SDS-PAGE and other assays.
Immunostaining of dOmi in Drosophila eye imaginal discs. Eye discs were dissected
from wandering 3rd instar larvae, and fixed with 3.7% formaldehyde for 30 minutes at
room temperature.
antibody
and
Antibody staining was performed using biotinylated secondary
streptavidin-horseradish
peroxidase,
and
visualized
using
the
Khan et al.
diaminobenzidine reaction. Polyclonal anti-Drosophila Omi was used at a dilution of
1:500.
In vitro ubiquitination assay. These experiments were performed essential as described
before 2.
Drosophila transgenes and heat shock treatment. The full-length dOmi or dOmiPDZ cDNAs were cloned into the pGMR plasmid under the control of the GMR
promoter by standard procedures.
previously described
3,4
Transgenic lines were established by methods
. For the experiments of Fig. 6B, transgenic flies were reared at
25°, transferred to 30° for 5 days, returned to 25° for 5 days, and adults were examined
10-14 hours after eclosion (based on timing and the normal time-dependent darkening of
their body color).
UV-induced cell death. In all the experiments, UV-induced cell death was performed at
200mJ/cm2 using a UV Stratalinker 1800 (Stratagene), and cells were lysed using
standard techniques 4 h after UV irradiation, for isolation of cytosol and mitochondria.
References
1.
Jones, JM, Datta, P, Srinivasula, SM, Ji, W, Gupta, S, Zhang, Z et al., (2003) Loss
of Omi mitochondrial protease activity causes the neuromuscular disorder of
mnd2 mutant mice. Nature 425: 721-7.
2.
Olson, MR, Holley, CL, Yoo, SJ, Huh, JR, Hay, BA and Kornbluth, S, (2003)
Reaper is regulated by IAP-mediated ubiquitination. J Biol Chem 278: 4028-34.
3.
Rubin, GM and Spradling, AC, (1982) Genetic transformation of Drosophila with
transposable element vectors. Science 218: 348-53.
4.
Fujioka, M, Jaynes, JB, Bejsovec, A and Weir, M, (2000) Production of
transgenic Drosophila. Methods in Molecular Biology 136: 353-63.
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