The mobile lysates had been immunoprecipitated in the existence of 2 mM EGTA (BAPTA-treated cells)

The Morgue deletion proteins were also examined for their abilities to enhance the eye cell death induced by expression of a chimeric R/Grim protein. P[GMR-Gal4], P[UAS-R/Grim] flies exhibit a moderate eye cell death phenotype that includes reduction in size and pigmentation of the adult compound eye. The presence of P[UAS-Morgue] enhances the level of eye cell death observed in P[GMR-Gal4], P[UAS-R/Grim] flies (Figure 1B; compare GFP to Morgue) in a manner similar to that observed for the morgue EP2367 chromosome (Wing et al. 2002).Table 1. Overexpression of Morgue results in lethality.Counts of heterozygote (balancer) and homozygote non-balancer (+) progeny derived from genetic crosses where P[da-Gal4] was used to drive expression of P[UAS-GFP] or P[UAS-Morgue]. Homozygous P[da-Gal4] or P[da-Gal4],P[UAS-GFP] flies are viable while P[daGal4],P[UAS-Morgue] homozygotes are completely lethal. Flies containing two copies of P[da-Gal4] and one copy of P[UAS-Morgue] or vice versa exhibit either complete or significant lethality. UAS-morgue1 and UAS-morgue2 represent independent insertions of P[UAS-Morgue].Table 2. Overexpresion of Morgue deletion (MorgueD) mutants exhibit variable viability.Wild-type Morgue as well as Morgue Gly421x mutants were co-expressed with Reaper in P[52A-Gal4], P[UAS-LacZ] flies and anti-?galactosidase immunostaining was used to label the midline cells in embryos. The results revealed that the Morgue Gly421x mutants exhibit a similar cell death-enhancing phenotype as native Morgue (Figure 2D; data now shown for P[UAS-MorgueG421A]). These data indicate that the conserved Gly421 residue is not specifically essential for enhancement of Grim-Reaper mediated cell death.Because the Morgue protein contains several putative protein interaction domains, these phenotypes likely involve interactions between Morgue and other factors. To identify these factors we performed in vivo co-immunoprecipitation experiments to identify Morgue-associated proteins in adult flies. P[UAS-3xFlag:Morgue] and P[UAS-Morgue:3xFlag] were generated that express full length wild-type Morgue proteins containing a 3xFLAG epitope tag at either the NH2- or COOH-terminal end. Two approaches were used to confirm that these lines both express active fusion proteins in a Gal4-dependent fashion. The first approach was to analyze the expression of FLAG:Morgue and Morgue:FLAG proteins. P[UAS-Morgue], P[UAS-3xFlag:Morgue], and P[UAS-Morgue:3xFlag] were crossed to P[da-Gal4] and P[elav-Gal4] strains. Protein extracts from the adult progeny were analyzed via anti-FLAG Western blots. Prominent bands were observed at the predicted molecular weight of ,60 kDa in FLAG:Morgueand Morgue:FLAG-expressing samples (Figure 3A). Anti-FLAG immunohistochemistry was also performed on embryos derived from crosses between P[elav-Gal4] and P[UAS-3xFlag:Morgue] or P[UAS-Morgue:3xFlag]. Strong immunostaining was observed for both 3xFLAG:Morgue and Morgue:3xFlag throughout the central and peripheral nervous system (Figure 3B and data not shown). These results confirm that the tagged Morgue proteins are appropriately expressed. The second approach to analyze the Morgue:FLAG proteins was functional. Over-expression of P[Morgue:3xFlag] was examined for lethality when expressed by P[da-Gal4]. Homozygous P[da-Gal4], P[UAS-Morgue:3xFlag] flies exhibited the same complete lethality as homozygous P[da-Gal4], P[UAS-Morgue] flies (Figure 3C). Both P[UAS-3xFlag:Morgue] and P[UASMorgue:3xFlag] were also analyzed for their ability to enhance the eye cell death phenotype of P[GMR-Gal4]/P[UAS-R/Grim] flies. Targeted expression of each fusion protein was shown to exhibit similar eye cell death enhancement as native Morgue (Figure 3D). In addition, similar to native Morgue, both P[3xFlag:morgue] and P[Morgue:3xFlag] enhanced P[UAS-Reaper]-induced cell death in the embryonic CNS midline (data not shown). Taken together, the data indicate that expression of each Morgue fusion protein can be targeted specifically and that Morgue functions are not impaired by the 3xFLAG tag. The Morgue:FLAG proteins can be expressed at high levels and are functional. They thus represent useful reagents for identifying Morgue-associated proteins.Effects of widespread expression of Morgue deletion mutants on fly viability. Counts of homozygous non-balancer (+) and heterozygote balancer progeny derived from genetic crosses where P[da-Gal4] was used to drive expression of P[UAS-GFP] or various P[UAS-MorgueD] strains. Homozygote (non-balancer) and heterozygote (with either a TM3 or both a CyO and a TM3 balancer) progeny derived from: P[da-Gal4], P[UAS-MorgueD]/TM3, P[da-Gal4], P[UASGFP]/TM3, or P[UAS-MorgueD]/CyO, P[da-Gal4]/TM3 parent flies were counted. Expression of MorgueDZF, MorgueDFB, MorgueDUEV, and MorgueDZF-FB resulted in reduced Morgue-induced lethality. In contrast, removal of the F box alone retained near complete Morgue-induced lethality. Note that if the homozygotes are fully viable, the expected percentage of viable flies is either 50% or 25% depending on whether one or two balancer chromosomes are present. Morgue (Figure 1B). Thus, the zinc finger, F-box, or UEV domain are not individually essential for Morgue’s ability to enhance eye cell death, as removal of each single domain does not dramatically alter the enhanced eye cell death phenotype.The invariance of the Gly421 in the catalytic site of the Morgue UEV domains suggests that this Glycine residue may have specific and crucial functions. To test this possibility we generated P[UASMorgueG421x] lines (Figure 2A) with a mis-sense point mutation of Gly421 to a Cysteine (G421C: the active catalytic residue in bona fide E2 conjugases), an Alanine (G421A: a small hydrophobic, non-polar amino acid chemically similar to Glycine), or a Serine (G421S: a polar residue present in other UEVs). The Morgue point mutant proteins were examined for their ability to induce lethality when expressed by P[da-Gal4], and their ability to enhance R/Grim eye cell death in P[GMR-Gal4], P[UAS-R/ Grim] flies. All three mutants exhibit complete or nearly complete lethality when expressed as homozygotes by P[da-Gal4] (Figure 2B). Thus, the invariant Gly421 residue is not essential for Morgue-induced lethality. Each of the Gly421x mutant Morgue proteins also exhibited a similar ability as native Morgue to enhance eye cell death in P[GMR-Gal4], P[UAS-R/Grim] flies (Figure 2C; data not shown for P[UAS-MorgueG421S]). The Morgue point mutants were also examined for their ability to enhance reaper-induced CNS midline cell death using the P[52a-Gal4] line.