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With Roc2, Cul5EY mutants develop normally and do not display any overt morphological defects. That Roc2 and Cul5 mutant animals are viable and display no obvious developmental defects is consistent with our analysis of Roc-Cullin interactions indicating that Roc2 and Cul5 bind exclusively to each other. Indeed, Roc2 and Cul5 accumulation in vivo is interdependent: Roc2 was undetectable in Cul5 mu
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A promoter. If the NH2-terminus is sufficient to confer Cullin binding specificity, all chimeras with the same NH2-terminal Roc sequence should bind the same Cullins, regardless of the RING domain to which they are fused. We transfected these constructs into S2 cells and determined Cullin binding by IP/immunoblot. Because we had already observed that Roc1a binds Cul1 and Cul3 but not Cul5, thatRoc
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Nally substitute for Roc2. We tested this by introducing a Roc1a transgene into the Roc2KG mutant background. Even in this genotype, Roc1a bound to Cul1 but not detectably to Cul5 (Fig. 8). Moreover, as we show in Fig. 6G, the pool of Cul5 available for binding Roc1a is greatly reduced in Roc2 mutant animals relative to wild type. Thus, the Cul1 and Cul5 E3 ligase complexes form independently of o
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Antibody. doi:10.1371/journal.pone.0002918.gdently of other Roc-Cullin complexes and that mutations of each gene would cause the same phenotype. In addition, this hypothesis predicts that the Roc2 mutant phenotype would be different than the phenotype we previously determined for Roc1a and Roc1b mutants [24,25]. The Roc2 locus is complex, with two protein coding exons separated by an intron greate
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Observations in vivo, we generated multiple AN2R and 2NAR transgenic lines and analyzed embryo extracts by IP-Western analysis as in Figure 1. We consistently obtained similar results as in S2 cells; AN2R bound no Cullin, while 2NAR bound Cul5 but not Cul1 as did normal Roc2 (Fig. 3A). In addition, 2NAR also bound Cul3 (Fig. 3B). Thus, the Roc NH2-terminus provides a strong determinant for Cullin
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T in vivo the Roc1b RING domain cannot productively interact (i.e. stimulate ubiquitylation of critical targets) with all of the E2s that Roc1a does. This may occur at the level of direct binding, such that there are some E2s that only bind the RING domain of Roc1a and not Roc1b, or at the level of stimulating ubiquitin transfer to substrate in the context of a fully assembled CDL. A similar obser
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Ubstitution of Lys-1149, to the Win1 MAPKKK to test whether Win1 plays a more important role in osmostress signaling than Wis4 (Samejima et al., 1997, 1998). The strain expressing the catalytically inactive Win1KM showed Spc1 activation equal to that in the wis4KM strain, and only the wis4KM win1KM double mutant failed to induce activation of Spc1 after osmostress (Figure 3B). The KM mutations in
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Ubstitution of Lys-1149, to the Win1 MAPKKK to test whether Win1 plays a more important role in osmostress signaling than Wis4 (Samejima et al., 1997, 1998). The strain expressing the catalytically inactive Win1KM showed Spc1 activation equal to that in the wis4KM strain, and only the wis4KM win1KM double mutant failed to induce activation of Spc1 after osmostress (Figure 3B). The KM mutations in