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Background  Cullin-dependent E3 ubiquitin ligases (CDL) are key regulators of protein destruction that participate in a wide range of cell biological processes. The Roc subunit of CDL contains an evolutionarily conserved RING domain that binds ubiquitin charged E2 and is essential for ubiquitylation.  Drosophila melanogaster  contains three highly related Roc proteins: Roc1a and Roc2, which are conserved in vertebrates, and Roc1b, which is specific to  Drosophila . Our previous genetic data analyzing  Roc1a  and  Roc1b  mutants suggested that Roc proteins are functionally distinct, but the molecular basis for this distinction is not known.    Methodology/Principal Findings  Using co-immunoprecipitation studies we show that  Drosophila  Roc proteins bind specific Cullins: Roc1a binds Cul1-4, Roc1b binds Cul3, and Roc2 binds Cul5. Through domain swapping experiments, we demonstrate that Cullin binding specificity is strongly influenced by the Roc NH 2 -terminal domain, which forms an inter-molecular β sheet with the Cullin. Substitution of the Roc1a RING domain with that of Roc1b results in a protein with similar Cullin binding properties to Roc1a that is active as an E3 ligase but cannot complement  Roc1a  mutant lethality, indicating that the identity of the RING domain can be an important determinant of CDL function. In contrast, the converse chimeric protein with a substitution of the Roc1b RING domain with that of Roc1a can rescue the male sterility of  Roc1b  mutants, but only when expressed from the endogenous  Roc1b  promoter. We also identified mutations of  Roc2  and  Cul5  and show that they cause no overt developmental phenotype, consistent with our finding that Roc2 and Cul5 proteins are exclusive binding partners, which others have observed in human cells as well.    Conclusions  The  Drosophila  Roc proteins are highly similar, but have diverged during evolution to bind a distinct set of Cullins and to utilize RING domains that have overlapping, but not identical, function  in vivo .

Identifying Determinants of Cullin Binding Specificity Among the Three Functionally Different Drosophila melanogaster Roc Proteins via Domain Swapping