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

Abstract

BackgroundCullin-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 FindingsUsing 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 NH2-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.ConclusionsThe 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.

Cullin-5 and cullin-2 play a role in the development of neuromuscular junction and the female germ line of Drosophila

“…Mutants of cul-5 have already been shown to have defects in germ line cells leading to compromised female fertility (Reynolds et al 2008;Kugler et al 2010) and we found cul-2 EY homozygous females to be completely sterile. So next we tested the roles of these genes in germ line development in the Drosophila ovary.…”

“…They also reported a total loss of function but viable allele, cul-5 EY , which did not produce any Cul-5 protein as detected in immunoblots (Reynolds et al 2008). When stained with anti-HRP, larvae of cul-5 EY in trans with the deficiency Df(3R)3450, showed a significant increase in bouton number (figure 4B) compared to controls (figure 4A).…”

“…This suggests that the loss-of-function mutation of cul-5 gives rise to branching defects too. The cul-5 EY mutation results from an insertion of a P-element in the second intron of the gene (Reynolds et al 2008). We generated a precise excision of the P-element, cul-5 exB .…”

“…We had previously used an insertional mutation upstream of Drosophila cul-5 to show that gain of function affects cell proliferation, cell fate determination and survival during sensory system development (Ayyub et al 2005). Later, Reynolds and co-workers identified a null allele of cul-5 and found that the total loss-of-function mutation does not affect viability but leads to reduced female fecundity (Reynolds et al 2008). Cul-5 has been recently implicated in germ line maintenance, follicular morphogenesis and regulation of cyst divisions within the ovary (Kugler et al 2010).…”

Squamous Cell Carcinoma-related Oncogene (SCCRO) Family Members Regulate Cell Growth and Proliferation through Their Cooperative and Antagonistic Effects on Cullin Neddylation

“…The complexity of the genome and its manipulation in mice convolutes in vivo analyses of gene function in mice. The relative simplicity of genome manipulation in Drosophila flies combined with their established role in assessing the activity of neddylation components makes Drosophila flies the ideal model for assessing functional redundancy among SCCRO and its paralogues (35)(36)(37)(38). In addition, the presence of a single homologue in each of the three SCCRO family subgroups facilitates genetic dissection of the pathway in flies.…”