Identifying determinants of cullin binding specificity among the three functionally different Drosophila melanogaster Roc proteins via domain swapping

Abstract

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 beta 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.

Figure 1. Roc-Cullin interactions in vivo.

A, Flag-Roc protein complexes were immunoprecipitated from extracts prepared from wild type (−), Flag-Roc1a, Flag-Roc1b, and Flag-Roc2 transgenic embryos, and co-precipitating Cullin proteins were detected by immunoblotting. B, Alignment of Drosophila Roc proteins, indicating how we designated NH₂-terminal and RING domains. Asterisks indicate the Zn⁺⁺ chelating residues of the RING domain that are essential for ligase function, and the carets indicate amino acids that make important contacts with Cullins. These assignments are based on the Cul1-Rbx1 structure of Zheng et al .

Figure 2. RING swap constructs and Cullin binding in S2 cells.

A, Schematic representation of the RING swap constructs. All constructs are expressed with the Roc1a promoter and regulatory regions, and the chimeric proteins contain an amino-terminal V5 tag. B, Control Roc1a and Roc1b or ANBR and BNAR constructs were transfected into S2 cells and cellular extracts were immunoprecipitated with anti-V5 antibodies and probed for the presence of Cul1 and Cul3 by immunoblotting. C, Control Roc1a and Roc2 or AN2R and 2NAR constructs were transfected into S2 cells and cellular extracts were immunoprecipitated with anti-V5 antibodies and probed for the presence of Cul1 and Cul5 by immunoblotting. D, AN2R and 2NAR chimeras were tested for interaction with Cul3 as in B, C.

Figure 3. RING Swap constructs and Cullin binding in embryos.

A, RING-swap chimeric proteins were immunoprecipitated from transgenic embryo extracts and tested for interaction with Cul1 and Cul5 by immunoblotting. Two independent transgenic lines of AN2R and 2NAR are shown. B, Two 2NAR transgenic lines were tested for interaction with Cul3.

Figure 4. ANBR does not rescue the lethality of Roc1a mutation.

A, Transgenes expressing chimeric ANBR proteins were tested for rescue of Roc1a^G1 lethality (see Methods for genetics). All F1 progeny contain a single copy of the transgene. The transgenes are expressed under control of the Roc1a regulatory sequences. The percentage of progeny with each genotype is indicated, as is the total number of progeny scored (n). B, V5 immunoblot of extracts prepared from adult males containing the indicated transgenes. (−) indicates non-transgenic wild type control.

Figure 5. BNAR rescues the Roc1b mutant male sterility.

A, Egg hatching was assessed for progeny of Roc1b^dc3 homozygous mutant males containing the indicated transgene. “Control” indicates Roc1b^dc3/+ genotype. (−) indicates no transgene; i.e. a Roc1b^dc3 homozygous mutant. 1A::1B indicates Roc1b driven by the Roc1a regulatory sequences. All other lines are under control of the Roc1b regulatory sequences. 500 eggs were analyzed for each line. B, V5 immunoblot of extracts prepared from adult males containing the indicated transgenes.

Figure 6. Ligase activity of RING swap proteins.

A, Coomassie stained gel of purified GST-Roc proteins used in the ligase assays. Bracket indicates GST-Roc proteins. Asterisk indicates GST. Lower bands are degradation products. B, All Drosophila Roc proteins and chimeras were assessed for ligase activity in a substrate free assay. Briefly, 250 ng of Roc protein (or control GST) were added to a ubiquitin ligase mixture containing UbcH5, ubiquitin, and ligase buffer and incubated for 45 minutes. “−” and “+” indicate the absence or presence of UbcH5 in the reaction, respectively. Ubiquitin conjugates were detected by Western using an anti-Ub antibody (bracket indicates poly-ubiquitin chains), and GST-Roc proteins were detected using an anti-GST antibody.

Figure 7. Analysis of Roc2 and Cul5 mutant alleles.

A, Schematic of the Roc2 locus. CG8234 and CG30035 are genes of unknown function as annotated by FlyBase (putative sugar transporters). B, Schematic of the Cul5 locus. Right angle arrows indicate start of transcription. Open arrow heads show the position of primers used for RT-PCR. Larger black triangles are P-element or piggyBac insertions. The boxes indicate exons, and the shaded regions represent the open reading frame. Dotted line indicates splicing. C, RT-PCR analysis of the Roc2 alleles. KG and pBac are homozygous for the insertions, and KG/pBac is a transheterozygote. Ribosomal protein 49 (rp49) was used as a positive control. D, RT-PCR analysis of the Cul5 allele. −RT indicates that no reverse transcriptase was added. E, Immunoblot comparing Roc2 protein levels in wild-type (w¹¹¹⁸) and homozygous Roc2^KG embryos. F, Immunoblot comparing Cul5 protein levels in wild-type and homozygous Cul5^EY embryos. In each case the embryos were derived from crosses between mutant mothers and fathers. G, Embryo extracts from Cul5 and Roc2 mutants were blotted with antibodies against the respective proteins.

Figure 8. Roc2 and Cul5 are exclusive binding partners.

A, Extracts were prepared from embryos with either a wild-type (Gen = +) or Roc2 mutant (Gen = Roc2^KG) background and the indicated transgene. The immunoprecipitated FLAG-Roc protein is indicated at the top. (−) indicates no transgene.