Andrew Willems

Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry

The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein–protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.

The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase

Many biological processes, such as development and cell cycle progression are tightly controlled by selective ubiquitin-dependent degradation of key substrates. In this pathway, the E3-ligase recognizes the substrate and targets it for degradation by the 26S proteasome. The SCF (Skp1–Cul1–F-box) and ECS (Elongin C–Cul2–SOCS box) complexes are two well-defined cullin-based E3-ligases. The cullin subunits serve a scaffolding function and interact through their C terminus with the RING-finger-containing protein Hrt1/Roc1/Rbx1, and through their N terminus with Skp1 or Elongin C, respectively. In Caenorhabditis elegans, the ubiquitin-ligase activity of the CUL-3 complex is required for degradation of the microtubule-severing protein MEI-1/katanin at the meiosis-to-mitosis transition. However, the molecular composition of this cullin-based E3-ligase is not known. Here we identified the BTB-containing protein MEL-26 as a component required for degradation of MEI-1 in vivo. Importantly, MEL-26 specifically interacts with CUL-3 and MEI-1 in vivo and in vitro, and displays properties of a substrate-specific adaptor. Our results suggest that BTB-containing proteins may generally function as substrate-specific adaptors in Cul3-based E3-ubiquitin ligases.

Cullin-based ubiquitin ligases: Cul3–BTB complexes join the family

Cullin‐based E3 ligases target substrates for ubiquitin‐dependent degradation by the 26S proteasome. The SCF (Skp1–Cul1–F‐box) and ECS (ElonginC–Cul2–SOCS box) complexes are so far the best‐characterized cullin‐based ligases. Their atomic structure has been solved recently, and several substrates have been described in different organisms. In addition to Cul1 and Cul2, higher eucaryotic genomes encode for three other cullins: Cul3, Cul4, and Cul5. Recent results have shed light on the molecular composition and function of Cul3‐based E3 ligases. In these complexes, BTB‐domain‐containing proteins may bridge the cullin to the substrate in a single polypeptide, while Skp1/F‐box or ElonginC/SOCS heterodimers fulfill this function in the SCF and ECS complexes. BTB‐containing proteins are evolutionary conserved and involved in diverse biological processes, but their function has not previously been linked to ubiquitin‐dependent degradation. In this review, we present these new findings and compare the composition of Cul3‐based ligases to the well‐defined SCF and ECS ligases.

Cdc53 Targets Phosphorylated G1 Cyclins for Degradation by the Ubiquitin Proteolytic Pathway

In budding yeast, cell division is initiated in late G1 phase once the Cdc28 cyclin-dependent kinase is activated by the G1 cyclins Cln1, Cln2, and Cln3. The extreme instability of the Cln proteins couples environmental signals, which regulate Cln synthesis, to cell division. We isolated Cdc53 as a Cln2-associated protein and show that Cdc53 is required for Cln2 instability and ubiquitination in vivo. The Cln2-Cdc53 interaction, Cln2 ubiquitination, and Cln2 instability all depend on phosphorylation of Cln2. Cdc53 also binds the E2 ubiquitin-conjugating enzyme, Cdc34. These findings suggest that Cdc53 is a component of a ubiquitin-protein ligase complex that targets phosphorylated G1 cyclins for degradation by the ubiquitin-proteasome pathway.

Suprafacial orientation of the SCFCdc4 dimer accommodates multiple geometries for substrate ubiquitination.

SCF ubiquitin ligases recruit substrates for degradation via F box protein adaptor subunits. WD40 repeat F box proteins, such as Cdc4 and beta-TrCP, contain a conserved dimerization motif called the D domain. Here, we report that the D domain protomers of yeast Cdc4 and human beta-TrCP form a superhelical homotypic dimer. Disruption of the D domain compromises the activity of yeast SCF(Cdc4) toward the CDK inhibitor Sic1 and other substrates. SCF(Cdc4) dimerization has little effect on the affinity for Sic1 but markedly stimulates ubiquitin conjugation. A model of the dimeric holo-SCF(Cdc4) complex based on small-angle X-ray scatter measurements reveals a suprafacial configuration, in which substrate-binding sites and E2 catalytic sites lie in the same plane with a separation of 64 A within and 102 A between each SCF monomer. This spatial variability may accommodate diverse acceptor lysine geometries in both substrates and the elongating ubiquitin chain and thereby increase catalytic efficiency.

Dcn1 Functions as a Scaffold-Type E3 Ligase for Cullin Neddylation

Cullin-based E3 ubiquitin ligases are activated through modification of the cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 regulates cullin neddylation and thus ubiquitin ligase activity. Here we describe the 1.9 A X-ray crystal structure of yeast Dcn1 encompassing an N-terminal ubiquitin-binding (UBA) domain and a C-terminal domain of unique architecture, which we termed PONY domain. A conserved surface on Dcn1 is required for direct binding to cullins and for neddylation. The reciprocal binding site for Dcn1 on Cdc53 is located approximately 18 A from the site of neddylation. Dcn1 does not require cysteine residues for catalytic function, and directly interacts with the Nedd8 E2 Ubc12 on a surface that overlaps with the E1-binding site. We show that Dcn1 is necessary and sufficient for cullin neddylation in a purified recombinant system. Taken together, these data demonstrate that Dcn1 is a scaffold-like E3 ligase for cullin neddylation.

Genome-wide surveys for phosphorylation-dependent substrates of SCF ubiquitin ligases

The SCF (Skp1-Cullin-F-box) family of ubiquitin ligases target numerous substrates for ubiquitin-dependent proteolysis, including cell cycle regulators, transcription factors, and signal transducers. Substrates are recruited to an invariant core SCF complex through one of a large family of substrate-specific adapter subunits called F-box proteins, each of which binds multiple specific substrates, often in a phosphorylation-dependent manner. The identification of substrates for SCF complexes has proven difficult, especially given the requirement of often complex phosphorylation events for substrate recognition. The archetype for such interactions is the binding of the yeast F-box protein Cdc4 to its various substrates by means of multiple motifs that weakly match an optimal consensus called the Cdc4 phosphodegron (CPD), which is phosphorylated by cyclin-dependent kinases (CDKs) and possibly other kinases. Provided phosphodegron recognition motifs and/or the targeting kinases for SCF substrates are delineated, it is possible to use genome-wide methods to identify new substrates. Here we describe two methods for the systematic retrieval of SCF substrates based on membrane arrays of synthetic phosphopeptides and on genome-wide kinase substrate profiles. In the first approach, which identifies substrates with strong matches to the CPD, a search of the predicted yeast proteome with the optimal CPD motif identified approximately 1100 matches. A phosphopeptide membrane array corresponding to each of these sequences is then probed with recombinant Cdc4, thereby identifying potential substrates. In the second approach, which identifies substrates that lack strong CPD motifs, a genome-wide set of recombinant CDK substrates is phosphorylated and directly assayed for binding to Cdc4. The proteins corresponding to these hits from each approach can then be subjected to the more stringent criteria of phosphorylation-dependent binding to Cdc4, ubiquitination by SCF(Cdc4)in vitro, and Cdc4-dependent protein instability in vivo. Both methods have identified novel substrates of Cdc4 and may, in principle, be used to identify numerous new substrates of other SCF and SCF-like complexes from yeast to humans.