Yeast E3 Research Articles

The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration SMURF1-UbcH7 K29 ubiquitination of p27 and cell migration

Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293 and HELA cells. But the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1, but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.

Structural insights into the regulation of the human E2∼SUMO conjugate through analysis of its stable mimetic

Protein SUMOylation is a ubiquitylation-like post-translational modification (PTM) that is synthesized through an enzymatic cascade involving an E1 (SAE1:SAE2), an E2 (UBC9), and various E3 enzymes. In the final step of this process, the small ubiquitin-like modifier (SUMO) is transferred from the UBC9∼SUMO thioester onto a lysine residue of a protein substrate. This reaction can be accelerated by an E3 ligase. As the UBC9∼SUMO thioester is chemically unstable, a stable mimetic is desirable for structural studies of UBC9∼SUMO alone and in complex with a substrate and/or an E3 ligase. Recently, a strategy for generating a mimetic of the yeast E2∼SUMO thioester by mutating alanine 129 of Ubc9 to a lysine has been reported. Here, we reproduce and further investigate this approach using the human SUMOylation system and characterize the resulting mimetic of human UBC9∼SUMO1. We show that substituting lysine for alanine 129, but not for other active-site UBC9 residues, results in a UBC9 variant that is efficiently auto-SUMOylated. The auto-modification is dependent on cysteine 93 of UBC9, suggesting that it proceeds via this residue, through the same pathway as that for SUMOylation of substrates. The process is also partially dependent on aspartate 127 of UBC9 and accelerated by high pH, highlighting the importance of the substrate lysine protonation state for efficient SUMOylation. Finally, we present the crystal structure of the UBC9-SUMO1 molecule, which reveals the mimetic in an open conformation and its polymerization via the noncovalent SUMO-binding site on UBC9. Similar interactions could regulate UBC9∼SUMO in some cellular contexts.

Molecular basis for the selective recognition and ubiquitination of centromeric histone H3 by yeast E3 ligase Psh1

Centromeres are chromosomal loci marked by histone variant CenH3 (centromeric histone H3) and essential for genomic stability and cell division. The budding yeast E3 ubiquitin ligase Psh1 selectively recognizes the yeast CenH3 (Cse4) for ubiquitination and controls the cellular level of Cse4 for proteolysis, but the underlying mechanism remains largely unknown. Here, we show that Psh1 uses a Cse4-binding domain (CBD, residues 1–211) to interact with Cse4-H4 instead of H3-H4, yielding a dissociation constant (Kd) of 27 nM. Psh1 recognizes Cse4-specific residues in the L1 loop and α2 helix to ensure Cse4 binding and ubiquitination. We map the Psh1-binding region of Cse4-H4 and identify a wide range of Cse4-specific residues required for the Psh1-mediated Cse4 recognition and ubiquitination. Further analyses reveal that histone chaperone Scm3 can impair Cse4 ubiquitination by abrogating Psh1-Cse4 binding. Together, our study reveals a novel Cse4-binding mode distinct from those of known CenH3 chaperones and elucidates the mechanism by which Scm3 competes with Psh1 for Cse4 binding.

Regulation of the endocytosis and prion-chaperoning machineries by yeast E3 ubiquitin ligase Rsp5 as revealed by orthogonal ubiquitin transfer

Attachment of the ubiquitin (UB) peptide to proteins via the E1-E2-E3 enzymatic machinery regulates diverse biological pathways, yet identification of the substrates of E3UB ligases remains a challenge. We overcame this challenge by constructing an "orthogonal UB transfer" (OUT) cascade with yeast E3 Rsp5 to enable the exclusive delivery of an engineered UB (xUB) to Rsp5 and its substrate proteins. The OUT screen uncovered new Rsp5 substrates in yeast, such as Pal1 and Pal2, which are partners of endocytic protein Ede1, and chaperones Hsp70-Ssb, Hsp82, and Hsp104 that counteract protein misfolding and control self-perpetuating amyloid aggregates (prions), resembling those involved in human amyloid diseases. We showed that prion formation and effect of Hsp104 on prion propagation are modulated by Rsp5. Overall, our work demonstrates the capacity of OUT to deconvolute the complex E3-substrate relationships in crucial biological processes such as endocytosis and protein assembly disorders through protein ubiquitination.

A selective transmembrane recognition mechanism by a membrane-anchored ubiquitin ligase adaptor

While it is well-known that E3 ubiquitin ligases can selectively ubiquitinate membrane proteins in response to specific environmental cues, the underlying mechanisms for the selectivity are poorly understood. In particular, the role of transmembrane regions, if any, in target recognition remains an open question. Here, we describe how Ssh4, a yeast E3 ligase adaptor, recognizes the PQ-loop lysine transporter Ypq1 only after lysine starvation. We show evidence of an interaction between two transmembrane helices of Ypq1 (TM5 and TM7) and the single transmembrane helix of Ssh4. This interaction is regulated by the conserved PQ motif. Strikingly, recent structural studies of the PQ-loop family have suggested that TM5 and TM7 undergo major conformational changes during substrate transport, implying that transport-associated conformational changes may determine the selectivity. These findings thus provide critical information concerning the regulatory mechanism through which transmembrane domains can be specifically recognized in response to changing environmental conditions.

Phase separation directs ubiquitination of gene-body nucleosomes.

The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle1. Although processive ubiquitination might-in principle-arise from Bre1 and Rad6 travelling with RNA polymeraseII2, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation3 as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated 'reaction chambers', with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.

Mms21 SUMO Ligase Activity Promotes Nucleolar Function in Saccharomyces cerevisiae.

The budding yeast E3 SUMO ligase Mms21, also known as Nse2, is a component of the Smc5/6 complex, which regulates sister chromatid cohesion, DNA replication, and repair. Our study shows that the mms21RINGΔ mutant exhibits (1) reduced ribosomal RNA production; (2) nuclear accumulation of ribosomal proteins; (3) elevated Gcn4 translation, indicating translational stress; and (4) upregulation of Gcn4 targets. Genes involved in ribosome biogenesis and translation are downregulated in the mms21RINGΔ mutant. We identified RPL19A as a novel genetic suppressor of the mms21RINGΔ mutant. Deletion of RPL19A partially suppresses growth defects in both smc5-6 and mms21RINGΔ mutants as well as nuclear accumulation of ribosome subunits in the mms21RINGΔ mutant. Deletion of a previously identified strong suppressor, MPH1, rescues both the accumulation of ribosome subunits and translational stress. This study suggests that the Smc5/6 complex supports nucleolar function.

Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6–Bre1

Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities.

Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases.

The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is not a unique case, and that several other yeast and human E3 ligases have sequence properties that may allow them to recognize substrates by a similar mechanism as San1.

Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity.

Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity.

Ubc9 Sumoylation Controls SUMO Chain Formation and Meiotic Synapsis in Saccharomyces cerevisiae

Posttranslational modification with the small ubiquitin-related modifier SUMO depends on the sequential activities of E1, E2, and E3 enzymes. While regulation by E3 ligases and SUMO proteases is well understood, current knowledge of E2 regulation is very limited. Here, we describe modification of the budding yeast E2 enzyme Ubc9 by sumoylation (Ubc9(*)SUMO). Although less than 1% of Ubc9 is sumoylated at Lys153 at steady state, a sumoylation-deficient mutant showed significantly reduced meiotic SUMO conjugates and abrogates synaptonemal complex formation. Biochemical analysis revealed that Ubc9(*)SUMO is severely impaired in its classical activity but promoted SUMO chain assembly in the presence of Ubc9. Ubc9(*)SUMO cooperates with charged Ubc9 (Ubc9~SUMO) by noncovalent backside SUMO binding and by positioning the donor SUMO for optimal transfer. Thus, sumoylation of Ubc9 converts an active enzyme into a cofactor and reveals a mechanism for E2 regulation that orchestrates catalytic (Ubc9~SUMO) and noncatalytic (Ubc9(*)SUMO) functions of Ubc9.

G Protein Mono-ubiquitination by the Rsp5 Ubiquitin Ligase

Emerging evidence suggests that ubiquitination serves as a protein trafficking signal in addition to its well characterized role in promoting protein degradation. The yeast G protein alpha subunit Gpa1 represents a rare example of a protein that undergoes both mono- and poly-ubiquitination. Whereas mono-ubiquitinated Gpa1 is targeted to the vacuole, poly-ubiquitinated Gpa1 is directed instead to the proteasome. Here we investigate the structural requirements for mono- and poly-ubiquitination of Gpa1. We find that variants of Gpa1 engineered to be unstable are more likely to be poly-ubiquitinated and less likely to be mono-ubiquitinated. In addition, mutants that cannot be myristoylated are no longer mono-ubiquitinated but are still polyubiquitinated. Finally, we show that the ubiquitin ligase Rsp5 is necessary for Gpa1 mono-ubiquitination in vivo and that the purified enzyme is sufficient to catalyze Gpa1 mono-ubiquitination in vitro. Taken together, these data indicate that mono- and poly-ubiquitination have distinct enzyme and substrate recognition requirements; whereas poly-ubiquitination targets misfolded protein for degradation, a distinct ubiquitination apparatus targets the fully mature, fully myristoylated G protein for mono-ubiquitination and delivery to the vacuole.

Histone H2B monoubiquitination in the chromatin of FLOWERING LOCUS C regulates flowering time in Arabidopsis.

Ubiquitination is one of many known histone modifications that regulate gene expression. Here, we examine the Arabidopsis thaliana homologs of the yeast E2 and E3 enzymes responsible for H2B monoubiquitination (H2Bub1). Arabidopsis has two E3 homologs (HISTONE MONOUBIQUITINATION1 [HUB1] and HUB2) and three E2 homologs (UBIQUITIN CARRIER PROTEIN [UBC1] to UBC3). hub1 and hub2 mutants show the loss of H2Bub1 and early flowering. By contrast, single ubc1, ubc2, or ubc3 mutants show no flowering defect; only ubc1 ubc2 double mutants, and not double mutants with ubc3, show early flowering and H2Bub1 defects. This suggests that ubc1 and ubc2 are redundant, but ubc3 is not involved in flowering time regulation. Protein interaction analysis showed that HUB1 and HUB2 interact with each other and with UBC1 and UBC2, as well as self-associating. The expression of FLOWERING LOCUS C (FLC) and its homologs was repressed in hub1, hub2, and ubc1 ubc2 mutant plants. Association of H2Bub1 with the chromatin of FLC clade genes depended on UBC1,2 and HUB1,2, as did the dynamics of methylated histones H3K4me3 and H3K36me2. The monoubiquitination of H2B via UBC1,2 and HUB1,2 represents a novel form of histone modification that is involved in flowering time regulation.

Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast

Three Upf proteins are essential to the nonsense-mediated mRNA decay (NMD) pathway. Although these proteins assemble on polysomes for recognition of aberrant mRNAs containing premature termination codons, the significance of this assembly remains to be elucidated. The Cys- and His-rich repeated N terminus (CH domain) of Upf1 has been implicated in its binding to Upf2. Here, we show that CH domain also plays a RING-related role for Upf1 to exhibit E3 ubiquitin ligase activity in yeast. Despite the sequence divergence from typical E3-RING fingers, the CH domain of yeast Upf1 specifically and directly interacted with the yeast E2 Ubc3. Interestingly, Upf1 served as a substrate for the in vitro self-ubiquitination, and the modification required its association with Upf3 rather than Upf2. Substitution of the coordinated Cys and His residues in the CH domain impaired not only self-ubiquitination of Upf1 but also rapid decay of aberrant mRNAs. These results suggest that Upf1 may serve as an E3 ubiquitin ligase upon its association with Upf3 and play an important role in signaling to the NMD pathway.

An emerging role for thioester‐linked polyubiquitin chains in protein degradation

The mechanisms of ubiquitin chain assembly are incompletely understood. The classical model proposes that ubiquitin is first added to a lysine residue within a substrate and then to the distal end of the growing ubiquitin chain. This model becomes less intuitively appealing when applied to substrates with long chains, because the newly added ubiquitin becomes structurally remote from original substrate. One hypothesis that tackles this structural dilemma proposes that ubiquitin chains are pre‐assembled on the catalytic cysteine of an E2 enzyme, prior to transfer onto a substrate. However, until recently no experimental evidence supported the existence of cysteine‐linked polyubiquitin chains in living cells.We found that Ubc7, a yeast E2 enzyme of the endoplasmic reticulum (ER) protein quality control system, is polyubiquitylated exclusively on its catalytic cysteine. The polyubiquitin polymer can then be transferred to a nearby lysine side chain. Moreover, thioester‐linked polybiquitylated Ubc7 undergoes proteasomal degradation when its levels exceed that of its binding partner Cue1, a transmembrane protein that tethers Ubc7 to the ER membrane. Unassembled, and thus mislocalized, Ubc7 is targeted to the proteasome by Ufd4, a HECT‐class E3 ligase. This ensures that no Ubc7 activity persists beyond the ER. Thus, cysteine‐linked polyubiquitin chains can function as an additional degradation signal.

Autoregulation of an E2 enzyme by ubiquitin-chain assembly on its catalytic residue

Cells have quality-control mechanisms to recognize non-native protein structures and either help the proteins fold or promote their degradation. Ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) work together to assemble polyubiquitin chains on misfolded or misassembled proteins, which are then degraded by the proteasome. Here, we find that Ubc7, a yeast E2, can itself undergo degradation when its levels exceed that of its binding partner Cue1, a transmembrane protein that tethers Ubc7 to the endoplasmic reticulum. Unassembled, and thus mislocalized, Ubc7 is targeted to the proteasome by Ufd4, a homologous to E6-AP C-terminus (HECT)-class E3. Ubc7 is autoubiquitinated by a novel mechanism wherein the catalytic cysteine, instead of a lysine residue, provides the polyubiquitin chain acceptor site, and this cysteine-linked chain functions as a degradation signal. The polyubiquitin chain can also be transferred to a lysine side chain, suggesting a mechanism for polyubiquitin chain assembly that precedes substrate modification.

Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast

Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets.

A High Throughput Screen to Identify Substrates for the Ubiquitin Ligase Rsp5

Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.

Crystal Structure of Human Dihydrolipoamide Dehydrogenase: NAD +/NADH Binding and the Structural Basis of Disease-causing Mutations

Human dihydrolipoamide dehydrogenase (hE3) is an enzymatic component common to the mitochondrial alpha-ketoacid dehydrogenase and glycine decarboxylase complexes. Mutations to this homodimeric flavoprotein cause the often-fatal human disease known as E3 deficiency. To catalyze the oxidation of dihydrolipoamide, hE3 uses two molecules: non-covalently bound FAD and a transiently bound substrate, NAD+. To address the catalytic mechanism of hE3 and the structural basis for E3 deficiency, the crystal structures of hE3 in the presence of NAD+ or NADH have been determined at resolutions of 2.5A and 2.1A, respectively. Although the overall fold of the enzyme is similar to that of yeast E3, these two structures differ at two loops that protrude from the proteins and at their FAD-binding sites. The structure of oxidized hE3 with NAD+ bound demonstrates that the nicotinamide moiety is not proximal to the FAD. When NADH is present, however, the nicotinamide base stacks directly on the isoalloxazine ring system of the FAD. This is the first time that this mechanistically requisite conformation of NAD+ or NADH has been observed in E3 from any species. Because E3 structures were previously available only from unicellular organisms, speculations regarding the molecular mechanisms of E3 deficiency were based on homology models. The current hE3 structures show directly that the disease-causing mutations occur at three locations in the human enzyme: the dimer interface, the active site, and the FAD and NAD(+)-binding sites. The mechanisms by which these mutations impede the function of hE3 are discussed.

Functional interaction of 13 yeast SCF complexes with a set of yeast E2 enzymes in vitro.

SCF complexes are multi-subunit ubiquitin ligases that, in concert with the E1 and E2 ubiquitination enzymes, catalyze the ubiquination of specific target proteins. Only three yeast SCFs have been reconstituted and characterized to date; each of these ubiquitinates its target protein with the E2 Cdc34. We have reconstituted and purified 1 known and 12 novel yeast SCF complexes, and explored the ability of these complexes to function with 5 different purified E2 enzymes; Ubc1, Cdc34, Ubc4, Ubc8 and Ubc11. We have found that the ubiquitination of Sic1 by the reconstituted SCF(Cdc4) complex was specifically catalyzed by two of the five E2 enzymes tested in vitro; Cdc34 and Ubc4. We also show that at least eight of the purified SCF complexes clearly ubiquitinated their F-box proteins in vitro, lending support for a regulatory mechanism in which F-box proteins catalyze their own destruction. The autoubiquitination of each F-box was in some cases catalyzed only by Cdc34, and in other cases preferentially catalyzed by Ubc4. Ubc4 thus interacts with multiple SCFs in vitro, and the interactions among SCF and E2 components of the ubiquitination machinery may allow further diversification of the roles of SCFs in vivo.