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Title: Skp1 Independent Function of Cdc53/Cul1 in F-box Protein Homeostasis}
Article:
Abstract:
Abundance of substrate receptor subunits of Cullin-RING ubiquitin ligases( CRLs) is tightly controlled to maintain the full repertoire of CRLs. Unbalanced levels can lead to sequestration of CRL core components by a few overabundant substrate receptors. Numerous diseases, including cancer, have been associated with misregulation of substrate receptor components, particularly for the largest class of CRLs, the SCF ligases. One relevant mechanism that controls abundance of their substrate receptors, the F-box proteins, is autocatalytic ubiquitylation by intact SCF complex followed by proteasome-mediated degradation. Here we describe an additional pathway for regulation of F-box proteins on the example of yeast Met30. This ubiquitylation and degradation pathway acts on Met30 that is dissociated from Skp1. Unexpectedly, this pathway required the cullin component Cdc53/Cul1 but was independent of the other central SCF component Skp1. We demonstrated that this non-canonical degradation pathway is critical for chromosome stability and effective defense against heavy metal stress. More importantly, our results assign important biological functions to a sub-complex of cullin-RING ligases that comprises Cdc53/Rbx1/Cdc34, but is independent of Skp1.
Introduction:
Ubiquitin dependent proteolysis controls many cellular processes including signal transduction and cell cycle progression. Ubiquitin is covalently linked to substrates in a multistep process that requires coordinated action of 3 classes of enzymes- E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase[1–5]. E3 ubiquitin ligases are the key players in this system as they mediate substrate specific covalent attachment of ubiquitin. Within the E3 ligase family, cullin-RING ligases( CRLs) comprise the largest class, and in this group the SCF ubiquitin ligases are one of the best-understood complexes[2, 6]. They are composed of yeast Cdc53( mammalian cullin-1), Skp1, Rbx1, and one of the multiple F-box proteins, which bind substrates and confer specificity to the complex[7, 8]. Amongst the SCF components, F-box proteins are relatively unstable in nature, which contributes to the dynamic assembly of a diverse repertoire of SCF complexes within the cell[9–13]. Accordingly, over expression of a single F-box protein in yeast can change the balanced distribution and diversity of available SCF complexes by sequestering cullin and Skp1 and thus block formation of functional SCF complexes with other F-box proteins[10, 11, 14]. Many F-box proteins control degradation of critical oncogenes and tumor suppressors and variation in their abundance has been linked to cancer[15, 16]. Thus, it is important to understand how cells maintain F-box protein homeostasis. F-box proteins are known to be regulated by autoubiquitylation where their degradation is dependent upon their incorporation into a functional SCF complex[10, 11]. The autocatalytic F-box protein degradation pathway is thought to be suppressed by substrate binding resulting in coordination of substrate availability with abundance of the corresponding assembled SCF complex[17, 18]. Additional degradation pathways for F-box proteins are likely as it is also important to restrict abundance of unbound F-box proteins to prevent substrate shielding effects that would compete with substrate recognition by fully assembled ligases. Indeed, mammalian Skp2 is targeted for degradation by the anaphase promoting complex or cyclosome[19, 20], Fbx5( Emi1) is degraded by SCFβTrCP/Slimb[21], and the level of the budding yeast F-box protein Dia2, which is required for genomic stability, is restricted by the HECT domain E3 ligase Tom1[22]. In Saccharomyces cerevisiae, Met30 is one of three essential F-box proteins. SCFMet30 coordinates metabolic pathways of sulfur containing compounds with cell cycle progression. The transcription factor Met4 is a key target of SCFMet30[23, 24]. Low levels of the methyl donor, S-adenosylmethionine cause a block in SCFMet30 dependent ubiquitylation of Met4, activating it and results in cell cycle arrest and transcription of methionine response genes[25]. SCFMet30 also represses expression of enzymes responsible for glutathione synthesis and is thus a key factor in response to heavy metal stress. Cadmium exposure induces active dissociation of Met30 from Skp1, thereby inhibiting SCFMet30 and inducing glutathione production and cell cycle arrest, which together protect cellular integrity[26–28]. Therefore, the SCFMet30 system generates unbound Met30 via heavy metal stress induced dissociation from Skp1. To prevent unwanted effects of excess unbound Met30, such as substrate sequestration, it seemed important, that a degradation mechanism exists in addition to autocatalysis that plays an integral role in maintaining Met30 homeostasis. Here we report such an additional mechanism for Met30 regulation in addition to autoubiquitylation. This degradation pathway targets Met30 that is detached from Skp1 and involves the SCF core components Cdc53 and Rbx1, as well as the cognate SCF ubiquitin conjugating enzyme Cdc34. Importantly, this ubiquitylation pathway does not require Skp1( Skp1-independent Cul1-dependent ubiquitylation) and suggests a function of Cdc53/Cul1 that is independent from association with Skp1.
Discussion:
We describe a novel pathway for regulation of F-box protein abundance in addition to autoubiquitylation that specifically targets F-box proteins that are dissociated from Skp1. The architectural theme of SCF ubiquitin ligases employs multiple F-box proteins that bind a common Skp1/Cdc53/Rbx1 core. This arrangement is effective in providing an array of diverse ubiquitin ligases. However, the modular design presents cells with the challenge of balancing the diversity and abundance of different SCF complexes when many F-box proteins, in the case of plants several hundred[45], compete for the shared SCF core components. Cycles of cullin neddylation and CAND1 association maintain a critical level of unoccupied SCF core complexes[9, 12, 46] while Skp1- F-box protein heterodimers are displaced to bind substrates and recruit them to the Cdc53/Rbx1 complex[47]. This CAND1/Nedd8 cycle maintains dynamic exchange of substrate adapters, but abundance of individual SCF ligases and overall SCF diversity is dictated by the distribution of F-box protein concentrations. It is thus critical to regulate F-box protein levels. In this study we characterize a ‘Skp1-free’ F-box protein degradation pathway that plays an important role in maintaining F-box protein homeostasis. F-box proteins that associate with Skp1 form functional ligases while those that do not, are recognized by the ‘Skp1-free’ F-box protein degradation pathway and degraded by the Cdc53/Rbx1 ligase thereby preventing competition between F-box proteins, limiting substrate shielding effects and ensuring representation even of low abundance F-box proteins in the cellular SCF repertoire. Degradation of F-box proteins that are not bound to Skp1 may also provide an important quality control mechanism to remove damaged F-box proteins. Such a mechanism may be critical for cells because F-box proteins incapable of forming active SCF ligases could maintain an intact substrate binding domain and thus shield their substrates from degradation. We describe the ‘Skp1-free’ F-box protein degradation pathway in detail for Met30, the substrate adaptor for SCFMet30 ubiquitin ligase, which negatively regulates transcription factor Met4 by proteolysis-independent ubiquitylation[23, 48, 49]. The ‘Skp1-free’ F-box protein degradation pathway is of particular importance for Met30. First, because Met4 ubiquitylation does not induce its degradation under normal growth conditions, Met4 remains associated with Met30 and therefore prevents the canonical F-box-protein degradation pathway through autoubiquitylation. Accordingly, we observed that the ‘Skp1-free’ degradation pathway is the predominant pathway that ensures turn over of excess Met30( Fig 5C). Second, heavy metal stress induces active dissociation of Met30 from Skp1 resulting in a burst of ‘Skp1-free’ Met30, which interferes with recovery from cadmium stress when the ‘Skp1-free’ degradation pathway is blocked( Fig 8B and 8C). Indications for a degradation pathway that targets F-box proteins that are not bound to Skp1 have been reported previously[14, 42]. In addition, we show that apart from Met30, the two other essential yeast F-box proteins—Cdc4 and Ctf13 are also degraded in the absence of Skp1( Fig 7A and 7B), suggesting that this pathway is a common mechanism to restrict F-box protein abundance outside the SCF complex. However, not all F-box proteins are subject to this degradation mechanism, because consistent with other reports[10] we found that deletion of the F-box region in Grr1 or inactivation of Skp1 stabilized Grr1( S5 Fig). The F-box protein degradation pathway we describe here is thus not universal and is probably functional for only those F-box proteins, which harbor the hydrophobic region adjacent to the F-box domain, which is paramount for Cdc53/Rbx1 ligase binding. In addition to advancing understanding of SCF ligase regulation, our results also demonstrate a function for Cdc53( cullin-1) independent from its adaptor Skp1. Not only was Met30 degradation active in the absence of Skp1 in vivo( Fig 3C), but in addition a Cdc53 mutant incapable of binding to Skp1 could fully complement the Met30 degradation defect of cdc53 mutants( Fig 4). In addition, Cdc53/Rbx1 can directly bind Met30 in the absence of Skp1 in vitro and binding depends on the degron region adjacent to the F-box motif( Fig 6). However, Met30 ubiquitylation in this minimal in vitro system with Cdc53/Rbx1, Cdc34, and E1 was very inefficient and required a long reaction time suggesting possible involvement of additional factors, inefficient protein folding in E. coli, or a post-translational modification lacking in this expression system. Further studies need to be conducted to explore these options in detail. The findings reported here illustrate a Skp1 independent function for the cullin Cdc53 in substrate ubiquitylation. Consistent with these results, a Skp1 independent cullin-1 based ubiquitylation event has been suggested previously in human cells where Rictor, a component of mTORC2 complex associates with Cullin-1 instead of Skp1, to form a functional E3 ubiquitin ligase that promotes ubiquitylation of SGK1[50]. Together, our findings shed light on the regulation and complexity of E3 ligases and suggest additional diversity in the cullin-RING family of ubiquitin ligases.
Protein ubiquitylation is the covalent attachment of the small protein ubiquitin onto other proteins and is a key regulatory pathway for most biological processes . The central components of the ubiquitylation process are the E3 ligases , which recognize substrate proteins . The best-studied E3 complexes are the SCF ligases , which are composed of 3 core components—Cdc53 , Skp1 , Rbx1—that assemble to the functional ligase complex by binding to one of the multiple substrate adaptors—the F-box proteins . Maintaining a balanced repertoire of diverse SCF complexes that represent the entire cellular panel of substrate adapters is challenging . Depending on the cell type , hundreds of different F-box proteins can compete for the single binding site on the common SCF core complex . Rapid degradation of F-box proteins helps in maintaining a critical level of unoccupied Cdc53/Skp1/Rbx1 core , complexes and alterations in levels of F-box proteins has been linked to diseases including cancer . Studying the yeast F-box protein Met30 as a model , we have uncovered a novel mechanism for degradation of F-box proteins . This pathway targets free F-box proteins and requires part of the SCF core . These findings add an additional layer to our understanding of regulation of multisubunit E3 ligase .