Cadmium binding by the F-box domain induces p97-mediated SCF complex disassembly to activate stress response programs.

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.

Cdc48/Shp1 participates in dissociation of protein complexes to regulate their activity.

The AAA-ATPase p97/Cdc48 is one of the most abundant proteins in eukaryotes, and owing to its multiple functions, is considered the swiss army knife of cells. Recent findings demonstrate that p97/Cdc48 and its cofactor p47/Shp1 control the heavy metal stress response by active, signal-triggered disassembly of a multisubunit ubiquitin ligase. Here we review this pathway and describe recently achieved mechanistic insight into the role of p47/Shp1 in this process.

Cdc48 cofactor Shp1 regulates signal-induced SCFMet30 disassembly.

Organisms can adapt to a broad spectrum of sudden and dramatic changes in their environment. These abrupt changes are often perceived as stress and trigger responses that facilitate survival and eventual adaptation. The ubiquitin-proteasome system (UPS) is involved in most cellular processes. Unsurprisingly, components of the UPS also play crucial roles during various stress response programs. The budding yeast SCFMet30 complex is an essential cullin-RING ubiquitin ligase that connects metabolic and heavy metal stress to cell cycle regulation. Cadmium exposure results in the active dissociation of the F-box protein Met30 from the core ligase, leading to SCFMet30 inactivation. Consequently, SCFMet30 substrate ubiquitylation is blocked and triggers a downstream cascade to activate a specific transcriptional stress response program. Signal-induced dissociation is initiated by autoubiquitylation of Met30 and serves as a recruitment signal for the AAA-ATPase Cdc48/p97, which actively disassembles the complex. Here we show that the UBX cofactor Shp1/p47 is an additional key element for SCFMet30 disassembly during heavy metal stress. Although the cofactor can directly interact with the ATPase, Cdc48 and Shp1 are recruited independently to SCFMet30 during cadmium stress. An intact UBX domain is crucial for effective SCFMet30 disassembly, and a concentration threshold of Shp1 recruited to SCFMet30 needs to be exceeded to initiate Met30 dissociation. The latter is likely related to Shp1-mediated control of Cdc48 ATPase activity. This study identifies Shp1 as the crucial Cdc48 cofactor for signal-induced selective disassembly of a multisubunit protein complex to modulate activity.

Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast.

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.

Proteomics Links Ubiquitin Chain Topology Change to Transcription Factor Activation.

A surprising complexity of ubiquitin signaling has emerged with identification of different ubiquitin chain topologies. However, mechanisms of how the diverse ubiquitin codes control biological processes remain poorly understood. Here, we use quantitative whole-proteome mass spectrometry to identify yeast proteins that are regulated by lysine 11 (K11)-linked ubiquitin chains. The entire Met4 pathway, which links cell proliferation with sulfur amino acid metabolism, was significantly affected by K11 chains and selected for mechanistic studies. Previously, we demonstrated that a K48-linked ubiquitin chain represses the transcription factor Met4. Here, we show that efficient Met4 activation requires a K11-linked topology. Mechanistically, our results propose that the K48 chain binds to a topology-selective tandem ubiquitin binding region in Met4 and competes with binding of the basal transcription machinery to the same region. The change to K11-enriched chain architecture releases this competition and permits binding of the basal transcription complex to activate transcription.

Set them free: F-box protein exchange by Cand1.

Cand1 (Cullin-associated and neddylation-dissociated protein 1) has long been known as a regulator of SCF ubiquitin ligases, but details remained puzzling due to conflicting results from in vitro and in vivo experiments. Three recent reports, one in Cell and two in Nature Communications, propose Cand1 as a protein exchange factor with interesting mechanism that reconciles Cand1 genetics and biochemistry.

Signal-induced disassembly of the SCF ubiquitin ligase complex by Cdc48/p97.

A large group of E3 ubiquitin ligases is formed by the multisubunit SCF complex, whose core complex (Rbx1/Cul1-Cdc53/Skp1) binds one of many substrate recruiting F-box proteins to form an array of SCF ligases with diverse substrate specificities. It has long been thought that ubiquitylation by SCF ligases is regulated at the level of substrate binding. Here we describe an alternative mechanism of SCF regulation by active dissociation of the F-box subunit. We show that cadmium stress induces selective recruitment of the AAA(+) ATPase Cdc48/p97 to catalyze dissociation of the F-box subunit from the yeast SCF(Met30) ligase to block substrate ubiquitylation and trigger downstream events. Our results not only provide an additional layer of ubiquitin ligase regulation but also suggest that targeted, signal-dependent dissociation of multisubunit enzyme complexes is an important mechanism in control of enzyme function.

Protein degradation and the stress response.

Environmental stresses are manifold and so are the responses they elicit. This is particularly true for higher eukaryotes where various tissues and cell types are differentially affected by the insult. Type and scope of the stress response can therefore differ greatly among cell types. Given the importance of the ubiquitin proteasome system (UPS) for most cellular processes, it comes as no surprise that the UPR plays a pivotal role in counteracting the effects of stressors. Here we outline contributions of the UPS to stress sensing, signaling, and response pathways. We make no claim to comprehensiveness but choose selected examples to illustrate concepts and mechanisms by which protein modification with ubiquitin and proteasomal degradation of key regulators ensures cellular integrity during stress situations.

Ubiquitin and transcription: The SCF/Met4 pathway, a (protein-) complex issue.

Ubiquitylation has emerged as an omnipresent factor at all levels of transcriptional regulation. A recent study that describes the yeast transcriptional activator Met4 as a functional component of the very same ubiquitin ligase that regulates its own activity highlights the close relation between transcription and the ubiquitin proteasome system.

A transcriptional activator is part of an SCF ubiquitin ligase to control degradation of its cofactors.

Multisubunit protein complexes pose a challenge to the coordinated regulation of individual components. We show how the yeast transactivating factor Met4 functions as a component of the SCF(Met30) ubiquitin ligase to synchronize its own activity with cofactor assembly. Cells maintain Met4 in a dormant state by a regulatory ubiquitin chain assembled by SCF(Met30). Nutritional and heavy-metal stress block Met4 ubiquitylation resulting in Met4 activation, which induces a stress-response program including cell-cycle arrest. Met4 relies on assembly with various cofactors for promoter binding. We report here that the stability of these DNA-binding cofactors is regulated by SCF(Met30). Remarkably, the transcriptional activator Met4 functions as a substrate-specificity factor in the context of SCF(Met30/Met4) to coordinate cofactor degradation with its own activity status. Our results establish an additional layer for substrate recruitment by SCF ubiquitin ligases and provide conceptual insight into coordinated regulation of protein complexes.

Physiologically relevant and portable tandem ubiquitin-binding domain stabilizes polyubiquitylated proteins.

Ubiquitylation of proteins can be a signal for a variety of cellular processes beyond the classical role in proteolysis. The different signaling functions of ubiquitylation are thought to rely on ubiquitin-binding domains (UBDs). Several distinct UBD families are known, but their functions are not understood in detail, and mechanisms for interpretation and transmission of the ubiquitin signals remain to be discovered. One interesting example of the complexity of ubiquitin signaling is the Saccharomyces cerevisiae transcription factor Met4, which is regulated by a single lysine-48 linked polyubiquitin chain that can directly repress activity of Met4 or induce degradation by the proteasome. Here we show that ubiquitin signaling in Met4 is controlled by its tandem UBD regions, consisting of a previously recognized ubiquitin-interacting motif and a novel ubiquitin-binding region, which lacks homology to known UBDs. The tandem arrangement of UBDs is required to protect ubiquitylated Met4 from degradation and enables direct inactivation of Met4 by ubiquitylation. Interestingly, protection from proteasomes is a portable feature of UBDs because a fusion of the tandem UBDs to the classic proteasome substrate Sic1 stabilized Sic1 in vivo in its ubiquitylated form. Using the well-defined Sic1 in vitro ubiquitylation system we demonstrate that the tandem UBDs inhibit efficient polyubiquitin chain elongation but have no effect on initiation of ubiquitylation. Importantly, we show that the nonproteolytic regulation enabled by the tandem UBDs is critical for ensuring rapid transcriptional responses to nutritional stress, thus demonstrating an important physiological function for tandem ubiquitin-binding domains that protect ubiquitylated proteins from degradation.

Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase.

The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

Proteomic revelation: SUMO changes partners when the heat is on.

Dynamic changes in the posttranslational modification of proteins govern most cellular signaling pathways. Work over the past decade has connected many of these processes with the covalent attachment of the small ubiquitin-like modifier (SUMO) protein to target proteins, but a global view of the dynamics of SUMOylation was missing. A system-level proteomics approach has now been used to describe quantitative changes in protein modification with the SUMO-2 paralog during the response to heat shock. The SUMOylation status of more than 700 proteins was monitored in HeLa cells during the induction of hyperthermic stress and the recovery period. A massive redistribution of SUMO-2 was observed that affected many biological pathways that are important for the heat shock response, including cell cycle regulation, transcription, translation, protein folding, and DNA repair. Collectively, these data suggest a wide-ranging role for SUMOylation in the cellular response to hyperthermic stress. The strategies that were developed to provide this global view of SUMOylation should guide future approaches to probing quantitative changes in protein modification.

Lipid and phospholipid profiling of biological samples using MALDI Fourier transform mass spectrometry.

Here we describe a study of the feasibility of lipid and phospholipid (PL) profiling using matrix assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) for two different applications. In this work PL profiles of different mammalian tissues as well as those of whole cell organisms were examined. In particular, comparative analysis of lipid and PL profiles of tissues from mice fed different diets was done and, in another application, MALDI FTMS was used to analyze PL profiles of genetically modified Saccharomyces cerevisiae. Computational sorting of the observed ions was done in order to group the lipid and PL ions from complex MALDI spectra. The PL profiles of liver tissues from mice fed different diets showed a cross correlation coefficient of 0.2580, indicating significant dissimilarity, and revealed more than 30 significantly different peaks at the 99.9% confidence level. Histogram plots derived from the spectra of wild type and genetically modified yeast resulted in a cross correlation coefficient 0.8941 showing greater similarity, but still revealing a number of significantly different peaks. Based on these results, it appears possible to use MALDI FTMS to identify PLs as potential biomarkers for metabolic processes in whole cells and tissues.

The yeast ubiquitin ligase SCFMet30: connecting environmental and intracellular conditions to cell division.

Ubiquitination regulates a host of cellular processes and is well known for its role in progression through the cell division cycle. In budding yeast, cadmium and arsenic stress, the availability of sulfur containing amino acids, and the intracellular concentration of S-adenosylmethionine are linked to cell cycle regulation through the ubiquitin ligase SCFMet30. Regulation is achieved by ubiquitination of the transcription factor Met4. Met4 activity is controlled by a regulatory K48-linked ubiquitin chain that is synthesized by Cdc34/SCFMet30. A ubiquitin-interacting-motif (UIM) present in Met4 prevents degradation of ubiquitinated Met4 allowing the ubiquitin chain to function as a reversible switch of Met4 activity. Here we discuss mechanisms of Met4 and SCFMet30 regulation in response to intracellular and environmental conditions, and describe the integration of these signals with cell cycle control.

A ubiquitin-interacting motif protects polyubiquitinated Met4 from degradation by the 26S proteasome.

Covalent attachment of ubiquitin to proteins regulates a host of cellular events by proteolysis dependent and independent mechanisms. A variety of protein domains that bind non-covalently to ubiquitin have been described and functionally linked to diverse cellular processes. Overall, however, the understanding and knowledge of the mechanisms by which ubiquitin-binding domains (UBDs) regulate these processes is limited. Here, we describe identification of a UBD in the yeast transcription factor Met4. Met4 activity, but not its stability, is regulated by polyubiquitination. We found that the UBD restricts the length of the polyubiquitin chain that is assembled on Met4, and prevents proteasomal recognition and degradation of polyubiquitinated Met4. Inactivation of the UBD allowed synthesis of longer ubiquitin chains on Met4 and transformed the normally stable polyubiquitinated Met4 into a short-lived protein. Our results demonstrate a function for UBDs in ubiquitin-chain synthesis and regulation of protein degradation.

A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking.

Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for two-step purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 m urea, by Ni(2+) chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein complexes, thereby expanding proteomic analyses to the description of weakly or transiently associated protein complexes.

The F-box protein Met30 is required for multiple steps in the budding yeast cell cycle.

The Saccharomyces cerevisiae ubiquitin ligase SCF(Met30) is essential for cell cycle progression. To identify and characterize SCF(Met30)-dependent cell cycle steps, we used temperature-sensitive met30 mutants in cell cycle synchrony experiments. These experiments revealed a requirement for Met30 during both G(1)/S transition and M phase, while progression through S phase was unaffected by loss of Met30 function. Expression of the G(1)-specific transcripts CLN1, CLN2, and CLB5 was very low in met30 mutants, whereas expression of CLN3 was unaffected. However, overexpression of Cln2 could not overcome the G(1) arrest. Interestingly, overexpression of Clb5 could induce DNA replication in met30 mutants, albeit very inefficiently. Increased levels of Clb5 could not, however, suppress the cell proliferation defect of met30 mutants. Consistent with the DNA replication defects, chromatin immunoprecipitation experiments revealed significantly lower levels of the replication factors Mcm4, Mcm7, and Cdc45 at replication origins in met30 mutants than in wild-type cells. These data suggest that Met30 regulates several aspects of the cell cycle, including G(1)-specific transcription, initiation of DNA replication, and progression through M phase.

Multiple pathways for suppression of mutants affecting G1-specific transcription in Saccharomyces cerevisiae.

In the budding yeast, Saccharomyces cerevisiae, control of cell proliferation is exerted primarily during G(1) phase. The G(1)-specific transcription of several hundred genes, many with roles in early cell cycle events, requires the transcription factors SBF and MBF, each composed of Swi6 and a DNA-binding protein, Swi4 or Mbp1, respectively. Binding of these factors to promoters is essential but insufficient for robust transcription. Timely transcriptional activation requires Cln3/CDK activity. To identify potential targets for Cln3/CDK, we identified multicopy suppressors of the temperature sensitivity of new conditional alleles of SWI6. A bck2Delta background was used to render SWI6 essential. Seven multicopy suppressors of bck2Delta swi6-ts mutants were identified. Three genes, SWI4, RME1, and CLN2, were identified previously in related screens and shown to activate G(1)-specific expression of genes independent of CLN3 and SWI6. The other four genes, FBA1, RPL40a/UBI1, GIN4, and PAB1, act via apparently unrelated pathways downstream of SBF and MBF. Each depends upon CLN2, but not CLN1, for its suppressing activity. Together with additional characterization these findings indicate that multiple independent pathways are sufficient for proliferation in the absence of G(1)-specific transcriptional activators.

Regulation and recognition of SCFGrr1 targets in the glucose and amino acid signaling pathways.

SCFGrr1, one of several members of the SCF family of E3 ubiquitin ligases in budding Saccharomyces cerevisiae, is required for both regulation of the cell cycle and nutritionally controlled transcription. In addition to its role in degradation of Gic2 and the CDK targets Cln1 and Cln2, Grr1 is also required for induction of glucose- and amino acid-regulated genes. Induction of HXT genes by glucose requires the Grr1-dependent degradation of Mth1. We show that Mth1 is ubiquitinated in vivo and degraded via the proteasome. Furthermore, phosphorylated Mth1, targeted by the casein kinases Yck1/2, binds to Grr1. That binding depends upon the Grr1 leucine-rich repeat (LRR) domain but not upon the F-box or basic residues within the LRR that are required for recognition of Cln2 and Gic2. Those observations extend to a large number of Grr1-dependent genes, some targets of the amino acid-regulated SPS signaling system, which are properly regulated in the absence of those basic LRR residues. Finally, we show that regulation of the SPS targets requires the Yck1/2 casein kinases. We propose that casein kinase I plays a similar role in both nutritional signaling pathways by phosphorylating pathway components and targeting them for ubiquitination by SCFGrr1.