Booher, R.N., Deshaies, R.J. and Kirschner, M.W. (1993). Properties of Saccharomyces cerevisiae wee1 and its differential regulation of p34CDC28 in response to G1 and G2 cyclins. EMBO J. 12, 3417-3426.

Wee1 is a protein kinase that negatively regulates p34cdc2 kinase activity. We have identified a Saccharomyces cerevisiae wee1 homolog encoded by the SWE1 gene. SWE1 overexpression arrests cells in G2 with short spindles whereas deletion of SWE1 did not alter the cell cycle but did eliminate the G2 delay observed in mih1- mutants. Swe1 immunoprecipitates were capable of tyrosine phosphorylating and inactivating p34CDC28 complexed with Clb2, a G2-type cyclin, but not p34CDC28 complexed with Cln2, a G1-type cyclin, consistent with the inability of Swe1 overexpression to inhibit the G1/S transition. These results suggest that specific cyclin subunits target p34CDC28 for distinct regulatory controls which may be important for ensuring proper p34CDC28 function during the cell cycle.

Deshaies, R.J., Chau, V., and Kirschner, M.W. (1995). Ubiquitination of the G1 cyclin Cln2p by a Cdc34p-dependent pathway. EMBO J. 14, 303-312.

Recombinant G1 cyclin Cln2p can bind to and stimulate the protein kinase activity of p34CDC28 (Cdc28p) in an extract derived from cyclin- depleted and G1-arrested Saccharomyces cerevisiae cells. Upon activating Cdc28p, Cln2p is extensively phosphorylated and conjugated with multiubiquitin chains. Ubiquitination of Cln2p in vitro requires the Cdc34p ubiquitin-conjugating enzyme, Cdc28p, protein phosphorylation and unidentified factors in yeast extract. Ubiquitination of Cln2p by Cdc34p contributes to the instability of Cln2p in vivo, as the rate of Cln2p degradation is reduced in cdc34ts cells. These results provide a molecular framework for G1 cyclin instability and suggest that a multicomponent, regulated pathway specifies the selective ubiquitination of G1 cyclins.

Deshaies, R.J. and Kirschner, M.W. (1995). G1 cyclin-dependent activation of p34CDC28 (Cdc28p) in vitro. Proc. Natl. Acad. Sci. USA 92,1182-1186.

In Saccharomyces cerevisiae, transient accumulation of G1 cyclin/p34CDC28 (Cdc28p) complexes induces cells to traverse the cell cycle Start checkpoint and commit to a round of cell division. To investigate posttranslational controls that modulate Cdc28p activity during the G1 phase, we have reconstituted cyclin-dependent activation of Cdc28p in a cyclin-depleted G1 extract. A glutathione S-transferase- G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase. Activation of Cdc28p by GST-Cln2p requires ATP, crude yeast cytosol, and the conserved Thr-169 residue that serves in other organisms as a substrate for phosphorylation by cyclin-dependent protein kinase-activating kinase. This assay may be useful for distinguishing genes that promote directly the posttranslational assembly of active Cln2p/Cdc28p kinase complexes from those that stimulate the accumulation of active complexes via a positive-feedback loop that governs synthesis of G1 cyclins.

Gerber, M.R., Farrell, A., Deshaies, R.J., Herskowitz, I. and Morgan, D.O. (1995). CDC37 is required for association of the protein kinase CDC28 with G1 and mitotic cyclins. Proc. Natl. Acad. Sci. USA 92, 4651-4655.

Studies of the temperature-sensitive cdc37-1 mutant of Saccharomyces cerevisiae suggest that Cdc37 is required for passage through the G1 phase of the cell cycle, but its precise function is not known. We have investigated the role of Cdc37 in the regulation of the cyclin- dependent protein kinase Cdc28. We find that G1 arrest in the cdc37-1 mutant is accompanied by a decrease in the Cdc28 activity associated with the G1 cyclin Cln2. This defect appears to be caused by a decrease in the binding of Cdc28 and Cln2. cdc37-1 mutants also exhibit a defect in the binding and activation of Cdc28 by the mitotic cyclin Clb2. Thus Cdc37 may be a regulator that is required for the association of Cdc28 with multiple cyclins.

Banerjee, A., Deshaies, R.J., and Chau, V. (1995). Characterization of a dominant negative mutant of the cell cycle ubiquitin-conjugating enzyme Cdc34. J. Biol. Chem. 270, 26209-26215.

The yeast Saccharomyces cerevisiae CDC34 gene encodes a ubiquitin- conjugating enzyme that is required for the cell cycle G1/S transition. We show here that a dominant negative Cdc34 protein is generated by simultaneously replacing both Cys95 and Leu99 with Ser residues. Cys95 is an essential catalytic residue that forms a transient thiol ester with ubiquitin during catalysis, and Leu99 is highly conserved among all known ubiquitin-conjugating enzymes. Mutants that encode either an alanine or a serine at one or both of these two positions are inactive. Of these eight mutants, overexpression of CDC34-C95S,L99S in wild type strains was found to block cell growth. Although cells overexpressing Cdc34-C95S,L99S do not exhibit the characteristic multibudded phenotype of cdc34 temperature-sensitive or null mutants, this blockade is relieved by simultaneous overxpression of wild type Cdc34. Purified Cdc34-C95S,L99S protein can be shown to inhibit in vitro ubiquitination of the Cdc34-specific substrate, Cln2 protein. We suggest that Cdc34- C95S,L99S selectively sequesters a subset of Cdc34 substrates or regulators. These findings have implications for the structure/function relationships of ubiquitin-conjugating enzymes, and suggest a general method for identifying components and substrates of specific ubiquitination pathways of eukaryotes.

Deshaies, R.J. (1995) Make it or break it: the role of ubiquitin-dependent proteolysis in cellular regulation. Trends Cell Bio. 5, 428-434.

Effective regulation of the concentration of a protein in the cell requires rapid protein degradation. Until recently, it was widely believed that intracellular proteolysis was largely confined to the turnover of damaged, or otherwise abnormal, proteins. Recently, however, the role of protein degradation in cellular regulation has gained centre stage, and ubiquitin/proteasome-dependent proteolysis has been shown to play a key role in processes as diverse as embryonic development, transcription, and the cell cycle.

Deshaies, R.J. (1995). The self-destructive personality of a cell cycle in transition. Curr. Opin. Cell Biol. 7, 781-789.

The transition from G1 to S phase, sister chromatid separation in anaphase, and the exit from mitosis are driven by the destruction of cell cycle regulatory proteins by distinct ubiquitin-dependent proteolytic pathways. The components and targets of these key degradation pathways are now becoming clear. Genetic and biochemical dissections of these extremely specific and well regulated destruction pathways are providing fundamental insights into the mechanisms of control of the cell division cycle.

King, R., Peters, J, Deshaies, R.J., and Kirschner, M. (1996). How proteolysis drives the cell cycle. Science 274, 1652-1659.

Oscillations in the activity of cyclin-dependent kinases (CDKs) promote progression through the eukaryotic cell cycle. This review examines how proteolysis regulates CDK activity-by degrading CDK activators or inhibitors-and also how proteolysis may directly trigger the transition from metaphase to anaphase. Proteolysis during the cell cycle is mediated by two distinct ubiquitin-conjugation pathways. One pathway, requiring CDC34, initiates DNA replication by degrading a CDK inhibitor. The second pathway, involving a large protein complex called the anaphase-promoting complex or cyclosome, initiates chromosome segregation and exit from mitosis by degrading anaphase inhibitors and mitotic cyclins. Proteolysis therefore drives cell cycle progression not only by regulating CDK activity, but by directly influencing chromosome and spindle dynamics.

Deshaies, R.J. (1997). Phosphorylation and proteolysis: partners in the regulation of cell division in budding yeast. Curr. Opin. Genet. Dev. Biol. 7, 7-16.

The budding yeast cell cycle oscillates between states of low and high cyclin B/cyclin-dependent kinase (CLB/CDK) activity. Remarkably, the two transitions that link these states are governed by ubiquitin-mediated proteolysis. The transition from low to high CLB activity is triggered by degradation of the CLB/CDK inhibitor SIC1, and the complementary excursion is propelled by the proteolytic destruction of CLBs. The extracellular environment controls this two-state circuit by regulating G1 cyclin/CDK activity, which is directly required for SIC1 proteolysis. Thus, stable oscillations of chromosome replication and segregation in budding yeast are propagated by the interplay between protein phosphorylation and protein degradation.

Verma, R., Feldman, R. M. R., and Deshaies, R. J. (1997). SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities. Mol. Biol.Cell 8, 1427-1437.

Traversal from G1 to S-phase in cycling cells of budding yeast is dependent on the destruction of the S-phase cyclin/CDK inhibitor SIC1. Genetic data suggest that SIC1 proteolysis is mediated by the ubiquitin pathway and requires the action of CDC34, CDC4, CDC53, SKP1, and CLN/CDC28. As a first step in defining the functions of the corresponding gene products, we have reconstituted SIC1 multiubiquitination in DEAE-fractionated yeast extract. Multiubiquitination depends on cyclin/CDC28 protein kinase and the CDC34 ubiquitin-conjugating enzyme. Ubiquitin chain formation is abrogated in cdc4ts mutant extracts and assembly restored by the addition of exogenous CDC4, suggesting a direct role for this protein in SIC1 multiubiquitination. Deletion analysis of SIC1 indicates that the N-terminal 160 residues are both necessary and sufficient to serve as substrate for CDC34-dependent ubiquitination. The complementary C- terminal segment of SIC1 binds to the S-phase cyclin CLB5, indicating a modular structure for SIC1.

Feldman, R.M.R., Correll, C.C., Kaplan, K.B., and Deshaies, R.J. (1997). A complex of Cdc4, Skp1, and Cdc53/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1. Cell 91/B>, 221-230.

In S. cerevisiae, the G1/S transition requires Cdc4p, Cdc34p, Cdc53p, Skp1p, and the Cln/Cdc28p cyclin-dependent kinase (Cdk). These proteins are thought to promote the proteolytic inactivation of the S-phase Cdk inhibitor Sic1p. We show here that Cdc4p, Cdc53p, and Skp1p assemble into a ubiquitin ligase complex named SCFCdc4p. When mixed together, SCFCdc4p subunits, E1 enzyme, the E2 enzyme Cdc34p, and ubiquitin are sufficient to reconstitute ubiquitination of Cdk-phosphorylated Sic1p. Phosphorylated Sic1p substrate is specifically targeted for ubiquitination by binding to a Cdc4p/Skp1p subcomplex. Taken together, these data illuminate the molecular basis for the G1/S transition in budding yeast and suggest a general mechanism for phosphorylation- targeted ubiquitination in eukaryotes.

Verma, R., Annan, R., Huddleston, M., Carr, S., Reynard, G., and Deshaies, R.J. (1997). Phosphorylation of Sic1p by G1 cyclin/Cdk is required for its degradation and entry into S phase. Science 278, 455-460.

G1 cyclin-dependent kinase (Cdk)-triggered degradation of the S-phase Cdk inhibitor Sic1p has been implicated in the transition from G1 to S phase in the cell cycle of budding yeast. A multidimensional electrospray mass spectrometry technique was used to map G1 Cdk phosphorylation sites in Sic1p both in vitro and in vivo. A Sic1p mutant lacking three Cdk phosphorylation sites did not serve as a substrate for Cdc34p-dependent ubiquitination in vitro, was stable in vivo, and blocked DNA replication. Moreover, purified phosphoSic1p was ubiquitinated in cyclin-depleted G1 extract, indicating that a primary function of G1 cyclins is to tag Sic1p for destruction. These data suggest a molecular model of how phosphorylation and proteolysis cooperate to bring about the G1/S transition in budding yeast.

Henchoz, S., Chi, Y., Catarin, B., Herskowitz, I., Deshaies, R. J., and Peter, M. (1997). Phosphorylation and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast. Genes Dev. 11, 3046-3060.

Cyclin-dependent kinase inhibitors (CKIs) play key roles in controlling the eukaryotic cell cycle by coordinating cell proliferation and differentiation. Understanding the roles of CKIs requires knowledge of how they are regulated both through the cell cycle and in response to extracellular signals. Here we show that the yeast CKI, Far1p, is controlled by ubiquitin-dependent proteolysis. Wild-type Far1p was stable only in the G1 phase of the cell cycle. Biochemical and genetic evidence indicate that its degradation required the components of the G1-S ubiquitination system, Cdc34p, Cdc4p, Cdc53p, and Skp1p. We isolated a mutant form of Far1p (Far1p-22) that was able to induce cell cycle arrest in the absence of alpha-factor. Cells that overexpress Far1-22p arrested in G1 as large unbudded cells with low Cdc28p-Clnp kinase activity. Wild-type Far1p, but not Far1-22p, was readily ubiquitinated in vitro in a CDC34- and CDC4-dependent manner. Far1-22p harbors a single amino acid change, from serine to proline at residue 87, which alters phosphorylation by Cdc28p-Cln2p in vitro. Our results show that Far1p is regulated by ubiquitin-mediated proteolysis and suggest that phosphorylation of Far1p by the Cdc28p-Clnp kinase is part of the recognition signal for ubiquitination.

Lyapina, S. L., Correll, C. C., Kipreos, E. T., and Deshaies, R. J. (1998). Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein. Proc. Natl. Acad. Sci. USA 95, 7451-7456.

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin- dependent kinase inhibitor SIC1. SCF is composed of three proteins- ySKP1, CDC53 (Cullin), and the F-box protein CDC4-that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, we isolated hSKP1 in a two- hybrid screen with hCUL1, the closest human homologue of CDC53. Here, we show that hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2-hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins.

Shou, W., Seol, J.H., Shevchenko, A., Baskerville, C., Moazed, D., Chen, Z.W.S., Jang, J., Shevchenko, A., Charbonneau, H., and Deshaies, R.J. (1999). Exit from mitosis triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 97 233-244.

Exit from mitosis in budding yeast requires a group of essential proteins--including the GTPase Tem1 and the protein phosphatase Cdc14-- that downregulate cyclin-dependent kinase activity. We identified a mutation, net1-1, that bypasses the lethality of tem1 delta. NET1 encodes a novel protein, and mass spectrometric analysis reveals that it is a key component of a multifunctional complex, denoted RENT (for regulator of nucleolar silencing and telophase), that also contains Cdc14 and the silencing regulator Sir2. From G1 through anaphase, RENT localizes to the nucleolus, and Cdc14 activity is inhibited by Net1. In late anaphase, Cdc14 dissociates from RENT, disperses throughout the cell in a Tem1-dependent manner, and ultimately triggers mitotic exit. Nucleolar sequestration may be a general mechanism for the regulation of diverse biological processes.

Straight, A.F., Shou, W., Dowd, G.J., Turck, C.W., Deshaies, R.J., Johnson, A.D., and Moazed, D. (1999). Net1, a Sir2-associated nucleolar protein required for rDNA silencing and nucleolar integrity. Cell 97 245-256.

The Sir2 protein mediates gene silencing and repression of recombination at the rDNA repeats in budding yeast. Here we show that Sir2 executes these functions as a component of a nucleolar complex designated RENT (regulator of nucleolar silencing and telophase exit). Net1, a core subunit of this complex, preferentially cross-links to the rDNA repeats, but not to silent DNA regions near telomeres or to active genes, and tethers the RENT complex to rDNA. Net1 is furthermore required for rDNA silencing and nucleolar integrity. During interphase, Net1 and Sir2 colocalize to a subdomain within the nucleous, but at the end of mitosis a fraction of Sir2 leaves the nucleolus and disperses as foci throughout the nucleus, suggesting that the structure of rDNA silent chromatin changes during the cell cycle. Our findings suggest that a protein complex shown to regulate exit from mitosis is also involved in gene silencing.

Seol, J.H., Feldman, R.M.R., Zachariae, W., Shevchenko, A., Correll, C.C., Lyapina, S., Chi, Y., Galova, M., Claypool, J., Sandmeyer, S., Nasmyth, K., Shevchenko, A., and Deshaies, R.J. (1999). Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. Genes Dev. 13 1614-1626.

SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.

Freed, E., Lacey, K.R., Huie, P., Lyapina, S.A., Deshaies, R.J., Stearns, T., and Jackson, P.K. (1999). Components of an SCF ubiquitin ligase localize to the centrosome and regulate the centrosome duplication cycle. Genes Dev. 13, 2242-2257.

Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, we show that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. We identify candidate centrosomal F-box proteins, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle.

Deshaies, R.J. (1999). SCF and cullin/RING H2-based ubiquitin ligases. Annu. Rev. Cell Dev. Biol. 15, 435-467.

Protein degradation is deployed to modulate the steady-state abundance of proteins, and to switch cellular regulatory circuits from one state to another by abrupt elimination of control proteins. In eukaryotes, the bulk of the protein degradation that occurs in the cytoplasm and nucleus is carried out by the 26S proteasome. In turn, most proteins are thought to be targeted to the 26S proteasome by covalent attachment of a multiubiquitin chain. Ubiquitination of proteins requires a multienzyme system. A key component of ubiquitination pathways, the ubiquitin ligase, controls both the specificity and timing of substrate ubiquitination. In this article, I focus on a conserved ubiquitin ligase complex known as SCF that plays a key role in marking a variety of regulatory proteins for destruction by the 26S proteasome.

Deshaies, R.J. and Jack, T. (1999). Cell multiplication. Peering in and peering out: regulation of and by the cell cyle. Curr. Opin. Cell Biol. 11, 705-7.

Verma, R., and Deshaies, R.J. (2000). A proteasome howdunit: the case of the missing signal. Cell, 101, 341-344.

Reynard, G. J., Reynolds, W., Verma, R., and Deshaies, R. J. (2000). CKS1 is required for G1 cyclin/CDK activity in budding yeast. Mol. Cell Biol. 20, 5858-5864.

p13suc1/Cks proteins have been implicated in the regulation of cyclin/cyclin-dependent kinase (CDK) activity. However, the mechanism by which p13suc1/Cks influences the function of cyclin/CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G1 cyclin/CDK complexes. Cln2 and Cdc28 subunits co­expressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2/Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G1 cyclin/CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The level of Cln2/Cdc28 and Cln3/Cdc28 protein kinase activity is severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G1 cyclins, which depends upon Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G1 cyclin/CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G1 phase in budding yeast.

Deshaies, R.J., and Meyerowitz, E.M. (2000). COP1 patrols the night beat. Nature Cell Biol. 2, E102-E104.

Light regulates the behavior of many organisms. New data indicate that the greening of plants is enabled by light-dependent stabilization of a transcription factor that is rapidly degraded in darkness. Thus, photomorphogenesis joins cell division and circadian rhythm as another critical biological process that is governed by proteolysis.

Verma, R., Chen, S., Feldman, R., Schieltz, D., Yates, J., and Deshaies, R.J. (2000). Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell 11, 3425-3439.

Ubiquitin-dependent proteolysis is catalyzed by the 26S proteasome, a dynamic complex of 32 different proteins whose mode of assembly and mechanism of action are poorly understood, in part due to the difficulties encountered in purifying the intact complex. Here we describe a one-step affinity method for purifying intact 26S proteasomes, 19S regulatory caps, and 20S core particles from budding yeast cells. Affinity-purified 26S proteasomes hydrolyze both model peptides and the ubiquitinated Cdk inhibitor Sic1. Affinity purifications performed in the absence of ATP or presence of the poorly hydrolyzable analog ATP-gamma-S unexpectedly revealed that a large number of proteins, including subunits of the skp1-cullin-F-box protein ligase (SCF) and anaphase-promoting complex (APC) ubiquitin ligases, copurify with the 19S cap. To identify these proteasome-interacting proteins, we used a recently developed method that enables the direct analysis of the composition of large protein complexes (DALPC) by mass spectrometry. Using DALPC, we identified more than 24 putative proteasome-interacting proteins, including Ylr421c (Daq1), which we demonstrate to be a new subunit of the budding yeast 19S cap, and Ygr232w (Nas6), which is homologous to a subunit of the mammalian 19S cap (PA700 complex). Additional PIPs include the heat shock proteins Hsp70 and Hsp82, the deubiquitinating enzyme Ubp6, and proteins involved in transcriptional control, mitosis, tubulin assembly, RNA metabolism, and signal transduction. Our data demonstrate that nucleotide hydrolysis modulates the association of many proteins with the 26S proteasome, and validate DALPC as a powerful tool for rapidly identifying stoichiometric and substoichiometric components of large protein assemblies.

Blondel, M., Galan, J.M., Chi, Y., Lafourcade, C., Longaretti, C., Deshaies, R.J., and Peter, M. (2000). Nuclear-specific degradation of Far1 is controlled by the localization of the F-box protein Cdc4. EMBO J. 19, 6085-97.

Far1 is a bifunctional protein that is required to arrest the cell cycle and establish cell polarity during yeast mating. Here we show that SCF(Cdc4) ubiquitylates Far1 in the nucleus, which in turn targets the multi-ubiquitylated protein to 26S proteasomes most likely located at the nuclear envelope. In response to mating pheromones, a fraction of Far1 was stabilized after its export into the cytoplasm by Ste21/Msn5. Preventing nuclear export destabilized Far1, while conversely cytoplasmic Far1 was stabilized, although the protein was efficiently phosphorylated in a Cdc28-Cln-dependent manner. The core SCF subunits Cdc53, Hrt1 and Skp1 were distributed in the nucleus and the cytoplasm, whereas the F-box protein Cdc4 was exclusively nuclear. A cytoplasmic form of Cdc4 was unable to complement the growth defect of cdc4-1 cells, but it was sufficient to degrade Far1 in the cytoplasm. Our results illustrate the importance of subcellular localization of F-box proteins, and provide an example of how an extracellular signal regulates protein stability at the level of substrate localization.

Annan, R.S, Huddleston, M.J., Verma, R., Deshaies, R.J., and Carr, S.A. (2001). A multidimensional electrospray MS-based approach to phosphopeptide mapping. Anal. Chem. 73, 393-404.

A new, multidimensional electrospray MS-based strategy for phosphopeptide mapping is described which eliminates the need to radiolabel protein with 32P or 33P. The approach utilizes two orthogonal MS scanning techniques, both of which are based on the production of phosphopeptide-specific marker ions at m/z 63 and/or 79 in the negative ion mode. These scan methods are combined with liquid chromatography-electrospray mass spectrometry and nanoelectrospray MS/MS to selectively detect and identify phosphopeptides in complex proteolytic digests. Low-abundance, low-stoichiometry phosphorylation sites can be selectively determined in the presence of an excess of nonphosphorylated peptides, even in cases where the signal from the phosphopeptide is indistinguishable from background in the conventional MS scan. The strategy, which has developed and refined in our laboratory over the past few years, is particularly well suited to phosphoproteins that are phosphorylated to varying degrees of stoichiometry on multiple sites. Sensitivity and selectivity of the method are demonstrated here using model peptides and a commercially available phosphoprotein standard. In addition, the strategy is illustrated by the complete in vitro and in vivo phosphopeptide mapping of Sic1p, a regulator of the G1/S transition in budding yeast.

Lippincott J., Shannon K.B., Shou W., Deshaies R.J., Li R. (2001). The Tem1 small GTPase controls actomyosin and septin dynamics during cytokinesis. Journal of Cell Science 114, 1379-86.

Cytokinesis in budding yeast involves an actomyosin-based ring which assembles in a multistepped fashion during the cell cycle and constricts during cytokinesis. In this report, we have investigated the structural and regulatory events that occur at the onset of cytokinesis. The septins, which form an hour-glass like structure during early stages of the cell cycle, undergo dynamic rearrangements prior to cell division: the hourglass structure splits into two separate rings. The contractile ring, localized between the septin double rings, immediately undergoes contraction. Septin ring splitting is independent of actomyosin ring contraction as it still occurs in mutants where contraction fails. We hypothesize that septin ring splitting may remove a structural barrier for actomyosin ring to contract. Because the Tem1 small GTPase (Tem1p) is required for the completion of mitosis, we investigated its role in regulating septin and actomyosin ring dynamics in the background of the net1-1 mutation, which bypasses the anaphase cell cycle arrest in Tem1-deficient cells. We show that Tem1p plays a specific role in cytokinesis in addition to its function in cell cycle progression. Tem1p is not required for the assembly of the actomyosin ring but controls actomyosin and septin dynamics during cytokinesis.

Traverso E.E., Baskerville C., Liu Y., Shou, W., James P., Deshaies R.J., and Charbonneau H. (2001). Characterization of the net1 cell cycle-dependent regulator of the Cdc14 phophatase from budding yeast. J Biol. Chem. 276, 21924-21931.

In the budding yeast Saccharomyces cerevisiae, the multifunctional protein Net1 is implicated in regulating the cell cycle function of the Cdc14 protein phosphatase. Genetic and cell biological data suggest that during interphase and early mitosis Net1 holds Cdc14 within the nucleolus where its activity is suppressed. Upon its transient release from Net1 at late anaphase, active Cdc14 promotes exit from mitosis by dephosphorylating targets in the nucleus and cytoplasm. Herein, we present evidence supporting the proposed role of Net1 in regulating Cdc14 and exit from mitosis. We show that the N-terminal fragment Net1(1-600) directly binds Cdc14 in vitro and is a highly specific competitive inhibitor of its activity (Ki = 3nM) with five different substrates including the physiologic targets Swi5 and Sic1. An analysis of truncation mutants indicates that the Cdc14-binding site is located within a segment of Net1 containing residues 1-341. We propose that Net1 inhibits by occluding the active site of Cdc14 because it acts as a competitive inhibitor, binds to a site located within the catalytic domain (residues 1-374), binds with reduced affinity to a Cdc14C283S mutant in which an active site Cys is replaced, and is displaced by tungstate, a transition state analog known to bind in the catalytic site of protein tyrosine phosphatases.

Seol, J.H., Shevchenko, A., Shevchenko, A., and Deshaies, R.J. (2001). Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly. Nat. Cell Biol. 4, 384-91.

SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs). Here we report the identification, using sequential rounds of epitope tagging, affinity purification and mass spectrometry, of 16 Skp1 and Cdc53-associated proteins in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2). Rav1, Rav2 and Skp1 form a complex that we have named 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases are conserved throughout eukaryotes, and have been implicated in tumour metastasis and multidrug resistance, and here we show that RAVE promotes glucose-triggeredassembly of the V-ATPase holoenzyme. Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here. Thus, our results provide a distinct view of the interactions that link proteins into a comprehensive cellular network.

Galan, J.M., Wiederkehr, A., Seol, J.H., Haguenauer-Tsapis, R., Deshaies, R.J., Riezman, H., and Peter, M. (2001). Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast. Mol. Cell Biol. 9, 3105-17.

Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-boxproteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p inSaccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted withSkp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the onlymajor partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.

Chi, Y., Huddleston, M.J., Zhang, X., Young, R.A., Annan, R.S., Carr, S.A., and Deshaies, R.J. (2001). Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase. Genes Dev. 15, 1078-1092.

The budding yeast transcriptional activator Gcn4 is rapidly degraded in an SCF(Cdc4)-dependent manner in vivo. Upon fractionation of yeast extracts to identify factors that mediate Gcn4 ubiquitination, we found that Srb10 phosphorylates Gcn4 and thereby marks it for recognition by SCF(Cdc4) ubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Gcn4 is almost completely stabilized in srb10Delta pho85Delta cells, or upon mutation of all Srb10 phosphorylation sites within Gcn4, suggesting that the Pho85 and Srb10 cyclin-dependent kinases (CDKs) conspire to limit the accumulation of Gcn4. The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat-stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in resting wild-type cells, its nuclear exclusion is partially compromised in srb10 mutant cells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. We propose that Srb10 also inhibits gene expression by promoting the rapid degradation or nuclear export of specific transcription factors. Simultaneous down-regulation of both transcriptional regulatory proteins and RNA polymerase may enhance the potency and specificity of transcriptional inhibition by Srb10.

Schwechheimer C., Serino G., Callis J., Crosby W.L., Lyapina S., Deshaies R.J., Gray W.M., Estelle M., Deng X.W. (2001). Interactions of the COP9 Signalosome with the E3 ubiquitin ligase SCFTIR1 in mediating auxin response. Science 292, 1379-82.

The COP9 signalosome is an evolutionary conserved multi-protein complex of unknown function that acts as a negative regulator of photomorphogenic seedling development in Arabidopsis. In this report, we show that plants with reduced COP9 signalosome levels had decreased auxin-response similar to loss-of-function mutants of the E3 ubiquitin ligase SCFTIR1. Furthermore, we found that the COP9 signalosome and SCFTIR1 interacted in vivoe and that the COP9 signalosome was required for efficient degradation of PSIAA6, a cnadidate substrate of SCFTIR1. Thus, the COP9 signalosome may play an important role in mediating E3 ubiquitin ligase-mediated responses.

Lyapina S, Cope G, Shevchenko A, Serino G, Tsuge T, Zhou C, Wolf DA, Wei N, Shevchenko A, and Deshaies RJ. (2001). Promotion of NEDD8-CUL1 conjugate cleavage by COP9 Signalosome. Science 292, 1382-1385.

SCF ubiquitin ligases control various processes by marking regulatory proteins for ubiquitin-dependent proteolysis. To illuminate how SCF complexes are regulated, we sought proteins that interact with the human SCF component CUL1. The COP9 signalosome (CSN), a suppressor of plant photomorphogenesis, associated with multiple cullins and promoted cleavage of the ubiquitin-like protein NEDD8 from Schizosaccharomyces pombe CUL1 in vivo and in vitro. Multiple NEDD8-modified proteins uniquely accumulated in CSN-deficient S. pombe cells. We propose that the broad spectrum of activities previously attributed to CSN subunits--including repression of photomorphogenesis, activation of JUN, and activation of p27 nuclear export--underscores the importance of dynamic cycles of NEDD8 attachment and removal in biological regulation.

Mah, A.S., Jang, J., and Deshaies R.J. (2001). Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex. Proc. Natl. Acad. Sci. USA, 98:7325-30.

Exit from mitosis in budding yeast requires inactivation of cyclin-dependent kinases through mechanisms triggered by the protein phosphatase Cdc14. Cdc14 activity, in turn, is regulated by a group of proteins, the mitotic exit network (MEN), which includes Lte1, Tem1, Cdc5, Cdc15, Dbf2/Dbf20, and Mob1. The direct biochemical interactions between the components of the MEN remain largely unresolved. Here, we investigate the mechanisms that underlie activation of the protein kinase Dbf2. Dbf2 kinase activity depended on Tem1, Cdc15, and Mob1 in vivo. In vitro, recombinant protein kinase Cdc15 activated recombinant Dbf2, but only when Dbf was bound to Mob1. Conserved phosphorylation sites Ser-374 and Thr-544, present in the human, Caenorhabditis elegans, and Drosophila melanogaster relatives of Dbf2, were required for DBF2 function in vivo, and activation of Dbf2-Mob1 by Cdc15 in vitro. Whereas Cdc15 phosphorylated Dbf2, Dbf2-Mob1, and Dbf2(S374A/T544A)-Mob1, the pattern of phosphate incorporation into Dbf2 was substantially altered by either the S374A T544A mutations or omission of Mob1. Thus, Cdc15 promotes the exit from mitosis by directly switching on the kinase activity of Dbf2. We propose that Mob1 promotes this activation process by enabling Cdc15 to phosphorylated late the critical Ser-374 and Thr-444 phosphoacceptor sites of Dbf2.

Zhou C., Seibert V., Rhee E., Geyer R., Cope G., Lyapina S., Deshaies R.J., Wolf D.A. (2001). The fission yeast COP9/signalosome is involved in cullin modification by ubiquitin-related Ned8p. BMC Biochemistry 2:7.

Background The function of the fission yeast cullins Pcu1p and Pcu4p requires modification by the ubiquitin-related peptide Ned8p. A recent report by Lyapina et al. shows that the COP9/signalosome (CSN), a multifunctional eight subunit complex, regulates Ned8p modification of Pcu1p. Disruption of caa1/csn, which encodes subunit 1 of the putative S. pombe CSN, results in accumulation of Pcu1p exclusively in the modified form. However, it remained unclear whether this reflects global control of all cullins by the entire CSN complex. Results We demonstrate that multiple CSN subunits control Ned8p modification of Pcu3p, another fission yeast cullin, which, in complex with the RING domain protein Pip1p, forms a ubiquitin ligase that functions in cellular stress response. Pcu3p is modified by Ned8p on Lys 729 and accumulates exclusively in the neddylated form in cells lacking the CSN subunits 1, 3, 4, and 5. These CSN subunits co-elute with Pcu3p in gel filtration fractions corresponding to 550 kDa and specifically bind both native and Ned8p-modified Pcu3p in vivo. While CSN does not influence the subcellular localization of Pcu3p, Pcu3p-associated in vitro ubiquitin ligase activity is stimulated in the absence of CSN. Conclusions Taken together, our data suggest that CSN is a global regulator of Ned8p modification of multiple cullins and potentially other proteins involved in cellular regulation.

Sakamoto, K.M., Kim, K.B., Kumagai, A., Mercurio, F., Crews, C.M. and Deshaies R.J. (2001). Protacs: chimeric molecules that target proteins to the SCF complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. USA 98:8554-9.

The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex known as SCF (Skp1/Cullin/F-box complex containing Hrt1). We sought to artificially target a protein to the SCF complex for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compound or "Protac-1" (Protein Targeting Chimeric molecule #1) was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the IkBa phosphopeptide that is recognized by the F-box protein b-TRCP, while the other domain is comprised of ovalicin. We show that MetAP-2 can be tethered to SCFb-TRCP, ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.

Shou, W., Sakamoto KM, Keener J, Morimoto KW, Traverso EE, Azzam R, Hoppe GJ, Feldman RMR, DeModena J, Moazed D, Charbonneau H, Nomura M, and Deshaies RJ (2001). Net1 stimulates RNA polymerase I transcription and regulates nucleolar structure independently of controlling mitotic exit. Mol. Cell 8:45-55.

The budding yeast RENT complex, consisting of at least three proteins (Net1, Cdc14, Sir2), is anchored to the nucleolus by Net1. RENT controls mitotic exit, nucleolar silencing, and nucleolar localization of Nop1. Here, we report two new functions of Net1. First, Net1 directly binds Pol I and stimulates rRNA synthesis both in vitro and in vivo. Second, Net1modulates nucleolar structure by regulating rDNA morphology and proper localization of multiple nucleolar antigens, including Pol I. Importantly, we show that the nucleolar and previously-described cell cycle functions of the RENT complex can be uncoupled by a dominant mutant allele of CDC14. The independent functions of Net1 link a key event in the cell cycle to nucleolar processes that are fundamental to cell growth.

Verma, R., McDonald, W.H., Yates, J.R. III, and Deshaies, R.J. (2001). Selective degradation of ubiquitinated Sic1 by purified 26S proteasome yields fully active S phase cyclin-Cdk. Mol. Cell 8:439-448.

Selective degradation of single subunits of multimeric complexes by the ubiquitin pathway underlies multiple regulatory switches, including those involving cyclins and Cdk inhibitors. The machinery that segregates ubiquitinated proteins from unmodified partners prior to degradation remains undefined. We report that ubiquitinated Sic1 (Ub-Sic1) embedded within inactive S phase cyclin-Cdk (S-Cdk) complexes was rapidly degraded by purified 26S proteasomes, yielding active S-Cdk. Mutant proteasomes that failed to degrade Ub-Sic1 nevertheless partially activated S-Cdk in an ATP-dependent manner. Whereas Ub-Sic1 was degraded within ~2 min, spontaneous dissociation of Ub-Sic1 from S-Cdk was ~200-fold slower. We propose that 26S proteasome has the intrinsic capability to extract, unfold, and degrade ubiquitinated proteins, while releasing bound partners untouched. Activation of S-Cdk reported herein represents a complete reconstitution of the regulatory switch underlying the G1/S transition in budding yeast.

Wu, G., Lyapina, S., Das, I., Li, Jinhe, Li., Gurney, M., Chui, I., Deshaies, R.J., Kitajewski, J. (2001). SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-medidated protein degradation.MCB 21:7403-15.

Notch receptors and their ligands play important roles in both normal animal development and pathogenesis. We show here that the F-box/WD40 repeat protein SEL-10 negatively regulates Notch receptor activity by targeting the intracellular domain of Notch receptors for ubiquitin-mediated protein degradation. Blocking of endogenous SEL-10 activity was done by expression of a dominant-negative form containing only the WD40 repeats. In the case of Notch1, this block leads to an increase in Notch signaling stimulated by either an activated form of the Notch1 receptor or Jagged1-induced signaling through Notch1. Expression of dominant-negative SEL-10 leads to stabilization of the intracellular domain of Notch1. The Notch4 intracellulardomain bound to SEL-10, but its activity was not increased as a result of dominant-negative SEL-10 expression. SEL-10 bound Notch4 via the WD40 repeats and bound preferentially to a phosphorylated form of Notch4 incells. We mapped the region of Notch4 essential for SEL-10 binding to the C-terminal region downstream of the ankyrin repeats. When this C-terminal fragment of Notch4 was expressed in cells, it was highly labile but could be stabilized by the expression of dominant-negative SEL-10. Ubiquitination of Notch1 and Notch4 intracellulardomains in vitro was dependent on SEL-10. Although SEL-10 interacts with the intracellular domains of both Notch1 and Notch4, these proteins respond differently to interference with SEL-10 function. Thus, SEL-10 functions to promote the ubiquitination of Notch proteins; however, the fates of these proteins may differ.

Deshaies, R.J. and Ferrell, J.E Jr. (2001). Multisite phosphorylation and the countdown to S phase. Cell. 107:819-822.

Remarkably, SCFCdc4 ubiquitin ligase binds and ubiquitinates Sic1 decorated with six, but not five, phosphates (Nash et al., 2001). This numerical wizardry suggests how analog inputs can be rectified to digital outputs. Unraveling the counting mechanism promises to generate new insights into the architecture of protein nanoprocessors.

Deshaies, R.J., Seol, J.H., McDonald, W.H., Cope, G., Lyapina, S., Shevchenko, A., Shevchenko A., Verma, R. and Yates, J.R. (2002). Charting the protein complexome in yeast by mass spectrometry. Mol. Cell. Prot. 1:3-10.

It has become evident over the past few years that many complex cellular processes, including control of the cell cycle and ubiquitin-dependent proteolysis, are carried out by sophisticated multisubunit protein machines that are dynamic in abundance, post-translational modification state, and composition. To understand better the nature of the macromolecular assemblages that carry out the cell cycle and ubiquitin-dependent proteolysis, we have used mass spectrometry extensively over the past few years to characterize both the composition of various protein complexes and the modification states of their subunits. In this article we review some of our recent efforts, and describe a promising new approach for using mass spectrometry to dissect protein interaction networks.

Shou, W., and Deshaies, R.J. (2002). Multiple telophase arrest bypassed (tab) mutants alleviate the essential requirement for Cdc15 in exit from mitosis in S cerevisiae. BMC Genet 3,4.

Background

The Mitotic Exit Network (MEN) proteins including the protein kinase Cdc15 and the protein phosphatase Cdc14 are essential for exit from mitosis in Saccharomyces cerevisiae. To identify downstream targets of the MEN, we sought telophase arrest bypassed (tab) mutations that bypassed the essential requirement for CDC15. Previous studies identified net1tab2-1 and CDC14TAB6-1 as mutations in the RENT complex subunits Net1 and Cdc14, respectively, and revealed that the MEN acts by promoting release of Cdc14 from its nucleolar Net1 anchor during anaphase. However, the remaining tab mutants were not characterized.

Results

Fourteen out of fifteen tab mutants were mapped to three recessive (tab1-tab3) and three dominant (TAB5-TAB7) linkage groups. We show that net1tab2-1 enables growth of tem1, cdc15, dbf2 dbf20, and mob1, but not cdc5 or cdc14, arguing that whereas the essential task of the first four genes is to promote exit from mitosis, CDC5 possesses additional essential function(s). net1tab2-1 but not CDC14TAB6-1 resulted in a high rate of chromosome loss, indicating that Net1 promotes accurate chromosome segregation in addition to its multiple known roles. Finally, TAB1 was shown to be MTR10, a gene encoding nuclear transport receptor/adaptor. In some of the tab mutants including mtr10tab1-1, defective nuclear export of the ribosomal protein Rpl11b was observed. Furthermore, the transport-defective -31 allele of the karyopherin SRP1, but not the transport competent -49 allele, exhibited a tab phenotype.

Conclusions

Transport-defective mutations in two karyopherins can bypass cdc15, suggesting that the function of the MEN is to promote mitotic exit by regulating nuclear transport.

Shou, W., Azzam, R., Chen, S.L., Huddleston, M.J., Baskerville, C., Charbonneau, H., Annan, R.S., Carr, S.A., and Deshaies, R.J. (2002). Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex. BMC Mol Biol. 3,3.

Background

In S. cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis. In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex. When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus.

Results

Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase. Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14. Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m')) lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow
normally and exhibit only minor defects in releasing Cdc14 during anaphase. However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene).

Conclusions

We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets. Our study suggests that Cdc5/polo is unusually promiscuous and highlights the need to validate Cdc5/polo in vitro phosphorylation sites by direct in vivo mapping experiments.

Loughrey, Chen S., Huddleston, M.J., Shou, W., Deshaies, R.J., Annan, R.S., and Carr, S.A. (2002). Mass spectrometry-based methods for phosphorylation site mapping of hyperphosphorylated proteins applied to Net1, a regulator of exit from mitosis in yeast. Mol. Cell. Prot. 1:186-96.

Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1.Cdc14 complex in vitro by phosphorylation of Net1. We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides
in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies.

Deshaies, R.J., Seol, J.H., McDonald, W.H., Cope, G., Lyapina, S., Shevchenko, A., Shevchenko, A., Verma, R., and Yates, J.R., 3rd (2002). Charting the protein complexome in yeast by mass spectrometry. Mol. Cell. Prot. 1:3-10.

It has become evident over the past few years that many complex cellular processes, including control of the cell cycle and ubiquitin-dependent proteolysis, are carried out by sophisticated multisubunit protein machines that are dynamic in abundance, post-translational modification state, and composition. To understand better the nature of the macromolecular assemblages that carry out the cell cycle and ubiquitin-dependent proteolysis, we have used mass spectrometry extensively over the past few years to characterize both the composition of various protein complexes and the modification states of their subunits. In this article we review some of our recent efforts, and describe a promising new approach for using mass spectrometry to dissect protein interaction networks.

Verma, R., Aravind, L., Oania, R., McDonald, W. H., Yates, J. R. III, Koonin, E.V., and Deshaies, R.J. (2002). Role of Rpn11 Metalloprotease in Deubiquitination and Degradation by the 26S Proteasome. Science, 298:611-614.

The 26S proteasome mediates degradation of ubiquitin-conjugated proteins. Although ubiquitin is recycled from proteasome substrates, the molecular basis of deubiquitination at the proteasome and its relationship to substrate degradation remain unknown. The Rpn11 subunit of the proteasome lid subcomplex contains a highly conserved Jab1/MPN domain-associated metalloisopeptidase (JAMM) motif - EXnHXHX10D. Mutation of the predicted active site histidines to alanine (rpn11AXA) was lethal and stabilized ubiquitin pathway substrates in yeast. Rpn11AXA mutant proteasomes assembled normally, but failed to either deubiquitinate or degrade ubiquitinated Sic1 in vitro. Our findings reveal an unexpected coupling between substrate deubiquitination and degradation, and suggest a unifying rationale for the presence of the lid in eukaryotic proteasomes.

Cope, G.A., Suh, G.S.B., Aravind, L., Schwarz, S.E., Zipursky, S.L., Koonin, E.V., and Deshaies, R.J. (2002). Role of Predicted Metalloprotease Motif of Jab1/Csn5 in Cleavage of NEDD8 from CUL1. Science, 298:608-611.

COP9 Signalosome (CSN) cleaves the ubiquitin-like protein Nedd8 from the Cul1 subunit of SCF ubiquitin ligases. The Jab1/MPN domain metalloenzyme (JAMM) motif in the Jab1/Csn5 subunit was found to underlie CSN's Nedd8 isopeptidase activity. JAMM is found in proteins from archaea, bacteria and eukaryotes, including the Rpn11 subunit of the 26S proteasome. Metal chelators and point mutations within JAMM abolished CSN-dependent cleavage of Nedd8 from Cul1, yet had little effect on CSN complex assembly. Optimal SCF activity in yeast and both viability and proper photoreceptor cell (R cell) development in Drosophila melanogaster required an intact Csn5 JAMM domain. We propose that JAMM isopeptidases play important roles in a variety of physiological pathways.

Park, C.J., Song, S., Lee, P.R., Shou, W., Deshaies, R.J., and Lee, K.S. (2003). Loss of CDC5 Function in Saccharomyces Cerevisiae Leads to Defects in Swe1p Regulation and Bfa1p/Bub2p-Independent Cytokinesis. Genetics, 163:21-33.

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain. Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G(2)/M transition. Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1Delta defect, suggesting that Cdc5p functions at a point upstream of Swe1p. In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect. Close examination of these mutants revealed delayed septin assembly at
the incipient bud site and loosely organized septin rings at the mother-bud neck. Components in the mitotic exit network (MEN) play important roles in normal cytokinesis. However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p. Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.

Petroski, M.D., and Deshaies, R.J. (2003). Context of multiubiquitin chain attachment influences the rate of Sic1 degradation. Mol. Cell 11:1435-1444.

The ubiquitin-dependent targeting of proteins to the proteasome is an essential mechanism for regulating eukaryotic protein stability. Here we define the minimal signal for the degradation of the S phase CDK inhibitor Sic1. Of 20 lysines scattered throughout Sic1, 6 N-terminal lysines serve as major ubiquitination sites. Sic1 lacking these lysines (K0N) is stable in vivo, but readdition of any one restores turnover. Nevertheless, ubiquitin chains attached at different N-terminal lysines specify degradation in vitro at markedly different rates. Moreover, although K0N can be ubiquitinated by SCFCdc4/Cdc34 in vitro in the absence (but not in the presence) of S-CDK, it is degraded slowly. Our results reveal that a single multiubiquitin chain can sustain a physiological turnover rate, but that chain position plays an unexpectedly significant role in the rate of proteasomal proteolysis.

Petroski, M.D, Deshaies, R.J. (2003). Redundant degrons ensure the rapid destruction of sic1 at the g1 /s transition of the budding yeast cell cycle. Cell Cycle 2:410-1.

Cope, G.A., Deshaies, R.J. (2003). COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases. Cell 114:663-71.

COP9 Signalosome (CSN) is a fascinating protein complex whose biochemical and physiological functions are only beginning to be understood. It is conserved throughout eukaryotes and is critical to the proper development of all multicellular organisms in which its function has been explored. Recent work suggests that CSN plays a key role in sustaining the activity of SCF and other cullin-based ubiquitin ligases, which may account for its essential roles in development. Here, we summarize what is known about CSN, and discuss hypotheses for how CSN promotes the activity of SCF ubiquitin ligases.

Lipford, J.R., Deshaies, R.J. (2003). Diverse roles for ubiquitin-dependent proteolysis in transcriptional activation. Nat. Cell Biol. 5:845-50.

A growing literature points to a fundamental role for the ubiquitin­proteasome degradation system (UPS) in transcription. Four recent publications add significant insight to our understanding of the connections between these processes. Each provides evidence that some aspect of the UPS can stimulate the activity of transcriptional activators. UPS might promote transcription by several mechanisms, and in some cases, even the final step of the UPS - proteolysis - might enhance activator function.

Sakamoto, K.M., Kim, K.B., Verma, R., Ransick, A., Stein, B., Crews, C.M., Deshaies, R.J. (2003). Development of protacs to target cancer-promoting proteins for ubiquitination and degradation. Mol. Cell Proteomics 2:1350-1358.

The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target proteins function. We previously demonstrated that a normally stable protein, MetAP-2, could be artificially targeted to an SCF ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (Proteolysis Targeting Chimeric Molecule). This Protac consisted of an SCFb-TRCPbinding phosphopeptide derived from IkBa linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer.

Ambroggio, X.I., Rees, D.C., and Deshaies, R.J. (2004). JAMM: A metalloprotease-like zinc site in the proteasome and signalosome. PLoS Biol. 2:0113-0119.

The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif in Rpn11 and Csn5 underlies isopeptidase activities intrinsic to the proteasome and signalosome, respectively. We show here that the archaebacterial protein AfJAMM possesses the key features of a zinc metalloprotease, yet with a distinct fold. The histidine and aspartic acid of the conserved EX(n)HS/THX(7)SXXD motif coordinate a zinc, whereas the glutamic acid hydrogen-bonds an aqua ligand. By analogy to the active site of thermolysin, we predict that the glutamic acid serves as an acid-base catalyst and the second serine stabilizes a tetrahedral intermediate. Mutagenesis of Csn5 confirms these residues are required for Nedd8 isopeptidase activity. The active site-like architecture specified by the JAMM motif motivates structure-based approaches to the study of JAMM domain proteins and the development of therapeutic proteasome and signalosome inhibitors.

Graumann J, Dunipace LA, Seol JH, McDonald WH, Yates JR 3rd, Wold BJ, Deshaies RJ. (2004). Applicability of Tandem Affinity Purification MudPIT to Pathway Proteomics in Yeast. Mol. Cell Proteomics 3:226-237.

A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification (TAP) tag. TAP-MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/Snf2p, the catalytic subunit of the Swi/Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP-MudPIT analysis of Rtt102p revealed subunits of both the Swi/Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process (i.e. "pathway proteomics") by systematic application of TAP-MudPIT.

Schneekloth JS Jr, Fonseca FN, Koldobskiy M, Mandal A, Deshaies R, Sakamoto K, Crews CM. (2004). Chemical genetic control of protein levels: Selective in vivo targeted degradation. J. Am. Chem. Soc. 126:3748-54.

Genetic loss of function analysis is a powerful method for the study of protein function. However, some cell biological questions are difficult to address using traditional genetic strategies often due to the lack of appropriate genetic model systems. Here, we present a general strategy for the design and syntheses of molecules capable of inducing the degradation of selected proteins in vivo via the ubiquitin-proteasome pathway. Western blot and fluorometric analyses indicated the loss of two different targets: green fluorescent protein (GFP) fused with FK506 binding protein (FKBP12) and GFP fused with the androgen receptor (AR), after treatment with PROteolysis TArgeting Chimeric moleculeS (PROTACS) incorporating a FKBP12 ligand and dihydrotestosterone, respectively. These are the first in vivo examples of direct small molecule-induced recruitment of target proteins to the proteasome for degradation upon addition to cultured cells. Moreover, PROTAC-mediated protein degradation offers a general strategy to create "chemical knockouts," thus opening new possibilities for the control of protein function.

Wertz IE, O'Rourke KM, Zhang Z, Dornan D, Arnott D, Deshaies RJ, Dixit VM. (2004). Human De-etiolated-1 regulates c-Jun by assembling a CUL4A ubiquitin ligase. Science 303:1371-4.

Arabidopsis thaliana De-etiolated-1 (AtDET1) is a highly conserved protein, with orthologs in vertebrate and invertebrate organisms. AtDET1 negatively regulates photomorphogenesis, but its biochemical mechanism and function in other species are unknown. We report that human DET1 (hDET1) promotes ubiquitination and degradation of the proto-oncogenic transcription factor c-Jun by assembling a multisubunit ubiquitin ligase containing DNA Damage Binding Protein-1 (DDB1), cullin 4A (CUL4A), Regulator of Cullins-1 (ROC1), and constitutively photomorphogenic-1. Ablation of any subunit by RNA interference stabilized c-Jun and increased c-Jun-activated transcription. These findings characterize a c-Jun ubiquitin ligase and define a specific function for hDET1 in mammalian cells.

 
Verma R, Oania R, Graumann J, Deshaies RJ. (2004). Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system. Cell 118:99-110.

Recruitment of ubiquitinated proteins to the 26S proteasome lies at the heart of the ubiquitin-proteasome system (UPS). Genetic studies suggest a role for the multiubiquitin chain binding proteins (MCBPs) Rad23 and Rpn10 in recruitment, but biochemical studies implicate the Rpt5 ATPase. We addressed this issue by analyzing degradation of the ubiquitinated Cdk inhibitor Sic1 (UbSic1) in vitro. Mutant rpn10Delta and rad23Delta proteasomes failed to bind or degrade UbSic1. Although Rpn10 or Rad23 restored UbSic1 recruitment to either mutant, rescue of degradation by Rad23 uncovered a requirement for the VWA domain of Rpn10. In vivo analyses confirmed that Rad23 and the multiubiquitin binding domain of Rpn10 contribute to Sic1 degradation. Turnover studies of multiple UPS substrates uncovered an unexpected degree of specificity in their requirements for MCBPs. We propose that recruitment of substrates to the proteasome by MCBPs provides an additional layer of substrate selectivity in the UPS.

Azzam R, Chen SL, Shou W, Mah AS, Alexandru G, Nasmyth K, Annan RS, Carr SA, Deshaies RJ. (2004). Phosphorylation by cyclin B-Cdk underlies release of mitotic exit activator Cdc14 from the nucleolus. Science 305:516-9.

Budding yeast protein phosphatase Cdc14 is sequestered in the nucleolus in an inactive state during interphase by the anchor protein Net1. Upon entry into anaphase, the Cdc14 early anaphase release (FEAR) network initiates dispersal of active Cdc14 throughout the cell. We report that the FEARnetwork promotes phosphorylation of Net1 by cyclin-dependent kinase (Cdk) complexed with cyclin B1 or cyclin B2. These phosphorylations appear to be required for FEAR and sustain the proper timing of late mitotic events. Thus, a regulatory circuit exists to ensure that the arbiter of the mitotic state, Cdk, sets in motion events that culminate in exit from mitosis.

Verma, R., Peter, N.R., Tochtrop, G.P., Sakamoto, K.M., D'Onofrio, M., Varadan, R., Fushman, D., Deshaies, R.J., and King, R.W. (2004). Inhibition of proteasome-dependent degradation by a small molecule that binds the ubiquitin chain. Science 306:117-120.

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys48-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system.

Petroski, M.D. and Deshaies, R.J. (2005). Function and regulation of cullin-RING ubiquitin ligases. Nat. Rev. Mol. Cell. Biol. 6:9-20.

Cullin-RING complexes comprise the largest known class of ubiquitin ligases. Owing to the great diversity of their substrate-receptor subunits, it is possible that there are hundreds of distinct cullin-RING ubiquitin ligases in eukaryotic cells, which establishes these enzymes as key mediators of post-translational protein regulation. In this review, we focus on the composition, regulation and function of cullin-RING ligases, and describe how these enzymes can be characterized by a set of general principles.

Mayor, T., Lipford, J.R., Graumann, J., Smith, G.T., Deshaies, R.J. (2005). Analysis of poly-ubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets. Mol. Cell Proteomics 4:741-751.

The poly-ubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1, or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10 cells. Poly-ubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H6-ubiquitin) were first enriched on a poly-ubiquitin binding protein affinity resin. This material was then denatured and subjected to immobilized metal ion affinity chromatography (IMAC) to retrieve H6-ubiquitin and proteins to which it may be covalently linked. Using this approach we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10) and identified 54 proteins that were uniquely recovered from rpn10 cells. These include two known targets of the UPS: the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild type and mutant cells has the resolving power to identify even an extremely inabundant transcriptional regulatory protein, and should be generally applicable to mapping enzyme-substrate networks in the UPS.

Lipford, J.R., Smith, G.T., Chi, Y., and Deshaies, R.J. (2005). A putative stimulatory role for activator turnover in gene expression. Nature 438:113-116.

The ubiquitin-proteasome system (UPS) promotes the destruction of target proteins by attaching to them a ubiquitin chain that is recognized by the 26S proteasome. The UPS influences most cellular processes, and its targets include transcriptional activators that are primary determinants of gene expression. Emerging evidence indicates that non-proteolytic functions of the UPS might stimulate transcriptional activity. Here we show that the proteolysis of some transcriptional activators by the UPS can stimulate their function. We focused on the role of UPS-dependent proteolysis in the function of inducible transcriptional activators in yeast, and found that inhibition of the proteasome reduced transcription of the targets of the activators Gcn4, Gal4 and Ino2/4. In addition, mutations in SCF(Cdc4), the ubiquitin ligase for Gcn4 (ref. 5), or mutations in ubiquitin that prevent degradation, also impaired the transcription of Gcn4 targets. These transcriptional defects were manifested despite the enhanced abundance of Gcn4 on cognate promoters. Proteasome inhibition also decreased the association of RNA polymerase II with Gcn4, Gal4 and Ino2/4 targets, as did mutations in SCF(Cdc4) for Gcn4 targets. Expression of a stable phospho-site mutant of Gcn4 (ref. 7) or disruption of the kinases that target Gcn4 for turnover alleviated the sensitivity of Gcn4 activity to defects in the UPS.

Mah, A.S., Elia, A.E.H., Devgan, G., Ptacek, J., Schutkowski, M., Snyder, M., Yaffe, M.B., and Deshaies, R.J. (2005). Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening. BMC Biochemistry 6:22.

Background: The mitotic exit network (MEN) is a group of proteins that form a signaling cascadethat is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified.

Results: Using a combination of peptide library selection, phosphorylation of opitmal peptide variants, and screening of a phosphosite array, we found that Dbf2-Mob1 preferentially phosphorylated serine over threonine and required an arginine three residues upstream of the phosphorylated serine in its substrate. This requirement for arginine in peptide substrates could not be substituted with the similarly charged lysine. This specificity determined for peptide
substrates was also evident in many of the proteins phosphorylated by Dbf2-Mob1 in a proteome
chip analysis.

Conclusion: We have determined by peptide library selection and phosphosite array screening that the protein kinase Dbf2-Mob1 preferentially phosphorylated substrates that contain an RXXS motif. A subsequent proteome microarray screen revealed proteins that can be phosphorylated by Dbf2-Mob1 in vitro. These proteins are enriched for RXXS motifs, and may include substrates that mediate the function of Dbf2-Mob1 in mitotic exit and cytokinesis. The relatively low degree of sequence restriction at the site of phosphorylation suggests that Dbf2 achieves specificity by docking its substrates at a site that is distinct from the phosphorylation site.

Mayor, T. and Deshaies, R.J. (2005). Two-step affinity purification of multiubiquitylated proteins from Saccharomyces cerevisiae. Methods Enzymol. 399:385-392.

In budding yeast and higher eukaryotic genomes, there are, respectively, 50 and up to 400 or more distinct genes that encode for ubiquitin-ligases, and approximately 15-90 genes that encode for ubiquitin isopeptidases (TM and RJD, Semple et al., 2003). This puts ubiquitylation on par with phosphorylation as the most common reversible posttranslational modifications in eukaryotic cells. A key challenge that has met with limited success to date is to identify the proteins that are the substrates for this large collection of enzymes. To begin to address this daunting challenge, we sought to identify ubiquitylated proteins that are potential substrates of the 26S proteasome. Here, we describe a two-step affinity purification protocol that uses a budding yeast strain that expresses hexahistidine-tagged ubiquitin. In the first step, native cell lysate was chromatographed on a UBA domain-containing matrix that binds preferentially to K48-linked multiubiquitin chains. Free ubiquitin and presumably monoubiquitylated proteins did not bind this column, whereas proteins that are potential substrates of the proteasome were enriched. In the second step, UBA domain-binding proteins were subjected to immobilized metal ion affinity chromatography (IMAC) under denaturing conditions on magnetic nickel beads, resulting in >3000-fold enrichment of ubiquitin conjugates relative to crude cell extract.

Verma, R. and Deshaies, R.J. (2005). Assaying degradation and deubiquitination of a ubiquitinated substrate by purified 26S proteasomes. Methods Enzymol. 398:391-399.

The 26S proteasome is a multisubunit complex that catalyzes ATP-dependent proteolysis of cellular proteins. It eliminates misfolded proteins, as well as labile regulatory proteins, thereby serving a central role in maintaining cellular homeostasis. The bulk of the known substrates of the 26S proteasome are earmarked for proteolysis by covalent modification with a multiubiquitin chain, which is recognized by specific receptors. Once targeted, the substrate is deubiquitinated and degraded by the 26S proteasome. This chapter describes assays that monitor ATP- and ubiquitin-dependent proteolysis of the S-Cdk inhibitor Sic1.

Petroski, M.D. and Deshaies, R.J. (2005). In vitro reconstitution of SCF substrate ubiquitination with purified proteins. Methods Enzymol. 398, 143-158.

The development of in vitro systems to monitor ubiquitin ligase activity with highly purified proteins has allowed for new insights into the mechanisms of protein ubiquitination to be uncovered. This chapter describes the methodologies employed to reconstitute ubiquitination of the budding yeast cyclin-dependent kinase inhibitor Sic1 by the evolutionarily conserved ubiquitin ligase SCF(Cdc4) and its ubiquitin-conjugating enzyme Cdc34. Based on our experience in reconstituting Sic1 ubiquitination, we suggest some parameters to consider that should be generally applicable to the study of different SCF complexes and other ubiquitin ligases.

Petroski, M.D. and Deshaies, R.J. (2005). Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34. Cell 123, 1107-1120.

Ubiquitin chains linked via lysine 48 (K48) of ubiquitin mediate recognition of ubiquitinated proteins by the proteasome. However, the mechanisms underlying polymerization of this targeting signal on a substrate are unknown. Here we dissect this process using the cyclin-dependent kinase inhibitor Sic1 and its ubiquitination by the cullin-RING ubiquitin ligase SCF(Cdc4) and the ubiquitin-conjugating enzyme Cdc34. We show that Sic1 ubiquitination can be separated into two steps: attachment of the first ubiquitin, which is rate limiting, followed by rapid elongation of a K48-linked ubiquitin chain. Mutation of an acidic loop conserved among Cdc34 orthologs has no effect on attachment of the first ubiquitin onto Sic1 but compromises the processivity and linkage specificity of ubiquitin-chain synthesis. We propose that the acidic loop favorably positions K48 of a substrate-linked ubiquitin to attack SCF bound Cdc34 approximately ubiquitin thioester and thereby enables processive synthesis of K48-linked ubiquitin chains by SCF-Cdc34.

Cope, G.A. and Deshaies, R.J. (2006). Targeted silencing of JabI /Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels. BMC Biochemistry 7, 1.

Background: SCF ubiquitin ligases target numerous proteins for ubiquitin dependent proteolysis, including p27 and cyclin E. SCF and other cullin-RING ligases (CRLs) are regulated by the ubiquitin-like protein Nedd8 that covalently modifies the cullin subunit. The removal of Nedd8 is catalyzed by the Jab1/MPN domain metalloenzyme (JAMM) motif within the Csn5 subunit of the Cop9 Signalosome.

Results: Here, we conditionally knock down Csn5 expression in HEK293 human cells using a doxycycline-inducible shRNA system. Cullin levels were not altered in CSN-deficient human cells, but the levels of multiple F-box proteins were decreased. Molecular analysis indicates that this decrease was due to increased Cul1- and proteasome-dependent turnover. Diminished F-box levels resulted in reduced SCF activity, as evidenced by accumulation of two substrates of the F-box protein Fbw7, cyclin E and c-myc, in Csn5-depleted cells.

Conclusion: We propose that deneddylation of Cul1 is required to sustain optimal activity of SCF ubiquitin ligases by repressing 'autoubiquitination' of F-box proteins within SCF complexes, thereby rescuing them from premature degradation.

Sharon, M., Taverner, T., Ambroggio, X.I., Deshaies, R.J., and Robinson, C.V. (2006). Structural organization of the 19S proteasome lid: Insights from MS of intact complexes. PLoS Biol 4, e267.

The 26S proteasome contains a 19S regulatory particle that selects and unfolds ubiquitinated substrates for degradation in the 20S catalytic particle. To date there are no high-resolution structures of the 19S assembly, nor of the lid or base subcomplexes that constitute the 19S. Mass spectra of the intact lid complex from Saccharomyces cerevisiae show that eight of the nine subunits are present stoichiometrically and that a stable tetrameric subcomplex forms in solution. Application of tandem mass spectrometry to the intact lid complex reveals the subunit architecture, while the coupling of a cross-linking approach identifies further interaction partners. Taking together our results with previous analyses we are able to construct a comprehensive interaction map. In summary, our findings allow us to identify a scaffold for the assembly of the particle and to propose a regulatory mechanism that prevents exposure of the active site until assembly is complete. More generally, the results highlight the potential of mass spectrometry to add crucial insight into the structural organization of an endogenous, wild-type complex.

Petroski, M.D., Kleiger, G., and Deshaies, R.J. (2006). Evaluation of a diffusion-driven mechanism for substrate ubiquitination by the SCF-Cdc34 ubiquitin ligase complex. Mol Cell 24, 523-534.

Release of ubiquitin-charged Cdc34 from the SCF ubiquitin ligase followed by diffusion-driven collision with substrate has been proposed to underlie ubiquitination of the canonical SCF substrate Sic1. Cdc34 F72V, reported to be defective in dissociation from SCF, served as key validation. Here, we test predictions of this "hit-and-run" hypothesis. We find that Cdc34 F72V is generally defective in SCF-mediated activation but, contrary to expectation, does not compete with wild-type Cdc34 in vitro or in vivo and can fulfill the physiological role of Cdc34 with only moderate delay in Sic1 turnover. Whereas a hit-and-run mechanism might explain how Cdc34 can transfer ubiquitin to the ends of growing ubiquitin chains on SCF-bound substrates, molecular modeling suggests that an E2 docked to SCF can do so without dissociating. We propose that interactions between Cdc34 approximately Ub and SCF directly activate ubiquitin transfer within a substrate-SCF-Cdc34 approximately Ub ternary complex.

Ghaboosi, N. and Deshaies, R.J. (2007). A conditional yeast e1 mutant blocks the ubiquitin-proteasome pathway and reveals a role for ubiquitin conjugates in targeting rad23 to the proteasome. Mol. Biol. Cell 18, 1953-63.

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin-proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo.

Mayor, T., Graumann, J., Bryan, J., Maccoss, M.M., and Deshaies, R.J. (2007). Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway. Mol. Cell. Proteomics 6, 1885-1895.

The ubiquitin-proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally-perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q-values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10) or only its UIM poly-ubiquitin binding domain (uim). Twenty seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10 cells, whereas only one-fifth as many accumulated in uim cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates, but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS.

Haririnia, A., Verma, R., Purohit, N., Twarog, M.Z., Deshaies, R.J., Bolon, D., and Fushman, D. (2008). Mutations in the hydrophobic core of ubiquitin differentially affect its recognition by receptor proteins. J. Mol. Biol. 375, 979-996.

Ubiquitin (Ub) is one of the most highly conserved signaling proteins in eukaryotes. In carrying out its myriad functions, Ub conjugated to substrate proteins interacts with dozens of receptor proteins that link the Ub signal to various biological outcomes. Here we report mutations in conserved residues of Ub's hydrophobic core that have surprisingly potent and specific effects on molecular recognition. Mutant Ubs bind tightly to the Ub-associated domain of the receptor proteins Rad23 and hHR23A but fail to bind the Ub-interacting motif present in the receptors Rpn10 and S5a. Moreover, chains assembled on target substrates with mutant Ubs are unable to support substrate degradation by the proteasome in vitro or sustain viability of yeast cells. The mutations have relatively little effect on Ub's overall structure but reduce its rigidity and cause a slight displacement of the C-terminal beta-sheet, thereby compromising association with Ub-interacting motif but not with Ub-associated domains. These studies emphasize an unexpected role for Ub's core in molecular recognition and suggest that the diversity of protein-protein interactions in which Ub engages placed enormous constraints on its evolvability.

Goh, A.M., Walters, J.K., Elsasser, S., Verma, R., Deshaies, R.J., Finley, D., and Howley, P.M. (2008). Components of the ubiquitin-proteasome pathway compete for surfaces on Rad32 family proteins. BMC Biochem. 9, 4.

Background: The delivery of ubiquitinated proteins to the proteasome for degradation is a key step in the regulation of the ubiquitin-proteasome pathway, yet the mechanisms underlying this step are not understood in detail. The Rad23 family of proteins is known to bind ubiquitinated proteins through its two ubiquitin-associated (UBA) domains, and may participate in the delivery of ubiquitinated proteins to the proteasome through docking via the Rad23 ubiquitin-like (UBL) domain.

Results: In this study, we investigate how the interaction between the UBL and UBA domains may modulate ubiquitin recognition and the delivery of ubiquitinated proteins to the proteasome by autoinhibition. We have explored a competitive binding model using specific mutations in the UBL domain. Disrupting the intramolecular UBL-UBA domain interactions in HHR23A indeed potentiates ubiquitin-binding. Additionally, the analogous surface on the Rad23 UBL domain overlaps with that required for interaction with both proteasomes and the ubiquitin ligase Ufd2. We have found that mutation of residues on this surface affects the ability of Rad23 to deliver ubiquitinated proteins to the proteasome.

Conclusion: We conclude that the competition of ubiquitin-proteasome pathway components for surfaces on Rad23 is important for the role of the Rad23 family proteins in proteasomal targeting.

Alexandru, G., Graumann, J., Smith, G.T., Kolawa, N.J., Fang, R., and Deshaies, R.J. (2008). UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIFIa turnover. Cell 134, 804-816.

p97 is an ATP-dependent chaperone that plays an important role in endoplasmicreticulum associated degradation, but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed 'network proteomics' to show that p97 assembles with all of the thirteen mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1(alpha) (HIF1[alpha]). Depletion of p97 leads to accumulation of endogenous HIF1(alpha) and increased expression of a HIF1(alpha) target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.

Saha, A. and Deshaies, R. (2008). Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Mol Cell 32, 21-31.

Conjugation of ubiquitin-like protein Nedd8 to cullin (i.e. neddylation) is essential for the function of cullin-RING ubiquitin ligases (CRLs). Here we show that neddylation stimulates recruitment of ubiquitin-conjugating enzyme (E2) esterified with ubiquitin (E2~Ub), helps bridge the ~50 Å gap between E2 and substrate bound to SCF to enable their reaction, and facilitates formation of amide bonds in E2's active site. Together, these effects potently stimulate transfer of ubiquitin to substrate. We propose that the initiator ubiquitin spans the gap, and the impact of neddylation on transfer of subsequent ubiquitins by the E2 Cdc34 arises from improved E2 recruitment and enhanced amide bond formation in the E2 active site. The combined effects of neddylation greatly enhance the probability that a substrate molecule acquires >4 ubiquitins in a single encounter with a CRL. The surprisingly diverse effects of Nedd8 conjugation underscore the complexity of CRL regulation and suggest that modification of other ubiquitin ligases with ubiquitin or ubiquitin-like proteins may likewise have major functional consequences.

Rodriguez-Gonzalez A, Cyrus K, Salcius M, Kim K, Crews CM, Deshaies RJ, Sakamoto KM. (2008). Targeting steroid hormone receptors for ubiquitination and degradation in breast and prostate cancer. Oncogene 27, 7201-7211.

Proteolysis targeting chimeric molecules (Protacs) target proteins for destruction by exploiting the ubiquitin-dependent proteolytic system of eukaryotic cells. We designed two Protacs that contain the peptide 'degron' from hypoxia-inducible factor-1alpha, which binds to the Von-Hippel-Lindau (VHL) E3 ubiquitin ligase complex, linked to either dihydroxytestosterone that targets the androgen receptor (AR; Protac-A), or linked to estradiol (E2) that targets the estrogen receptor-alpha (ERalpha; Protac-B). We hypothesized that these Protacs would recruit hormone receptors to the VHL E3 ligase complex, resulting in the degradation of receptors, and decreased proliferation of hormone-dependent cell lines. Treatment of estrogen-dependent breast cancer cells with Protac-B induced the degradation of ERalpha in a proteasome-dependent manner. Protac-B inhibited the proliferation of ERalpha-dependent breast cancer cells by inducing G(1) arrest, inhibition of retinoblastoma phosphorylation and decreasing expression of cyclin D1, progesterone receptors A and B. Protac-B treatment did not affect the proliferation of estrogen-independent breast cancer cells that lacked ERalpha expression. Similarly, Protac-A treatment of androgen-dependent prostate cancer cells induced G(1) arrest but did not affect cells that do not express AR. Our results suggest that Protacs specifically inhibit the proliferation of hormone-dependent breast and prostate cancer cells through degradation of the ERalpha and AR, respectively.

Mohl DA, Huddleston MJ, Collingwood TS, Annan RS, Deshaies RJ. (2009). Dbf2-Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis. J Cell Biol 184, 527-539.

Exit from mitosis is characterized by a precipitous decline in cyclin-dependent kinase (Cdk) activity, dissolution of mitotic structures, and cytokinesis. In Saccharomyces cerevisiae, mitotic exit is driven by a protein phosphatase, Cdc14, which is in part responsible for counteracting Cdk activity. Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown. In this study, we show that a MEN component, protein kinase Dbf2-Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity. Our results define a mechanism by which the MEN promotes exit from mitosis.

Machida, K., Tsukamoto, H., Mkrtchyan, H., Duan, L., Dynnyk, A., Liu, H.M., Asahina, K., Govindarajan, S., Ray, R., Ou, J.H., Seki, E., Deshaies, R., Miyake, K., Lai, M.M. (2009). Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog. Proc. Nat. Acad. Sci. USA 106, 1548-1553.

Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by hepatitis C virus (HCV). However, the molecular mechanism of this synergy remains unclear. Here, we provide the first evidence that Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor formation by alcohol-induced endotoxemia. We also identify Nanog, the stem/progenitor cell marker, as a novel downstream gene up-regulated by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive LPS injection, but concomitant transduction of Nanog shorthairpin RNA abrogates this outcome. Taken together, our study demonstrates a TLR4-dependent mechanism of synergistic liver disease by HCV and alcohol and an obligatory role for Nanog, a TLR4 downstream gene, in HCV-induced liver oncogenesis enhanced by alcohol.

Oh, Y.M., Kwon, Y.E., Kim, J.M., Bae, S.J., Lee, B.K., Yoo, S.J., Chung, C.H., Deshaies R.J., Seol, J.H. (2009) Chfr is linked to tumour metastasis through the downregulation of HDAC1. Nat. Cell Biol. 11, 295-302.

Chfr is a ubiquitin ligase that functions in the mitotic checkpoint by delaying entry into metaphase in response to mitotic stress1,2. It has been suggested that Chfr is a tumour suppressor as Chfr is frequently silenced in human cancers3. To better understand how Chfr activity relates to cell-cycle progression and tumorigenesis, we sought to identify Chfr-interacting proteins using affinity purification combined with mass spectrometry. Histone deacetylase 1 (HDAC1), which represses transcription by deacetylating histones, was newly isolated as a Chfr-interacting protein. Chfr binds and downregulates HDAC1 by inducing its polyubiquitylation, both in vitro and in vivo. Ectopic expression of Chfr in cancer cells that normally do not express it results in downregulation of HDAC1, leading to upregulation of the Cdk inhibitor p21CIP1/WAF1 and the metastasis suppressors KAI1 and E?cadherin. Coincident with these changes, cells arrest in the G1 phase of the cell cycle and become less invasive. Collectively, our data suggest that Chfr functions as a tumour suppressor by regulating HDAC1.

Deshaies, R.J. (2009). Drug discovery: Fresh target for cancer therapy (News & Views). Nature 458, 709-710.

The Byzantine system for degrading proteins inside cells is already the target of a successful anticancer drug. A compound that inhibits another part of this system also shows promise in models of cancer in mice.

Deshaies, R.J. and Joazeiro, C.A.P. (2009). RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399-434.

E3 ligases confer specificity to ubiquitination by recognizing target substrates and mediating transfer of ubiquitin from an E2 ubiquitinconjugating enzyme to substrate. The activity of most E3s is specified by a RING domain, which binds to an E2<ubiquitin thioester and activates discharge of its ubiquitin cargo. E2-E3 complexes can either monoubiquitinate a substrate lysine or synthesize polyubiquitin chains assembled via different lysine residues of ubiquitin. These modifications can have diverse effects on the substrate, ranging from proteasome-dependent proteolysis to modulation of protein function, structure, assembly, and/or localization. Not surprisingly, RING E3-mediated ubiquitination can be regulated in a number of ways.

RING-based E3s are specified by over 600 human genes, surpassing the 518 protein kinase genes. Accordingly, RING E3s have been linked to the control of many cellular processes and to multiple human diseases. Despite their critical importance, our knowledge of the physiological partners, biological functions, substrates, and mechanism of action for most RING E3s remains at a rudimentary stage.

Kleiger, G., Saha, A., Lewis, S., Kuhlman, B., and Deshaies, R.J. (2009). Rapid E2-E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. Cell 139, 957-968.

Degradation by the ubiquitin-proteasome system requires assembly of a polyubiquitin chain upon substrate. However, the structural and mechanistic features that enable template-independent processive chain synthesis are unknown. We show that chain assembly by ubiquitin ligase SCF and ubiquitin-conjugating enzyme Cdc34 is facilitated by the unusual nature of Cdc34-SCF transactions: Cdc34 binds SCF with nanomolar affinity, nevertheless the complex is extremely dynamic. These properties are enabled by rapid association driven by electrostatic interactions between the acidic tail of Cdc34 and a basic 'canyon' in the Cul1 subunit of SCF. Ab initio docking between Cdc34 and Cul1 predicts intimate contact between the tail and the basic canyon, an arrangement confirmed by cross-linking and kinetic analysis of mutants. Basic canyon residues are conserved in both Cul1 paralogs and orthologs, suggesting that the same mechanism underlies processivity for all cullin-RING ubiquitin ligases. We discuss different strategies by which processive ubiquitin chain synthesis may be achieved.

Kleiger G, Hao B, Mohl DA, Deshaies RJ. (2009). The acidic tail of the CDC34 ubiquitin-conjugating enzyme functions in both binding to and catalysis with ubiquitin ligase SCFCDC4. J. Biol. Chem. 284, 36012-36023.

Ubiquitin ligases, together with their cognate ubiquitin conjugating enzymes, are responsible for the ubiquitylation of proteins, a process that regulates a myriad of eukaryotic cellular functions. The first cullin-RING ligase (CRL) discovered, yeast SCF(Cdc4), functions with the conjugating enzyme Cdc34 to regulate the cell cycle. Cdc34 orthologs are notable for their highly acidic C-terminal extension. Here we confirm that the Cdc34 acidic C-terminal tail has a role in Cdc34 binding to SCF(Cdc4) and makes a major contribution to the sub-micromolar Km of Cdc34 for SCF(Cdc4). Moreover, we demonstrate that a key functional property of the tail is its acidity. Our analysis also uncovers an unexpected new function for the acidic tail in promoting catalysis. We demonstrate that SCF is functional when Cdc34 is fused to Cul1s C-terminus and that this fusion retains partial function even when the acidic tail has been deleted. The Cdc34-SCF fusion proteins that lack the acidic tail must interact in a fundamentally different manner than un-fused SCF and wild type Cdc34, demonstrating that distinct mechanisms of E2 recruitment to E3 - as is seen in nature - can sustain substrate ubiquitylation. Finally, a search of the yeast proteome uncovered scores of proteins containing highly acidic stretches of amino acids, hinting that electrostatic interactions may be a common mechanism for facilitating protein assembly.

Pierce, N., Kleiger, G., Shan, S., and Deshaies, R.J. (2009). Detection of sequential ubiquitination on a millisecond time-scale. Nature 462, 615-619.

The pathway by which ubiquitin chains are generated on substrate via a cascade of enzymes consisting of an E1, E2 and E3 remains unclear. Multiple distinct models involving chain assembly on E2 or substrate have been proposed. However, the speed and complexity of the reaction have precluded direct experimental tests to distinguish between potential pathways. Here we introduce new theoretical and experimental methodologies to address both limitations. A quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3s SCFCdc4 and SCFb-TrCP work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Our results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and illuminate the quantitative parameters that underlie the rate and pattern of ubiquitin chain assembly.

Radhakrishnan, S.K., Lee, C.S., Young, P., Beskow, A., Chan, J.Y., and Deshaies, R.J. (2010). Transcription factor Nrf1 mediates the proteasome recovery pathway after proteasome inhibition in mammalian cells. Mol. Cell 38, 17-28.

In Saccharomyces cerevisiae, chemical or genetic inhibition of proteasome activity induces new proteasome synthesis promoted by the transcription factor RPN4. This ensures that proteasome activity is matched to demand. This transcriptional feedback loop is conserved in mammals, but its molecular basis is not understood. Here we report that Nuclear factor erythroid derived 2-related factor 1 (Nrf1), a transcription factor of the cap 'n' collar basic leucine zipper family, but not the related Nrf2, is necessary for induced proteasome gene transcription in mouse embryonic fibroblasts (MEFs). Promoter-reporter assays revealed the importance of antioxidant response elements in Nrf1-mediated upregulation of proteasome subunit genes. Nrf1-/- MEFs were impaired in the recovery of proteasome activity after transient treatment with the covalent proteasome inhibitor YU101 and knockdown of Nrf1 in human cancer cells enhanced cell killing by YU101. Taken together, our results suggest that Nrf1-mediated proteasome homeostasis could be an attractive target for therapeutic intervention in cancer.

Aghajan, M., Jonai, N., Flick, K., Fu, F., Luo, M., Cai, X., Ouni, I., Pierce, N., Tang, X., Lomenick, B., Damoiseaux, R., Hao, R., Del Moral, P.M., Verma, R., Li, Y., Li, C., Houk, K.N., Jung, M.E., Zheng, N., Huang, L., Deshaies, R.J., Kaiser, P., and Huang, J. (2010). Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase. Nat. Biotechnol. 28, 738-742.

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.

Collins, G.A., Gomez, T.A., Deshaies, R.J., and Tansey, W.P. (2010). Combined chemical and genetic approach to inhibit proteolysis by the proteasome. Yeast 27, 965-974.

Regulated protein destruction by the proteasome is crucial for the maintenance of normal cellular homeostasis. Much of our understanding of proteasome function stems from the use of drugs that inhibit its activity. Curiously, despite the importance of proteasomal proteolysis, previous studies have found that proliferation of the yeast Saccharomyces cerevisiae is relatively resistant to the effects of proteasome inhibitors such as MG132, even in the presence of mutations that increase inhibitor levels in cells. We reasoned that part of the resistance of S. cerevisiae to proteasome inhibitors stems from the fact that most proteasome inhibitors preferentially target the chymotryptic activity of the proteasome, and that the caspase-like and tryptic sites within the 20S core could compensate for proteasome function under these conditions. To test this hypothesis, we generated a strain of yeast in which the gene encoding the drug efflux pump Pdr5 is deleted, and the tryptic and caspase-like proteasome activities are inactivated by mutation. We find that this strain has dramatically increased sensitivity to the proteasome inhibitor MG132. Under these conditions, treatment of yeast with MG132 blocks progression through the cell cycle, increases the accumulation of polyubiquitylated proteins and decreases the ability to induce transcription of certain genes. These results highlight the contribution of the caspase-like and tryptic activities of the proteasome to its function, and provide a strategy to potently block proteasomal proteolysis in yeast that has practical applications. 

Tyrell, A., Flick, K., Kleiger, G., Zhang, H., Deshaies, R.J., and Kaiser, P. (2010). Physiologically relevant and portable tandem ubiquitin-binding domain stabilizes polyubiquitylated proteins. Proc. Natl. Acad. Sci. USA 107, 19796-19801.

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.

Deshaies, R.J., Emberley, E.D., and Saha, A. (2010). Control of cullin-ring ubiquitin ligase activity by Nedd8. Subcell. Biochem., 54, 41-56.

The Culllin-RING ubiquitin ligase (CRL) family, which may number as many as 350 different enzymes, has an enormous impact on cellular regulation. CRL enzymes regulate cell biology by conjugating ubiquitin onto target proteins that are involved in a multitude of processes. In most cases this leads to degradation of the target, but in some cases CRL-dependent ubiquitination acts as a switch to activate or repress target function. The ubiquitin ligase activity of CRLs is controlled by cycles of attachment and removal of the ubiquitin-like protein Nedd8. Conjugation of Nedd8 onto the cullin subunit of CRLs promotes assembly of an intact CRL complex and switches on ubiquitin ligase activity. Conversely, removal of Nedd8 switches off ubiquitin ligase activity and initiates CRL disassembly. Continuous maintenance of CRL function in vivo requires the activities of both the Nedd8-conjugating and deconjugating enzymes, pointing to a critical role of complex dynamics in CRL function. Here, we review how the Nedd8 cycle controls CRL activity and how perturbations of this cycle can lead to disease.

Verma, R., Oania, R., Fang, R., Smith, G.T., and Deshaies, R.J. (2011). Cdc48/p97 Mediates UV-Dependent Turnover of RNA Pol II. Mol Cell. 41, 82-92.

Cdc48/p97 is an essential ATPase whose role in targeting substrates to the ubiquitin-proteasome system (UPS) remains unclear. Existing models posit that Cdc48 acts upstream of UPS receptors. To address this hypothesis, we examined the association of ubiquitin (Ub) conjugates with 26S proteasomes. Unexpectedly, proteasomes isolated from cdc48 mutants contain high levels of Ub conjugates, and mass spectrometry identified numerous nonproteasomal proteins, including Rpb1, the largest subunit of RNA Pol II. UV-induced turnover of Rpb1 depends upon Cdc48-Ufd1-Npl4, Ubx4, and the uncharacterized adaptor Ubx5. Ubiquitinated Rpb1, proteasomes, and Cdc48 accumulate on chromatin in UV-treated wild-type cells, and the former two accumulate to higher levels in mutant cells, suggesting that degradation of Rpb1 is facilitated by Cdc48 at sites of stalled transcription. These data reveal an intimate coupling of function between proteasomes and Cdc48 that we suggest is necessary to sustain processive degradation of unstable subunits of some macromolecular protein complexes.

Chou, T.F. and Deshaies, R.J. (2011). Quantitative cell-based protein degradation assays to identify and classify drugs that target the ubiquitin-proteasome system. J. Biol. Chem. 286, 16546-16554.

We have generated a set of dual reporter human cell lines and devised a chase protocol to quantify proteasomal degradation of an ubiquitin fusion degradation (UFD) substrate, an ubiquitin ligase CRL2VHL substrate, and an ubiquitin independent substrate. Well characterized inhibitors that target different aspects of the ubiquitin proteasome system (UPS) can be distinguished by their distinctive patterns of substrate stabilization, enabling assignment of test compounds as inhibitors of either the proteasome, ubiquitin chain formation or perception, cullin RING ubiquitin ligase (CRL) activity, or the UFD/p97 pathway. We confirmed that degradation of the UFD but not the CRL2VHL or ubiquitin independent substrates depends on p97 activity. We optimized our suite of assays to establish conditions suitable for high-throughput screening (HTS) and then validated their performance by screening against 160 cell-permeable protein kinase inhibitors. This screen identified Syk Inhibitor III as an irreversible p97/VCP inhibitor (IC50 1.7 uM) that acts through cysteine 522 within the D2 ATPase domain. Our work establishes an HTS compatible pipeline for identification and classification of small molecules, cDNAs, or siRNAs that target components of the UPS.

Saha, A., Lewis, S., Kleiger, G., Kuhlman, B., and Deshaies, R.J. (2011). Essential role for ubiquitin-ubiquitin-conjugating enzyme interaction in ubiquitin discharge from Cdc34 to substrate. Mol. Cell 42, 75-83.

During ubiquitin conjugation, the thioester bond that links "donor" ubiquitin to ubiquitin-conjugating enzyme (E2) undergoes nucleophilic attack by the ?-amino group of an acceptor lysine, resulting in formation of an isopeptide bond. Models of ubiquitination have envisioned the donor ubiquitin to be a passive participant in this process. However, we show here that the I44A mutation in ubiquitin profoundly inhibits its ability to serve as a donor for ubiquitin chain initiation or elongation, but can be rescued by computationally predicted compensatory mutations in the E2 Cdc34. The donor defect of ubiquitin-I44A can be partially suppressed either by using a low pKa amine (hydroxylamine) as the acceptor or by performing reactions at higher pH, suggesting that the discharge defect arises in part due to inefficient deprotonation of the acceptor lysine. We propose that interaction between Cdc34 and the donor ubiquitin organizes the active site to promote efficient ubiquitination of substrate.

Lee, J.E., Sweredoski, M.J., Graham, R.L., Kolawa, N.J., Smith, G.T., Hess, S., and Deshaies, R.J. (2010). The steady-state repertoire of human SCF Ubiquitin ligase complexes does not require ongoing Nedd8 conjugation. Mol Cell Proteomics 10, 6460 1-9.

The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin- like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1- RING catalytic core of SCF and the cellular repertoire of FBPs. To test this hypothesis, we determined the cellular composition of SCF complexes and evaluated the impact of Nedd8 conjugation on this steady-state. At least 42 FBPs assembled with Cul1 in HEK 293 cells, and the levels of Cul1-bound FBPs varied by over two orders of magnitude. Unexpectedly, quantitative mass spectrometry revealed that blockade of Nedd8 conjugation led to a modest increase, rather than a decrease, in the overall level of most SCF complexes. We suggest that multiple mechanisms including FBP dissociation and turnover cooperate to maintain the cellular pool of SCF ubiquitin ligases.

Chou, T.F., Brown, S.J., Minond, D., Nordin, B.E., Li, K., Jones, A.C., Chase, P., Porubsky, P.R., Stoltz, B.M., Schoenen, F.J., Patricelli, M.P., Hodder, P., Rosen, H., and Deshaies, R.J. (2011) Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways. Proc. Natl. Acad. Sci. U. S. A. 108, 4834-4839.

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N(2),N(4)-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.

 Gomez, T.A., Kolawa, N., Gee, M., Sweredoski, M.J., and Deshaies, R.J. (2011) Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1. BMC Biol. 9, 33.

BACKGROUND: The proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin (Ub) chain appendage. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn10, Rpn13, or Rpt5, or are shuttled to the proteasome by Rad23, Dsk2, or Ddi1. The latter proteins share an Ub association domain (UBA) for binding poly-Ub chains and an Ub-like-domain (UBL) for binding to the proteasome. It has been proposed that shuttling receptors dock on the proteasome via Rpn1, but the precise nature of the docking site remains poorly defined.

RESULTS: To shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in Rpn1 that disrupt its binding to UBA-UBL proteins. Here we demonstrate that delivery of Ub conjugates and docking of Ddi1 (and to a lesser extent Dsk2) to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved D517 residue of Rpn1. Moreover, degradation of the Ddi1-dependent proteasome substrate, Ufo1, is blocked in rpn1-D517A yeast cells. By contrast, Rad23 recruitment to the proteasome is not affected by rpn1-D517A.

CONCLUSIONS: These studies provide insight into the mechanism by which the UBA-UBL protein Ddi1 is recruited to the proteasome to enable Ub-dependent degradation of its ligands. Our studies suggest that different UBA-UBL proteins are recruited to the proteasome by distinct mechanisms.

Rabut, G., Le Dez, G., Verma, R., Makhnevych, T., Knebel, A., Kurz, T., Boone, C., Deshaies, R.J. and Peter, M. (2011). The TFIIH Subunit Tfb3 Regulates Cullin Neddylation. Mol Cell. 43, 488-495.

Cullin proteins are scaffolds for the assembly of multisubunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING domain of Hrt1 nor required the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient neddylation involves the RING domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 and the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING domain protein Tfb3 controls activation of a subset of cullins.

Sohn CH, Lee JE, Sweredoski MJ, Graham RL, Smith GT, Hess S, Czerwieniec G, Loo JA, Deshaies RJ, Beauchamp JL. (2012). Click chemistry facilitates formation of reporter ions and simplified synthesis of amine-reactive multiplexed isobaric tags for protein quantification. J Am Chem Soc. 134, 2672-80.

We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d(5)-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.

Besten Wd, Verma R, Kleiger G, Oania RS, and Deshaies, RJ. (2012). NEDD8 links cullin-RING ubiquitin ligase function to the p97 pathway. Nat Struct Mol Biol. 19, 511-516.

The AAA+ ATPase p97 and its UBA-UBX cofactors are thought to extract ubiquitinated proteins from membranes or protein complexes as a prelude to their degradation. However, for many cofactors ubiquitinated targets have not yet been identified, leaving their biological function unclear. Previous analysis has linked the p97 pathway to cullin-RING ubiquitin ligases (CRLs); here we demonstrate that the human p97 cofactor UBXD7 mediates the p97-CRL interaction through its conserved ubiquitin-interacting motif (UIM). UBXD7 and its yeast ortholog, Ubx5, associate only with the active, NEDD8- or Rub1-modified form of cullins. Disruption of the Ubx5 UIM results in a loss of CRL binding and consequently impedes degradation of a Cul3 substrate. These results uncover an unexpected and conserved role for NEDD8 in linking CRL ubiquitin ligase function to the p97 pathway.