The self-destructive personality of a cell cycle in transition.

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.

COP1 patrols the night beat.

Light regulates the behaviour of many organisms. New data indicate that the greening of plants is facilitated 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.

Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly.

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-triggered assembly 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.

Make it or break it: the role of ubiquitin-dependent proteolysis in cellular regulation.

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.

A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.

We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation.

SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum.

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.

SEC11 is required for signal peptide processing and yeast cell growth.

Among the collection of temperature-sensitive secretion mutants of Saccharomyces cerevisiae, sec11 mutant cells are uniquely defective in signal peptide processing of at least two different secretory proteins. At 37 degrees C, the restrictive growth temperature, sec11 cells accumulate core-glycosylated forms of invertase and acid phosphatase, each retaining an intact signal peptide. In contrast, other sec mutant strains in which transport of core-glycosylated molecules from the endoplasmic reticulum is blocked show no defect in signal peptide cleavage. A DNA fragment that complements the sec11-7 mutation has been cloned. Genetic analysis indicates that the complementing clone contains the authentic SEC11 gene, and that a null mutation at the SEC11 locus is lethal. The DNA sequence of SEC11 predicts a basic protein (estimated pI of 9.5) of 167 amino acids including an NH2-terminal hydrophobic region that may function as a signal and/or membrane anchor domain. One potential N-glycosylation site is found in the 18.8-kD (Sec 11p) predicted protein. The mass of the SEC11 protein is very close to that found for two of the subunits of the canine and hen oviduct signal peptidases. Furthermore, the chromatographic behavior of the hen oviduct enzyme indicates an overall basic charge comparable to the predicted pI of the Sec11p.

Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast.

Genes that function in translocation of secretory protein precursors into the ER have been identified by a genetic selection for mutant yeast cells that fail to translocate a signal peptide-cytosolic enzyme hybrid protein. The new mutants, sec62 and sec63, are thermosensitive for growth and accumulate a variety of soluble secretory and vacuolar precursors whose electrophoretic mobilities coincide with those of the corresponding in vitro translated polypeptides. Proteolytic sensitivity of precursor molecules in extracts of mutant cells confirms that polypeptide translocation is blocked. Some form of interaction among the SEC61 (Deshaies, R. J., and R. Schekman. 1987. J. Cell Biol. 105:633-645), SEC62 and SEC63 gene products is suggested by the observation that haploid cells containing any pair of the mutations are inviable at 24 degrees C and show a marked enhancement of the translocation defect. The translocation defects of two mutants (sec62 and sec63) have been reproduced in vitro. sec63 microsomes display low and thermolabile translocation activity for prepro-alpha-factor (pp alpha F) synthesized with a cytosol fraction from wild type yeast. These gene products may constitute part of the polypeptide recognition or translocation apparatus of the ER membrane. Pulse-chase analysis of the translocation-defective mutants demonstrates that insertion of pp alpha F into the ER can proceed posttranslationally.

How proteolysis drives the cell cycle.

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.

Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase.

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.

Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in mediating auxin response.

The COP9 signalosome is an evolutionary conserved multiprotein complex of unknown function that acts as a negative regulator of photomorphogenic seedling development in Arabidopsis. Here, 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 vivo and that the COP9 signalosome was required for efficient degradation of PSIAA6, a candidate substrate of SCFTIR1. Thus, the COP9 signalosome may play an important role in mediating E3 ubiquitin ligase-mediated responses.

Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome.

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.

Phosphorylation and proteolysis: partners in the regulation of cell division in budding yeast.

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.

Genetic dissection of the early stages of protein secretion in yeast.

The earliest events in the export of secretory proteins from eukaryotic cells are their insertion into and transport across the membrane of the endoplasmic reticulum, followed by signal peptide cleavage and transfer of core oligosaccharides to specific asparagine residues. Much has been learned through reconstitution of these processes in vitro using cell-free extracts prepared from mammalian and yeast cells. Now, a combination of genetic, molecular and biochemical approaches are being employed to study the early stages of protein secretion in the yeast Saccharomyces cerevisiae.

Structural and functional dissection of Sec62p, a membrane-bound component of the yeast endoplasmic reticulum protein import machinery.

SEC62 is required for the import of secretory protein precursors into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae. The DNA sequence of SEC62 predicts a 32-kDa polypeptide with two potential membrane-spanning segments. Two antisera directed against different portions of the SEC62 coding region specifically detected a 30-kDa polypeptide in cell extracts. A combination of subcellular fractionation, detergent and alkali extraction, and indirect immunofluorescence studies indicated that Sec62p is intimately associated with the ER membrane. Protease digestion of intact microsomes and analysis of the oligosaccharide content of a set of Sec62p-invertase hybrid proteins suggested that Sec62p spans the ER membrane twice, displaying hydrophilic amino- and carboxy-terminal domains towards the cytosol. Sec62p-invertase hybrid proteins that lack the Sec62p C terminus failed to complement the sec62-l mutation and dramatically inhibited the growth of sec62-l cells at a normally permissive temperature. The inhibitory action of toxic Sec62p-invertase hybrids was partially counteracted by the overexpression of Sec63p. Taken together, these data suggest that the C-terminal domain of Sec62p performs an essential function and that the N-terminal domain associates with other components of the translocation machinery, including Sec63p.

Cks1 is required for G(1) cyclin-cyclin-dependent kinase activity in budding yeast.

p13(suc1) (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which 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 G(1) 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 G(1) 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 levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G(1) cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G(1) 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 G(1) phase in budding yeast.

Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast.

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-box proteins 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 in Saccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p 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 only major 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.

SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-mediated protein degradation.

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 intracellular domain 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 in cells. 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 intracellular domains 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.

SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities.

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.

Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes.

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.