Search

DNA-protein crosslinks (DPCs) are highly toxic DNA adducts, but whether dedicated DPC-repair mechanisms exist was until recently unknown. This has changed with discoveries made in yeast and Xenopus laevis that revealed a protease-based DNA-repair pathway specific for DPCs. Importantly, mutations in the gene encoding the putative human homologue of a yeast DPC protease cause a human premature ageing and cancer predisposition syndrome. Thus, DPC repair is a previously overlooked genome-maintenance mechanism that may be essential for tumour suppression.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions because they interfere with DNA transactions. The recent discovery of a yeast protease that processes DPCs proteolytically raises the question whether DPC proteases also exist in higher eukaryotes. We argue here that the yeast enzyme, Wss1 (weak suppressor of smt3), is a member of a protease family whose mammalian representative is Spartan (SprT-like domain-containing protein)/DVC1 (DNA damage protein targeting VCP). DPC proteases may thus be common to all eukaryotes where they function as novel guardians of the genome.

Modification by the ubiquitin-like protein SUMO affects hundreds of cellular substrate proteins and regulates a wide variety of physiological processes. While the SUMO system appears to predominantly target nuclear proteins and, to a lesser extent, cytosolic proteins, hardly anything is known about the SUMOylation of proteins targeted to membrane-enclosed organelles. Here, we identify a large set of structurally and functionally unrelated mitochondrial proteins as substrates of the SUMO pathway in yeast. We show that SUMO modification of mitochondrial proteins does not rely on mitochondrial targeting and, in fact, is strongly enhanced upon import failure, consistent with the modification occurring in the cytosol. Moreover, SUMOylated forms of mitochondrial proteins particularly accumulate in HSP70- and proteasome-deficient cells, suggesting that SUMOylation participates in cellular protein quality control. We therefore propose that SUMO serves as a mark for nonfunctional mitochondrial proteins, which only sporadically arise in unstressed cells but strongly accumulate upon defective mitochondrial import and impaired proteostasis. Overall, our findings provide support for a role of SUMO in the cytosolic response to aberrant proteins.

Summary Selective ubiquitin-dependent autophagy plays a pivotal role in the elimination of protein aggregates, assemblies, or organelles and counteracts the cytotoxicity of proteins linked to neurodegenerative diseases. Following substrate ubiquitylation, the cargo is delivered to autophagosomes involving adaptors like human p62 that bind ubiquitin and the autophagosomal ubiquitin-like protein Atg8/LC3; however, whether similar pathways exist in lower eukaryotes remained unclear. Here, we identify by a screen in yeast a new class of ubiquitin-Atg8 adaptors termed CUET proteins, comprising the ubiquitin-binding CUE-domain protein Cue5 from yeast and its human homolog Tollip. Cue5 collaborates with Rsp5 ubiquitin ligase, and the corresponding yeast mutants accumulate aggregation-prone proteins and are vulnerable to polyQ protein expression. Similarly, Tollip depletion causes cytotoxicity toward polyQ proteins, whereas Tollip overexpression clears human cells from Huntington's disease-linked polyQ proteins by autophagy. We thus propose that CUET proteins play a critical and ancient role in autophagic clearance of cytotoxic protein aggregates.

Cdc48 (also known as p97), a conserved chaperone-like ATPase, plays a strategic role in the ubiquitin system(1-3). Empowered by ATP-driven conformational changes(4), Cdc48 acts as a segregase by dislodging ubiquitylated proteins from their environment(1,2,5). Ufd1, a known co-factor of Cdc48, also binds SUMO (ref. 6), but whether SUMOylated proteins are subject to the segregase activity of Cdc48 as well and what these substrates are remains unknown. Here we show that Cdc48 with its co-factor Ufd1 is SUMO-targeted to proteins involved in DNA double-strand break repair. Cdc48 associates with SUMOylated Rad52, a factor that assembles the Rad51 recombinase on chromatin. By acting on the Rad52-Rad51 complex, Cdc48 curbs their physical interaction and displaces the proteins from DNA. Genetically interfering with SUMO-targeting or segregase activity leads to an increase in spontaneous recombination rates, accompanied by aberrant in vivo Rad51 foci formation in yeast and mammalian cells. Our data thus suggest that SUMO-targeted Cdc48 restricts the recombinase Rad51 by counterbalancing the activity of Rad52. We propose that Cdc48, through its ability to associate with co-factors that have affinities for ubiquitin and SUMO, connects the two modification pathways for protein degradation or other regulatory purposes.

Protein modification by conjugation to the ubiquitin-related protein SUMO (SUMOylation) regulates numerous cellular functions and is reversible. However, unlike typical posttranslational modifications, SUMOylation often targets and regulates proteins of functionally and physically linked protein groups, rather than individual proteins. Functional studies of protein-group SUMOylation are thus particularly challenging, as they require the identification of ideally all members of a modified protein group. Here, we describe mass spectrometric approaches to detect SUMOylated protein groups in Saccharomyces cerevisiae, yet the protocols can be readily adapted for studies of SUMOylation in mammalian cells.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a specialized activity of the ubiquitin–proteasome system that is involved in clearing the ER of aberrant proteins and regulating the levels of specific ER-resident proteins. Here we show that the yeast ER-SNARE Ufe1, a syntaxin (Qa-SNARE) involved in ER membrane fusion and retrograde transport from the Golgi to the ER, is prone to degradation by an ERAD-like mechanism. Notably, Ufe1 is protected against degradation through binding to Sly1, a known SNARE regulator of the Sec1–Munc18 (SM) protein family. This mechanism is specific for Ufe1, as the stability of another Sly1 partner, the Golgi Qa-SNARE Sed5, is not influenced by Sly1 interaction. Thus, our findings identify Sly1 as a discriminating regulator of SNARE levels and indicate that Sly1-controlled ERAD might regulate the balance between different Qa-SNARE proteins.

Ubiquitin-dependent protein degradation usually involves escort factors that target ubiquitylated substrates to the proteasome. A central element in a major escort pathway is Cdc48, a chaperone-like AAA ATPase that collects ubiquitylated substrates via alternative substrate-recruiting cofactors. Cdc48 also associates with Ufd2, an E4 multiubiquitylation enzyme that adds further ubiquitin moieties to preformed ubiquitin conjugates to promote degradation. Here, we show that E4 can be counteracted in vivo by two distinct mechanisms. First, Ufd3, a WD40 repeat protein, directly competes with Ufd2, because both factors utilize the same docking site on Cdc48. Second, Cdc48 also binds Otu1, a deubiquitylation enzyme, which disassembles multiubiquitin chains. Notably, Cdc48 can bind Otu1 and Ufd3 simultaneously, making a cooperation of both inhibitory mechanisms possible. We propose that the balance between the distinct substrate-processing cofactors may determine whether a substrate is multiubiquitylated and routed to the proteasome for degradation or deubiquitylated and/or released for other purposes.

The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.

The OLE pathway of yeast regulates the level of the ER-bound enzyme Delta9-fatty acid desaturase OLE1, thereby controlling membrane fluidity. A central component of this regulon is the transcription factor SPT23, a homolog of mammalian NF-kappaB. SPT23 is synthesized as an inactive, ER membrane-anchored precursor that is activated by regulated ubiquitin/proteasome-dependent processing (RUP). We now show that SPT23 dimerizes prior to processing and that the processed molecule, p90, retains its ubiquitin modification and initially remains tethered to its unprocessed, membrane-bound SPT23 partner. Subsequently, p90 is liberated from its partner for nuclear targeting by the activity of the chaperone-like CDC48(UFD1/NPL4) complex. Remarkably, this enzyme binds preferentially ubiquitinated substrates, suggesting that CDC48(UFD1/NPL4) is qualified to selectively remove ubiquitin conjugates from protein complexes.

Processing of integral membrane proteins in order to liberate active proteins is of exquisite cellular importance. Examples are the processing events that govern sterol regulation, Notch signaling, the unfolded protein response, and APP fragmentation linked to Alzheimer's disease. In these cases, the proteins are thought to be processed by regulated intramembrane proteolysis, involving site-specific, membrane-localized proteases. Here we show that two homologous yeast transcription factors SPT23 and MGA2 are made as dormant ER/nuclear membrane-localized precursors and become activated by a completely different mechanism that involves ubiquitin/proteasome-dependent processing. SPT23 and MGA2 are relatives of mammalian NF-κB and control unsaturated fatty acid levels. Intriguingly, proteasome-dependent processing of SPT23 is regulated by fatty acid pools, suggesting that the precursor itself or interacting partners are sensors of membrane composition or fluidity.

Two ubiquitin-conjugating enzymes, RAD6 and the heteromeric UBC13-MMS2 complex, have been implicated in post-replicative DNA damage repair in yeast. Here we provide a mechanistic basis for cooperation between the two enzymes. We show that two chromatin-associated RING finger proteins, RAD18 and RAD5, play a central role in mediating physical contacts between the members of the RAD6 pathway. RAD5 recruits the UBC13-MMS2 complex to DNA by means of its RING finger domain. Moreover, RAD5 association with RAD18 brings UBC13-MMS2 into contact with the RAD6-RAD18 complex. Interaction between the two RING finger proteins thus promotes the formation of a heteromeric complex in which the two distinct ubiquitin-conjugating activities of RAD6 and UBC13-MMS2 can be closely coordinated. Surprisingly, UBC13 and MMS2 are largely cytosolic proteins, but DNA damage triggers their redistribution to the nucleus. These findings suggest a mechanism by which the activity of this DNA repair pathway could be regulated.

The ubiquitin-like protein SMT3 fromSaccharomyces cerevisiaeand SUMO-1, its mammalian homolog, can be covalently attached to other proteins posttranslationally. Conjugation of ubiquitin requires the activities of ubiquitin-activating (E1) and -conjugating (E2) enzymes and proceeds via thioester-linked enzyme-ubiquitin intermediates. Herein we show that UBC9, one of the 13 different E2 enzymes from yeast, is required for SMT3 conjugationin vivo. Moreover, recombinant yeast and mammalian UBC9 enzymes were found to form thioester complexes with SMT3 and SUMO-1, respectively. This suggests that UBC9 functions as an E2 in a SMT3/SUMO-1 conjugation pathway analogous to ubiquitin-conjugating enzymes. The role of yeast UBC9 in cell cycle progression may thus be mediated through its SMT3 conjugation activity.

Ubiquitin-conjugating enzymes (UBC) catalyze the covalent attachment of ubiquitin to target proteins and are distinguished by the presence of a UBC domain required for catalysis. Previously identified members of this enzyme family are small proteins and function primarily in selective proteolysis pathways. Here we describe BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), a giant (528-kD) ubiquitin-conjugating enzyme from mice. BRUCE is membrane associated and localizes to the Golgi compartment and the vesicular system. Remarkably, in addition to being an active ubiquitin-conjugating enzyme, BRUCE bears a baculovirus inhibitor of apoptosis repeat (BIR) motif, which to this date has been exclusively found in apoptosis inhibitors of the IAP-related protein family. The BIR motifs of IAP proteins are indispensable for their anti–cell death activity and are thought to function through protein–protein interaction. This suggests that BRUCE may combine properties of IAP-like proteins and ubiquitin-conjugating enzymes and indicates that the family of IAP-like proteins is structurally and functionally more diverse than previously expected.

Summary Toxic DNA-protein crosslinks (DPCs) arise by ionizing irradiation and UV light, are particularly caused by endogenously produced reactive compounds such as formaldehyde, and also occur during compromised topoisomerase action. Although nucleotide excision repair and homologous recombination contribute to cell survival upon DPCs, hardly anything is known about mechanisms that target the protein component of DPCs directly. Here, we identify the metalloprotease Wss1 as being crucial for cell survival upon exposure to formaldehyde and topoisomerase 1-dependent DNA damage. Yeast mutants lacking Wss1 accumulate DPCs and exhibit gross chromosomal rearrangements. Notably, in vitro assays indicate that substrates such as topoisomerase 1 are processed by the metalloprotease directly and in a DNA-dependent manner. Thus, our data suggest that Wss1 contributes to survival of DPC-harboring cells by acting on DPCs proteolytically. We propose that DPC proteolysis enables repair of these unique lesions via downstream canonical DNA repair pathways.

The ubiquitin/proteasome system is the main eukaryotic nonlysosomal protein degradation system. Substrate selectivity of this pathway is thought to be mediated in part by members of a large family of ubiquitin-conjugating (E2) enzymes, which catalyze the covalent attachment of ubiquitin to proteolytic substrates. E2 enzymes have a conserved approximately 150-residue so-called UBC domain, which harbors the cysteine residue required for enzyme-ubiquitin thioester formation. Some E2 enzymes possess additional carboxyl-terminal extensions that are involved in substrate specificity and intracellular localization of the enzyme. Here we describe a novel family of E2 enzymes from higher eukaryotes (Drosophila, mouse, and man) that have amino-terminal extensions but lack carboxyl-terminal extensions. We have identified four different variants of these enzymes that have virtually identical UBC domains (94% identity) but differ in their amino-terminal extensions. In yeast, these enzymes can partially complement mutants deficient in the UBC4 E2 enzyme. This indicates that members of this novel E2 family may operate in UBC4-related proteolytic pathways.

Protein translocation into the mammalian endoplasmic reticulum requires the Sec61p complex, which consists of three membrane proteins. The alpha-subunit, the homologue of Sec61p of yeast, shows some similarity to SecYp, a key component of the protein export apparatus of bacteria. In Escherichia coli, SecYp is also associated with two other proteins (SecEp and band-1 protein). We have now determined the sequences of the beta- and gamma-subunits of the mammalian Sec61p complex. Sec61-gamma is homologous to SSS1p, a suppressor of sec61 mutants in Saccharomyces cerevisiae, and can functionally replace it in yeast cells. Moreover, Sec61-gamma and SSS1p are structurally related to SecEp of E. coli and to putative homologues in various other bacteria. At least two subunits of the Sec61/SecYp complex therefore seem to be key components of the protein translocation apparatus in all classes of organisms.

All living organisms are vulnerable to DNA damage. Cells respond to this hazard by activating a complex network of checkpoint and repair proteins to preserve genomic integrity. The DNA-encircling, ring-shaped heterotrimeric 9-1-1 complex, a relative of the replication protein PCNA, is a central coordinator of these events. 9-1-1 is loaded to damaged sites where it serves as a platform for the selective recruitment of checkpoint and repair proteins. In this Opinion article, 9-1-1 and proliferating cell nuclear antigen (PCNA) are compared and discussed in light of their respective structures and functions. We propose that the interaction partners of 9-1-1 possess specific 9-1-1-interaction boxes, which discriminate between 9-1-1 and PCNA thereby enabling specific interactions with individual 9-1-1 subunits.

The related transcription factors ACE1 of Saccharomyces cerevisiae and AMT1 of Candida glabrata are involved in copper metabolism by activating the transcription of copper metallothionein genes. ACE1 and AMT1 are 'copper-fist' transcription factors which possess a conserved cysteine-rich copper binding domain required for DNA binding. Here we report the identification of a nuclear protein from S. cerevisiae, MAC1, whose N-terminal region is highly similar to the copper and DNA binding domains of ACE1 and AMT1. Loss-of-function mutants of MAC1 have a defect in the plasma membrane Cu(II) and Fe(III) reductase activity, are slow growing, respiratory deficient, and hypersensitive to heat and exposure to cadmium, zinc, lead and H2O2. Conversely, a dominant gain-of-function mutant of MAC1 shows an elevated reductase activity and is hypersensitive to copper. We have identified two target genes of MAC1 whose altered expression in mutants of MAC1 can account for some of the observed mutant phenotypes. First, MAC1 is involved in basal level transcription of FRE1, encoding a plasma membrane component associated with both Cu(II) and Fe(III) reduction. Second, MAC1 is involved in the H2O2-induced transcription of CTT1, encoding the cytosolic catalase. This suggests that MAC1 may encode a novel metal-fist transcription factor required for both basal and regulated transcription of genes involved in Cu/Fe utilization and the stress response.

UBIQUITIN-conjugating enzymes function in selective proteolysis pathways and catalyse the covalent attachment of ubiquitin to proteolytic substrates1–4. Here we report the identification of an integral membrane ubiquitin-conjugating enzyme. This enzyme, UBC6, localizes to the endoplasmic reticulum (ER), with the cata-lytic domain facing the cytosol. ubc6 loss-of-function mutants sup-press the protein translocation defect caused by a mutation in SEC61, which encodes a key component of a multisubunit protein translocation apparatus of the ER5–11. The expression of the sec61 mutant phenotype requires both the activity of UBC6 and its localization at the ER membrane. This suggests that UBC6 may mediate selective degradation of ER membrane proteins and that the protein translocation defect of sec61 may be caused by proteolysis of components of a structurally distorted mutant translocation apparatus.

Selective ubiquitin-dependent autophagy mediates the disposal of superfluous cellular structures and clears cells of protein aggregates such as polyQ proteins linked to Huntington disease. Crucial selectivity factors of this pathway are ubiquitin-Atg8 receptors such as human SQSTM1/p62, which recognize ubiquitinated cargoes and deliver them to phagophores for degradation. Contrasting previous beliefs, we recently showed that ubiquitin-dependent autophagy is not restricted to higher eukaryotes but also exists in yeast with Cue5, harboring a ubiquitin-binding CUE domain, being a ubiquitin-Atg8 receptor. Notably, the human CUE domain protein TOLLIP is functionally similar to yeast Cue5, indicating that Cue5/TOLLIP (CUET) proteins represent a new and conserved class of autophagy receptors. Remarkably, both Cue5 in yeast and TOLLIP in human cells mediate efficient clearance of aggregation-prone polyQ proteins derived from human HTT/huntingtin. Indeed, TOLLIP is potentially more potent in polyQ clearance than SQSTM1/p62 in HeLa cells, suggesting that TOLLIP, also implicated in innate immunity, may be significant for human health and disease.

The ubiquitin/proteasome system is believed to be the major nonlysosomal proteolytic system of eukaryotic cells. It is present in the cytosol and the nucleus but apparently absent from the lumen of membrane-enclosed organelles, i. e., the endoplasmic reticulum, the Golgi and vesicular system, mitochondria, chloroplasts, and peroxisomes (for reviews see Finley and Chau, 1991; Jentsch, 1992a,b; Hershko and Ciechanover, 1992; Ciechanover, 1994; Hochstrasser, 1995; Smith et al., 1996). Substrates of this pathway include soluble proteins, subunits of oligomeric protein complexes, and integral membrane proteins. An important function of this pathway is the elimination of abnormal proteins (e. g., misfolded, misassembled) generated under normal and, in particular, stress conditions. Moreover, it is assumed that most naturally short-lived proteins of the cytosol and the nucleus are degraded by this pathway. Known substrates include proteins with important regulatory roles such as transcription factors, cell cycle regulators, and signal transducers.

A major pathway for protein degradation in eukaryotes is ubiquitin dependent. Substrate-specific ubiquitin-conjugating enzymes and accessory factors recognize specific signals on proteolytic substrates and attach ubiquitin to defined lysine residues of substrate proteins. Ubiquitin-protein conjugates are then degraded by the proteasome, a multicatalytic protease complex. This proteolytic pathway is highly selective and tightly regulated. It mediates the elimination of abnormal proteins and controls the half-lifes of certain regulatory proteins. Targets include transcriptional regulators, p53 and cyclins, pointing to a role of the ubiquitin system in the regulation of gene expression and growth control.

The covalent attachment of ubiquitin to cellular proteins is catalyzed by members of a family of ubiquitin-conjugating enzymes. These enzymes participate in a variety of cellular processes, including selective protein degradation, DNA repair, cell cycle control, and sporulation. In the yeast Saccharomyces cerevisiae, two closely related ubiquitin-conjugating enzymes, UBC4 and UBC5, have recently been shown to mediate the selective degradation of short-lived and abnormal proteins. We have now identified a third distinct member of this class of ubiquitin-conjugating enzymes, UBC1. UBC1, UBC4 and UBC5 are functionally overlapping and constitute an enzyme family essential for cell growth and viability. All three mediate selective protein degradation, however, UBC1 appears to function primarily in the early stages of growth after germination of spores. ubc1 mutants generated by gene disruption display only a moderate slow growth phenotype, but are markedly impaired in growth following germination. Moreover, yeast carrying the ubc1ubc4 double mutation are viable as mitotic cells, however, these cells fail to survive after undergoing sporulation and germination. This specific requirement for UBC1 after a state of quiescence suggests that degradation of certain proteins may be crucial at this transition point in the yeast life cycle.

Ubiquitin-conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins. Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control. We have cloned the genes for two novel ubiquitin-conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae. These enzymes mediate selective degradation of short-lived and abnormal proteins. UBC4 and UBC5 are closely related in sequence and complementing in function. Expression of UBC4 and UBC5 genes is heat inducible. UBC4 and UBC5 enzymes generate high mol. wt ubiquitin-protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation. UBC4 and UBC5 enzymes comprise a major part of total ubiquitin-conjugation activity in stressed cells. Turnover of short-lived proteins and canavanyl-peptides but not of long-lived proteins is markedly reduced in ubc4ubc5 mutants. Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response.

Cdc48 (p97), a conserved chaperone-like ATPase of eukaryotic cells, has attracted attention recently because of its wide range of cellular functions. Cdc48 is intimately linked to the ubiquitin pathway because its primary action is to segregate ubiquitinated substrates from unmodified partners. This ‘segregase' activity is crucial for certain proteasomal degradation pathways and for some nonproteolytic functions of ubiquitin. Cdc48 associates not only with different ‘substrate-recruiting cofactors' but also with distinct ‘substrate-processing cofactors'. The latter proteins control the degree of ubiquitination of bound substrates by shifting the polyubiquitination reaction into ‘forward', ‘neutral' or ‘reverse'. We discuss how Cdc48 might use this ‘gearbox activity' to control protein fate and propose a similar mode of action for the 19S cap of the proteasome.

The proteasome is a barrel-shaped protease that conceals its active sites within its central cavity. Proteasomes usually completely degrade substrates into small peptides, but in some cases, degradation yields biologically active protein fragments. Some transcription factors are generated from precursors by this activity, but the mechanism of proteasomal protein processing remains unclear. Here we show that proteasomal processing of the yeast NFkappaB-related transcription factors Spt23 and Mga2 is initiated by an internal cleavage, followed by bidirectional proteolysis of the polypeptides. Studies with protein fusions indicate that stable proteolytic fragments are generated if the protein contains tightly folded structures that prevent the complete degradation of the protein. Furthermore, we provide evidence that the ability of the proteasome to initiate proteolysis from an internal site and to proceed via bidirectional polypeptide degradation may be relevant for the complete degradation of proteins as well.

Post-translational modification with small ubiquitin-related modifier (SUMO) alters the function of many proteins, but the molecular mechanisms and consequences of this modification are still poorly defined. During a screen for novel SUMO1 targets, we identified the ubiquitin-conjugating enzyme E2-25K (Hip2). SUMO attachment severely impairs E2-25K ubiquitin thioester and unanchored ubiquitin chain formation in vitro. Crystal structures of E2-25K(1-155) and of the E2-25K(1-155)-SUMO conjugate (E2-25K(*)SUMO) indicate that SUMO attachment interferes with E1 interaction through its location on the N-terminal helix. The SUMO acceptor site in E2-25K, Lys14, does not conform to the consensus site found in most SUMO targets (PsiKXE), and functions only in the context of an alpha-helix. In contrast, adjacent SUMO consensus sites are modified only when in unstructured peptides. The demonstration that secondary structure elements are part of SUMO attachment signals could contribute to a better prediction of SUMO targets.

Modification of proteins by covalent attachment of ubiquitin and the ubiquitin‐like modifier SUMO are widespread regulatory events of all eukaryotic cells. SUMOylation has received much attention, because several identified targets play prominent roles, in particular, in cell signaling, gene expression, and DNA repair. Notably, only a very small fraction of a substrate is usually SUMOylated at steady‐state levels, which could be because modification is reversible and transient. Because of the low level of modification, SUMOylated proteins are often overlooked or sometimes misinterpreted as a less important fraction of a protein pool. Here we discuss procedures that can circumvent identification problems and describe methods for their verification.

Ubiquitin and ubiquitin-like modifiers (UBLs) form covalent complexes with other proteins by isopeptide formation between their carboxyl (C)-termini and ɛ-amino groups of lysine residues of acceptor proteins. A hallmark of UBLs is a protruding C-terminal tail with a terminal glycine residue, which is required for ATP-dependent conjugation. Recently, the highly conserved protein HUB1 (homologous to ubiquitin 1) has been reported to function as a UBL following C-terminal processing. HUB1 exhibits sequence similarity with ubiquitin but lacks a C-terminal tail bearing a glycine residue. Here we show that HUB1 can form SDS-resistant complexes with cellular proteins, but provide evidence that these adducts are not formed through covalent C-terminal conjugation of HUB1 to substrates. The adducts are still formed when the C-terminus of HUB1 was altered by epitope tagging, amino-acid exchange or deletion, or when cells were depleted of ATP. We propose that HUB1 may act as a novel protein modulator through the formation of tight, possibly noncovalent interactions with target proteins.

The OLE pathway of yeast regulates the abundance of the ER-bound enzyme Delta-9 fatty acid desaturase OLE1, thereby controlling unsaturated fatty acid pools and membrane fluidity. Previously, we showed that this pathway is exquisitely regulated by the ubiquitin/proteasome system. Activation of the pathway involves proteasomal processing of a membrane-bound transcription factor and the subsequent mobilization of the cleaved, ubiquitylated transcription factor from its partner molecule by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone-like enzyme. Here we report that the OLE1 protein itself is naturally short-lived and is degraded by ubiquitin/proteasome-dependent ER-associated degradation (ERAD). We found that CDC48(UFD1/NPL4) plays a second role in the OLE pathway by mediating ERAD of OLE1. Intriguingly, other ERAD substrates also require CDC48(UFD1/NPL4) for degradation, indicating that this enzyme is a novel, constitutive component of the ERAD machinery. We propose that CDC48(UFD1/NPL4) functions as a segregase that liberates ubiquitylated proteins from non-modified partners.

Cadmium is a potent poison for living cells. In man, chronic exposure to low levels of cadmium results in damage to kidneys and has been linked to neoplastic disease and ageing, and acute exposure can cause damage to a variety of organs and tissues. Cadmium reacts with thiol groups and can substitute for zinc in certain proteins, but the reason for its toxicity in vivo remains uncertain. In eukaryotes, an important selective proteolysis pathway for the elimination of abnormal proteins that are generated under normal or stress conditions is ATP-dependent and mediated by the ubiquitin system. Substrates of this pathway are first recognized by ubiquitin-conjugating enzymes (or auxiliary factors) which covalently attach ubiquitin, a small and highly conserved protein, to specific internal lysine residues of proteolytic substrates. Ubiquitinated substrates are then degraded by the proteasome, a multisubunit protease complex. Here we show that expression of this ubiquitin-dependent proteolysis pathway in yeast is activated in response to cadmium exposure and that mutants deficient in specific ubiquitin-conjugating enzymes are hypersensitive to cadmium. Moreover, mutants in the proteasome are hypersensitive to cadmium, suggesting that cadmium resistance is mediated in part by degradation of abnormal proteins. This indicates that a major reason for cadmium toxicity may be cadmium-induced formation of abnormal proteins.

Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin–protein ligase, E3. Here we show that efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4. This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3. Intriguingly, E4 defines a novel protein family that includes two human members and the regulatory protein NOSA from Dictyostelium required for fruiting body development. In yeast, E4 activity is linked to cell survival under stress conditions, indicating that eukaryotes utilize E4-dependent proteolysis pathways for multiple cellular functions.

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.

Eukaryotic proteins are tagged for degradation by addition of ubiquitin or single amino acids. Now Jentsch discusses a new prokaryotic protein tagging system reported by Keiler et al. ([p. 990][1]) in this issue of Science . In this novel tagging pathway, nascent polypeptides, translated from truncated mRNAs lacking stop codons, receive short COOH-terminal peptide tags encoded by a separate RNA, 10Sa RNA. [1]: /lookup/doi/10.1126/science.271.5251.990

Adenovirus E1A encodes two nuclear phosphoproteins that can transform primary rodent fibroblasts in culture. Transformation by E1A is mediated at least in part through binding to several cellular proteins, including the three members of the retinoblastoma family of growth inhibitory proteins. We report here the cloning of a novel murine cDNA whose encoded protein interacts with both adenovirus type 5 and type 12 E1A proteins. The novel E1A-interacting protein shares significant sequence homology with ubiquitin-conjugating enzymes, a family of related proteins that is involved in the proteasome-mediated proteolysis of short-lived proteins. Highest homology was seen with a Saccharomyces cerevisiae protein named UBC9. Importantly, the murine E1A-interacting protein complements a cell cycle defect of a S. cerevisiae mutant which harbors a temperature-sensitive mutation in UBC9. We therefore named this novel E1A-interacting protein mUBC9. We mapped the region of E1A that is required for mUBC9 binding and found that the transformation-relevant conserved region 2 of E1A is required for interaction.

Entry into a new cell cycle is triggered by environmental signals at a point called Start in G1 phase. A key regulator of this transition step in yeast is the CDC28 kinase together with its short-lived regulatory subunits called G1-cyclins or CLN proteins. To identify genes involved in G1-cyclin degradation, we employed a genetic screen by selecting for stable CLN1-beta-galactosidase fusion proteins. Surprisingly, one group of mutants was found to be allelic to GRR1, a gene previously described to be involved in glucose uptake, glucose repression, and divalent cation transport. In grr1 mutants, both CLN1 and CLN2 cyclins are significantly stabilized. A suppressor analysis indicated that G1-cyclin stabilization in grr1 was not a consequence of the nutrient uptake defect. This suggests that the GRR1 gene product is part of a common regulatory pathway linking two functions important for cell growth, nutrient uptake, and G1 cyclin-controlled cell division.

Attachment of ubiquitin to proteins is catalyzed by a family of ubiquitin-conjugating (UBC) enzymes. Although these enzymes are essential for many cellular processes; their molecular functions remain unclear because no physiological target has been identified for any of them. Here we show that four UBC proteins (UBC4, UBC5, UBC6, and UBC7) target the yeast MAT alpha 2 transcriptional regulator for intracellular degradation by two distinct ubiquitination pathways. UBC6 and UBC7 define one of the pathways and can physically associate. The UBC6/UBC7-containing complex targets the Deg1 degradation signal of alpha 2, a conclusion underscored by the finding that UBC6 is encoded by DOA2, a gene previously implicated in Deg1-mediated degradation. These data reveal an unexpected overlap in substrate specificity among diverse UBC enzymes and suggest a combinatorial mechanism of substrate selection in which UBC enzymes partition into multiple ubiquitination complexes.

Ubiquitin-dependent selective protein degradation serves to eliminate abnormal proteins and provides controlled short half-lives to certain cellular proteins, including proteins of regulatory function such as phytochrome, yeast MAT alpha 2 repressor, p53 and cyclin. Moreover, ubiquitin-dependent proteolysis is thought to play an essential role during development and in programmed cell death. We have cloned a gene from Drosophila melanogaster, UbcD1, coding for a protein with striking sequence similarity to the yeast ubiquitin-conjugating enzymes UBC4 and UBC5. These closely related yeast enzymes are known to be central components of a major proteolytic pathway of Saccharomyces cerevisiae. By doing a precise open reading frame replacement in the yeast genome we could show that the Drosophila UbcD1 enzyme can functionally substitute for yeast UBC4. UbcD1 driven by the UBC4 promoter rescues growth defects and temperature sensitivity of yeast ubc4 ubc5 double mutant cells. Moreover, expression of UbcD1 restores proteolysis proficiency in the ubc4 ubc5 double mutant, indicating that the Drosophila enzyme also mediates protein degradation. This structural and functional conservation suggests that the UbcD1-UBC4-UBC5 class of enzymes defines a major proteolytic pathway in probably all eukaryotes.

Here we review the genetic analysis of the ubiquitin system with an emphasis on the study of the functions of the enzymatic components. Additionally, we will compile recent data on ubiquitin-like proteins encoded by cellular and viral genes. Furthermore, we will present and discuss models for the mechanisms which might control the remarkable selectivity of this posttranslational modification system

All known functions of ubiquitin are mediated through its covalent attachment to other proteins. The post-translational formation of ubiquitin--protein conjugates is preceded by an ATP-requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin-activating enzyme, also known as E1. We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin-activating enzyme of the yeast Saccharomyces cerevisiae. UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide-binding sites. Expression of catalytically active UBA1 protein in E. coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin-activating enzyme. Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin--protein conjugates is essential for cell viability.

Mutants in the gene CDC34 of the yeast Saccharomyces cerevisiae are defective in the transition from G1 to the S phase of the cell cycle. This gene was cloned and shown to encode a 295-residue protein that has substantial sequence similarity to the product of the yeast RAD6 gene. The RAD6 gene is required for a variety of cellular functions including DNA repair and was recently shown to encode a ubiquitin-conjugating enzyme. When produced in Escherichia coli, the CDC34 gene product catalyzed the covalent attachment of ubiquitin to histones H2A and H2B in vitro, demonstrating that the CDC34 protein is another distinct member of the family of ubiquitin-conjugating enzymes. The cell cycle function of CDC34 is thus likely to be mediated by the ubiquitin-conjugating activity of its product.

Protein degradation in eukaryotes usually requires multiubiquitylation and subsequent delivery of the tagged substrates to the proteasome. Recent studies suggest the involvement of the AAA ATPase CDC48, its cofactors, and other ubiquitin binding factors in protein degradation, but how these proteins work together is unclear. Here we show that these factors cooperate sequentially through protein-protein interactions and thereby escort ubiquitin-protein conjugates to the proteasome. Central to this pathway is the chaperone CDC48/p97, which coordinates substrate recruitment, E4-catalyzed multiubiquitin chain assembly, and proteasomal targeting. Concomitantly, CDC48 prevents the formation of excessive multiubiquitin chain sizes that are surplus to requirements for degradation. In yeast, this escort pathway guides a transcription factor from its activation in the cytosol to its final degradation and also mediates proteolysis at the endoplasmic reticulum by the ERAD pathway.

The properties of two DNA methyltransferases, termed M. BsuRIa and M. BsuRIb, whose isolation was described in the preceding paper (Günthert, U., Freund, M., and Trautner, T. A. (1981) J. Biol. Chem. 256, 9340-9345) were compared. Both enzymes recognize the same target sequence in double-stranded DNA, leading to methylation of the internal cytosine: 5'GGCC. The enzymes have identical reaction constants with their substrates, DNA (km = 2.7 nM for the 5' GGCC sequence), and S-adenosyl-L-methionine (km = 0.7 microM). Initial rates of methyl group transfer were proportional to enzyme concentration over a range of 50-fold, indicating absence of aggregation. The enzymes are different in their ionic strength requirements using Tris-HCl, pH 8.4. M. BsuRIa is most active at 100 mM, M. BsuRIb at 440 mM. As measured by incorporation kinetics and heat inactivation, M. BsuRIa is the more stable enzyme of the two. Equilibrium dialysis was used to study the mode of methyl group transfer to the DNA with either enzyme. The data indicate that initially S-adenosyl-L-methionine binds to methyltransferase. This complex attaches to either modified or nonmodified DNA. The methyl group will then be transferred to a nonmodified target sequence, leading to the dissociation of enzyme and S-adenosyl-L-homocysteine from the DNA.

The RAD6 gene of the yeast Saccharomyces cerevisiae is required for a variety of cellular functions including DNA repair. The discovery that the RAD6 gene product can catalyse the covalent attachment of ubiquitin to other proteins suggests that the multiple functions of the RAD6 protein are mediated by its ubiquitin-conjugating activity.

Bacillus subtilis phage SPR codes for a DNA methyltransferase (Mtase) which methylates the 5' cytosine in the sequence GGCC and both cytosines in the sequence CCGG. A 2126-bp fragment of SPR DNA containing the Mtase gene has been sequenced. This fragment has only one significant open reading frame of 1347 bp, which corresponds to the Mtase gene. Within the sequence the Mtase promoter has been defined by S1 mapping. The size of the SPR Mtase predicted from the deduced amino acid composition is 49.9 kDal. This is in agreement with both the Mr of the purified enzyme and with that of the SPR Mtase gene product identified here by minicell technique. Base changes leading to mutants affected in Mtase activity were localized within the Mtase gene.

Ubiquitin-conjugating enzymes catalyze the covalent attachment of ubiquitin to cellular substrates. Here we describe the isolation of a novel ubiquitin-conjugating enzyme from human placenta and the cloning of the corresponding cDNA. DNA sequencing revealed that this gene, UbcH2, encodes a protein with significant sequence similarity to yeast UBC8. In contrast to a previous report (Qin, S., Nakajima, B., Nomura, M., and Arfin, S. M. (1991) J. Biol. Chem. 266, 15549-15554), we discovered that UBC8 is interrupted by a single intron bearing an unusual branch point sequence. The revised amino acid sequence of yeast UBC8 exhibits 54% amino acid sequence identity to human UbcH2. Moreover, full-length UbcH2 and UBC8 enzymes expressed from their cDNAs show similar enzymatic activities in vitro by catalyzing the ubiquitination of histones, suggesting that the two enzymes may fulfill similar functions in vivo. Interestingly, comparison of the enzymatic activities of a truncated UBC8 (Qin, S., Nakajima, B., Nomura, M. and Arfin, S. M. (1991) J. Biol. Chem. 266, 15549-15554) and of the full-length enzyme (this report) suggests, that the first 12 amino-terminal residues of UBC8 are required for ubiquitination of histones in vitro but not for thiolester formation with ubiquitin. This suggests that the NH2 terminus of UBC8 may be necessary either for substrate recognition or for the transfer of ubiquitin onto substrates. The UbcH2 gene is located on chromosome 7 and shows a complex expression pattern with at least five different mRNAs.

Covalent attachment of ubiquitin to cellular proteins is essential for cell viability and is catalysed by a set of distinct ubiquitin-conjugating enzymes. Individual members of this novel enzyme family mediate strikingly diverse functions, including DNA repair, cell cycle control, selective protein degradation and essential functions of the stress response.