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At least 19 recordsLinked to original sources

PubMed2000

Histidine-tagged ubiquitin substitutes for wild-type ubiquitin in Saccharomyces cerevisiae and facilitates isolation and identification of in vivo substrates of the ubiquitin pathway.

A general method for purification of any substrate of the ubiquitin pathway, the major eukaryotic proteolytic pathway, should utilize the common characteristic of covalent linkage of ubiquitin to substrate lysyl residues. The utility of a N-terminal histidine-tagged ubiquitin (HisUb) for in vivo conjugation and isolation of ubiquitinated proteins by metal chelation chromatography is conditioned by the requirement that HisUb conjugate to the same set of proteins as wild-type ubiquitin. Stringent in vivo tests with Saccharomyces cerevisiae strains expressing ubiquitins only from plasmids were performed to show that HisUb could substitute for wild-type ubiquitin. The utility of HisUb as a method for purification of proteins ubiquitinated in vivo was demonstrated by metal chelation chromatography of yeast extracts expressing HisUb and immunoblotting for Rpb1, the largest subunit of RNA polymerase II. A fraction of Rpb1 was present in the ubiquitinated form in vivo. The ability to use HisUb expression in transgenic organisms that retain expression of their endogenous ubiquitin genes was demonstrated through transgenic Arabidopsis thaliana expressing HisUb or its variant HisUbK48R. UbK48R is a version of ubiquitin capable of conjugation to proteins, but cannot serve as an attachment site for ubiquitin via the major in vivo interubiquitin linkage. Whereas transgenic plants expressing HisUb showed insignificant enrichment of ubiquitinated proteins, transgenic Arabidopsis lines expressing HisUbK48R gave a much better yield.

Arabidopsis↗

PubMed1993

Utility of polyhistidine-tagged ubiquitin in the purification of ubiquitin-protein conjugates and as an affinity ligand for the purification of ubiquitin-specific hydrolases.

The purification and biochemical characterization of protein substrates of the ubiquitin-dependent pathway of proteolysis is made difficult in part by the low steady state levels of ubiquitin-protein conjugates. We report here on the use of a polyhistidine-tagged ubiquitin molecule (HisUb) for the purification of ubiquitin-protein conjugates by metal chelate chromatography. When Escherichia coli extracts containing expressed HisUb were passed through a nitrilotriacetic acid-agarose column containing immobilized Ni2+ ions (Ni-NTA column), HisUb was retained. After washing to remove unbound and nonspecifically bound proteins, a pH 4.5 wash was used to elute highly purified HisUb. Purified HisUb and wild-type ubiquitin were tested for their ability to form Ni(2+)-binding ubiquitin-protein conjugates in a wheat germ in vitro conjugation reaction. In some experiments, wheat germ extracts were preincubated with iodoacetamide to inhibit ubiquitin activating and conjugating enzymes. Only those conjugation assays containing HisUb and an ATP-regenerating system not pretreated with iodoacetamide produced significant levels of multiple Ni(2+)-binding ubiquitin-protein conjugates. We also examined the potential of HisUb as an affinity ligand for the purification of higher plant ubiquitin-specific hydrolases. As a test, a crude lysate of E. coli expressing a yeast ubiquitin-specific hydrolase (Yuh1) was passed through a Ni-NTA column containing bound HisUb. Yuh1 was retained on the column and was specifically eluted when the column was equilibrated with buffer containing wild-type ubiquitin.

Amino Acid Sequence↗

PubMed1989

Binding sites of ubiquitin-protein ligase. Binding of ubiquitin-protein conjugates and of ubiquitin-carrier protein.

It was found previously that the enzyme ubiquitin-protein ligase (E3) contains specific protein substrate binding sites that are responsible for the selection of proteins for degradation by the ubiquitin system. In the present study, we have tried to gain more insight into the mode of action of E3 by the characterization of other binding sites of this enzyme. Following the ligation of ubiquitin to 125I-lysozyme, the conjugates produced are very tightly bound to E3, as indicated by size analysis on glycerol density gradient centrifugation. The strong binding of ubiquitin-protein conjugates to the enzyme may account for the apparently processive addition of multiple molecules of ubiquitin to the protein substrate. Both the protein substrate moiety and the ubiquitin moiety participate in the interaction of ubiquitin-protein conjugates with E3, as indicated by competition with specific agents and by the comparison of the binding of ubiquitin-conjugated protein to that of free protein. In addition to the binding of its substrates and products, E3 also appears to interact with some of the enzymes with which it acts in concert. When E3 is incubated with the ubiquitin-carrier protein E2, a complex is formed between the two enzymes as analyzed on glycerol gradients. The formation of an E2.E3 complex may facilitate the transfer of activated ubiquitin from E2 to the protein substrate bound to the ligase.

Adenosine Triphosphate↗

PubMedDate not supplied

Ubiquitin pools, ubiquitin mRNA levels, and ubiquitin-mediated proteolysis in aging human fibroblasts.

Senescent cells have less free ubiquitin and more ubiquitin-protein conjugates than do young cells. The ubiquitin-protein conjugates are heterogeneous in size but contain prominent bands at 106, 55, and 22 kDa. The age-related increase in ubiquitin-protein conjugates applies primarily to the 55-kDa band, while the 106-kDa and 22-kDa conjugates change little with age. Ubiquitin mRNA levels do not change with age, and the ability of cells to degrade two proteins that are good substrates for ubiquitin-mediated proteolysis is unaltered by aging. These results indicate that an increase in ubiquitin-protein conjugates does not necessarily reflect alterations in ubiquitin-mediated proteolysis. Furthermore, an overactive pathway of ubiquitin-mediated proteolysis does not appear to contribute to the proliferative arrest in senescent cells.

Actins↗

PubMed1997

Changes in ubiquitin immunoreactivity in developing rat brain: a putative role for ubiquitin and ubiquitin conjugates in dendrite outgrowth and differentiation.

The role of ubiquitin in development of the mammalian brain has been studied using a monoclonal antibody, RHUb1, specific for ubiquitin. Immunodevelopment of western blots of homogenate samples of the cerebral cortex, hippocampus and cerebellum prepared from animals of known postnatal age show marked developmental changes in conjugate level. Striking decreases in the level of a prominent conjugate of molecular weight 22,000, which is identified as ubiquitinated histone, are observed during the first postnatal week in the cerebral cortex and hippocampus, but not the cerebellum. A marked overall developmental decrease in the level of high-molecular-weight (> 40,000) ubiquitin conjugates which occurs predominantly during the third, but also the fourth, postnatal week is observed in all three regions. Immunocytochemical data obtained with the RHUb1 antibody show intense staining of neuronal perikarya, nuclei and dendrites in early postnatal cerebral cortex and hippocampus. Staining of pyramidal cell perikarya and dendrites is particularly prominent. The intensity of dendritic staining, particularly for the cerebral cortex, shows a striking decrease after postnatal day 14 and only faint dendritic staining is observed in the adult. In early postnatal cerebellum, immunoreactivity is predominantly nuclear, though some staining of the proximal regions of Purkinje cell dendrites is observed between postnatal days 4 and 19. As with the cerebral cortex and hippocampus, most of the ubiquitin reactivity is lost in adult animals. The loss of dendritic staining, particularly in the cerebral cortex, correlates with the decrease in the level of high-molecular-weight ubiquitin conjugates observed on the western blots. Immunodevelopment of western blots of a range of subcellular fractions prepared from developing rat forebrain shows that the developmental decrease in the level of high-molecular-weight ubiquitin conjugates is not uniform for all fractions. The decrease in conjugate level is most marked for the cell-soluble, mitochondrial and detergent-insoluble cytoskeletal fractions. Taken overall, the data suggest a role for ubiquitin in dendrite outgrowth and arborization, loss of dendritic ubiquitin immunoreactivity correlating with completion of these processes.

Age Factors↗

PubMed2001

Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins.

BACKGROUND: The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs. RESULTS: The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis. CONCLUSIONS: The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.

Amino Acid Sequence↗

PubMed2004

A RING finger ubiquitin ligase is protected from autocatalyzed ubiquitination and degradation by binding to ubiquitin-specific protease USP7.

Herpes simplex virus type 1 immediate-early regulatory protein ICP0 stimulates lytic infection and reactivation from latency, processes that require the ubiquitin E3 ligase activity mediated by the RING finger domain in the N-terminal portion of the protein. ICP0 stimulates the production of polyubiquitin chains by the ubiquitin-conjugating enzymes UbcH5a and UbcH6 in vitro, and in infected and transfected cells it induces the proteasome-dependent degradation of a number of cellular proteins including PML, the major constituent protein of PML nuclear bodies. However, ICP0 binds strongly to the cellular ubiquitin-specific protease USP7, a member of a family of proteins that cleave polyubiquitin chains and/or ubiquitin precursors. The region of ICP0 that is required for its interaction with USP7 has been mapped, and mutations in this domain reduce the functionality of ICP0. These findings pose the question: why does ICP0 include domains that are associated with the potentially antagonistic functions of ubiquitin conjugation and deconjugation? Here we report that although neither protein affected the intrinsic activities of the other in vitro, USP7 protected ICP0 from autoubiquitination in vitro, and their interaction can greatly increase the stability of ICP0 in vivo. These results demonstrate that RING finger-mediated autoubiquitination of ICP0 is biologically relevant and can be regulated by interaction with USP7. This principle may extend to a number of cellular RING finger E3 ubiquitin ligase proteins that have analogous interactions with ubiquitin-specific cleavage enzymes.

Animals↗

PubMed1995

Ubiquitin-mediated processing of NF-kappa B transcriptional activator precursor p105. Reconstitution of a cell-free system and identification of the ubiquitin-carrier protein, E2, and a novel ubiquitin-protein ligase, E3, involved in conjugation.

In most cases, the transcriptional factor NF-kappa B is a heterodimer consisting of two subunits, p50 and p65, which are encoded by two distinct genes of the Rel family. p50 is translated as a precursor of 105 kDa. The C-terminal domain of the precursor is rapidly degraded, forming the mature p50 subunit consisted of the N-terminal region of the molecule. The mechanism of generation of p50 is not known. It has been suggested that the ubiquitin-proteasome system is involved in the process; however, the specific enzymes involved and the mechanism of limited proteolysis, in which half of the molecule is spared, have been obscure. Palombella and colleagues (Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) Cell 78, 773-785) have shown that ubiquitin is required for the processing in a cell-free system of a truncated, artificially constructed, 60-kDa precursor. They have also shown that proteasome inhibitors block the processing both in vitro and in vivo. In this study, we demonstrate reconstitution of a cell-free processing system and demonstrate directly that: (a) the ubiquitin-proteasome system is involved in processing of the intact p105 precursor, (b) conjugation of ubiquitin to the precursor is an essential intermediate step in the processing, (c) the recently discovered novel species of the ubiquitin-carrier protein, E2-F1, that is involved in the conjugation and degradation of p53, is also required for the limited processing of the p105 precursor, and (d) a novel, approximately 320-kDa species of ubiquitin-protein ligase, is involved in the process. This novel enzyme is distinct from E6-AP, the p53-conjugating ligase, and from E3 alpha, the "N-end rule" ligase.

Animals↗

PubMed1998

Structure of ubiquitin-conjugating enzyme 9 displays significant differences with other ubiquitin-conjugating enzymes which may reflect its specificity for sumo rather than ubiquitin.

The three-dimensional structure of ubiquitin-conjugating enzyme 9 (Ubc9) has been obtained to a resolution of 2.8 A by molecular replacement followed by a combination of automated refinement and graphical intervention. Diffraction data were recorded on a single crystal in space group P43 with cell dimensions a = b = 73.9, c = 42. 9 A. The final model has an R factor of 21.3% for all data to 2.8 A. Only the N-terminal methionine, a two-residue N-terminal extension and a four-residue loop are not located by the final electron-density map. Ubc9 is now known to be the first sumo, a new ubiquitin-like protein, conjugating enzyme and does not conjugate ubiquitin. The structure of Ubc9 shows important differences compared with the structures of known ubiquitin-conjugating enzymes. At the N-terminal helix, the structural and sequence alignments are out of register by one amino acid giving Ubc9 a different recognition surface compared to ubiquitin-conjugating enzymes. This is coupled to a profound change in the electrostatic surface of the molecular face remote from the catalytic site. These differences may be important in recognition of other proteins in the Sumo conjugation pathway. The catalytic cysteine in Ubc9 has a positively charged lip and a negatively charged ridge nearby. Both these features seem confined to sumo-conjugating enzymes, and a sequence alignment of sumo and ubiquitin suggests how these might play a role in sumo/ubiquitin discrimination.

Amino Acid Sequence↗

PubMed1990

A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin.

Target protein multi-ubiquitination involving lysine 48 of ubiquitin (Ub) is known to occur during protein degradation in the ATP- and Ub-dependent proteolytic pathway (Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989) Science 243, 1576-1583). However, little is known about the enzymatic mechanism of multi-ubiquitination. We show that a purified Ub carrier protein, E2(25)K, catalyzes multi-Ub chain synthesis from purified Ub. Incubation of E2(25)K with Ub activating enzyme (E1), MgATP, and radiolabeled Ub (Mr = 8500) resulted in time dependent appearance of a "ladder" of radiolabeled Ub conjugates with molecular masses of 8.5n kDa, where n = 1, 2, 3, 4... (up to at least n = 10). The kinetics of this conjugative process were consistent with Ub2 acting as a steady-state intermediate. The putative Ub2 product of E2(25)K catalysis was purified and cleaved with a partially purified isopeptidase preparation. The sole cleavage product (Mr = 8500) had a tryptic digest identical to that of authentic Ub, confirming that the original conjugate was Ub2. Tryptic digestion of intact Ub2 gave products consistent with the existence of an isopeptide linkage between the COOH terminus of one Ub and Lys-48 of the other; this structure was confirmed by sequence analysis of the unique Ub2 tryptic fragment. Tryptic digestion of higher order Ubn adducts (n greater than or equal to 4) yielded fragments identical to those of Ub2, indicating that E2(25)K ligates successive Ub molecules primarily or exclusively via Lys-48. Although several other E2s supported synthesis of an apparent Ub2 adduct of undetermined linkage, only E2(25)K was capable of synthesizing multi-Ub chains from isolated Ub. Quantitative analysis of single turnovers showed that transfer from E2(25)K-Ub to Ub and Ub2 occurred with kappa 2 = 488 and 1170 M-1 min-1, respectively, at pH 7.3 and 37 degrees C. These results show that increasing the number of Ub molecules in a chain increases susceptibility to further ubiquitination by E2(25)K. Ub2 was a good substrate for activation by E1 and was readily transferred to E2(25)K. The labile E2(25)K-Ub2 adduct was catalytically active, and exhibited preference for Ub2 (versus Ub) as acceptor. These results suggest that E2(25)K may function as a multi-ubiquitinating enzyme in the Ub-dependent proteolytic pathway.

Amino Acid Sequence↗

PubMed2004

Bortezomib rapidly suppresses ubiquitin thiolesterification to ubiquitin-conjugating enzymes and inhibits ubiquitination of histones and type I inositol 1,4,5-trisphosphate receptor.

The proteasome inhibitor bortezomib is an emerging anticancer agent. Although the proteasome is clearly its locus of action, the early biochemical consequences of bortezomib treatment are poorly defined. Here, we show in cultured cells that bortezomib and other proteasome inhibitors rapidly inhibit free ubiquitin levels and ubiquitin thiolesterification to ubiquitin-conjugating enzymes. Inhibition of thiolesterification correlated with a reduction in the ubiquitination of certain substrates, exemplified by a dramatic decline in histone monoubiquitination and a decrease in the rate of inositol 1,4,5-trisphosphate receptor polyubiquitination. Thus, in addition to the expected effect of blocking the degradation of polyubiquitinated substrates, bortezomib can also inhibit ubiquitination. The effect of bortezomib on histone monoubiquitination may contribute to its therapeutic actions.

Animals↗

PubMed2001

Ubiquitin-associated (UBA) domains in Rad23 bind ubiquitin and promote inhibition of multi-ubiquitin chain assembly.

Rad23 is a DNA repair protein that promotes the assembly of the nucleotide excision repair complex. Rad23 can interact with the 26S proteasome through an N-terminal ubiquitin-like domain, and inhibits the assembly of substrate-linked multi-ubiquitin (multi-Ub) chains in vitro and in vivo. Significantly, Rad23 can bind a proteolytic substrate that is conjugated to a few ubiquitin (Ub) moieties. We report here that two ubiquitin-associated (UBA) domains in Rad23 form non-covalent interactions with Ub. A mutant that lacked either UBA sequence was capable of blocking the assembly of substrate-linked multi-Ub chains, although a mutant that lacked both UBA domains was significantly impaired. These studies suggest that the interaction with Ub is required for Rad23 activity, and that other UBA-containing proteins may have a similar function.

Amino Acid Sequence↗

PubMed1995

Increased ubiquitin expression suppresses the cell cycle defect associated with the yeast ubiquitin conjugating enzyme, CDC34 (UBC3). Evidence for a noncovalent interaction between CDC34 and ubiquitin.

The yeast ubiquitin (Ub) conjugating enzyme CDC34 plays a crucial role in the progression of the cell cycle from the G1 to S phase. In an effort to identify proteins that interact with CDC34 we undertook a genetic screen to isolate genes whose increased expression suppressed the cell cycle defect associated with the cdc34-2 temperature-sensitive allele. From this screen, the poly-Ub gene UBI4 was identified as a moderately strong suppressor. The fact that the overexpression of a gene encoding a single Ub protein could also suppress the cdc34-2 allele indicated that suppression was related to the increased abundance of Ub. Ub overexpression was found to suppress two other structurally unrelated cdc34 mutations, in addition to the cdc34-2 allele. In all three cases, suppression depended on the expression of Ub with an intact carboxyl terminus. Only the cdc34-2 allele, however, could be suppressed by Ub with an amino acid substitution at lysine 48 which is known to be involved in multi-Ub chain assembly. Genetic results showing allele specific suppression of cdc34 mutations by various Ub derivatives suggested a specific noncovalent interaction between Ub and CDC34. Consistent with this prediction, we have shown by chemical cross-linking the existence of a specific noncovalent Ub binding site on CDC34. Together, these genetic and biochemical experiments indicate that Ub suppression of these cdc34 mutations results from the combined contributions of Ub-CDC34 thiol ester formation and a noncovalent interaction between Ub and CDC34 and therefore suggest that the correct positioning of Ub on a surface of the ubiquitin conjugating enzyme is a requirement of enzyme function.

Alleles↗

PubMed1991

Axonal transport of two major components of the ubiquitin system: free ubiquitin and ubiquitin carboxyl-terminal hydrolase PGP 9.5.

Ubiquitin (Ub), a stress protein thought to target abnormal proteins for degradation, is present in abnormal structures that occur in neuronal perikarya and axons of degenerative diseases including Alzheimer disease. To begin to assess the role of the Ub system in the axon, we studied expression and axonal transport of Ub and other stress proteins, as well as of Ub carboxyl-terminal hydrolase PGP 9.5, in the rat visual system in normal conditions and following heat-shock (HS). In the retina, both the constitutive and inducible forms of HSPs 70 were expressed under normal conditions, while in the superior colliculus the inducible form was detected only following HS. Ub, PGP 9.5 and HSPs 70 were transported in the axon exclusively with the slow component b (SCb), known to carry cytoskeletal and cytoplasmic proteins. The exceedingly long time needed for stress proteins to reach distant axonal locales at the rate of SCb (approximately 3 mm/day) makes it unlikely that they could contribute significantly to the stress response at those sites.

Animals↗

PubMed2005

Lys6-modified ubiquitin inhibits ubiquitin-dependent protein degradation.

Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys(6) of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys(6) was the most readily labeled with sulfosuccinimidobiotin. Lys(6)-biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys(6)-biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys(6)-biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys(6) had similar phenotypes as Lys(6)-biotinylated ubiquitin. Lys(6) mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys(6)-modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys(6)-modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.

Adenosine Triphosphate↗

PubMed1994

Substrate properties of site-specific mutant ubiquitin protein (G76A) reveal unexpected mechanistic features of ubiquitin-activating enzyme (E1).

Ubiquitin-mediated proteolysis proceeds via the formation and degradation of ubiquitin-protein conjugates. Ubiquitin (Ub)-activating enzyme (E1) catalyzes the first, MgATP-dependent step in the conjugative reaction sequence. With wild type ubiquitin, the product of the E1 reaction is a ternary complex (E1-Ub-AMP-Ub) containing one thiol-linked ubiquitin (via the Ub COOH terminus, Gly-76) and one tightly bound ubiquitin adenylate. The thiol-linked ubiquitin is subsequently transferred to the thiol of a ubiquitin-conjugating enzyme (E2 protein); the latter adduct is the proximal donor of ubiquitin to the target protein. A mutant ubiquitin, bearing a Gly to Ala substitution at the COOH terminus (G76A-ubiquitin), was characterized as a substrate for E1. G76A-ubiquitin 1) supported PPi-ATP exchange poorly (500-fold decrease in kcat/K(m); 2) did not produce detectable AMP-Ub with native E1; 3) produced stoichiometric AMP-Ub with thiol-blocked E1; 4) gave a stoichiometric burst of ATP consumption (1 mol/mol E1) with either native or thiol-blocked E1; 5) supported E1-ubiquitin thiol ester formation with native E1; 6) supported several downstream reactions of the proteolytic pathway at approximately 20% of the rate of wild type ubiquitin. These results indicate that G76A-ubiquitin gives a binary E1 thiol ester complex with native E1, due to the failure of the E1-ubiquitin thiol ester to undergo another round of adenylate synthesis; thus AMP-Ub is detected only if adenylate to thiol transfer is prevented by alkylating E1. The inability of G76A-ubiquitin to support ternary complex formation has implications for E1 active site structure. In other experiments, occupancy of the nucleotide/adenylate site of E1, by either MgATP or AMP-Ub, was found to stimulate ubiquitin transthiolation between E1 and E2 proteins. The intermediacy of ubiquitin adenylate thus provides a previously unrecognized catalytic advantage in the E1 mechanism.

Adenosine Monophosphate↗

PubMed1997

A ubiquitin-specific protease that efficiently cleaves the ubiquitin-proline bond.

Ubiquitin is a small eukaryotic protein that is synthesized naturally as one of several fusion proteins, which are processed by ubiquitin-specific proteases to release free ubiquitin. The expression of heterologous proteins as fusions to ubiquitin in either prokaryotic or eukaryotic hosts often dramatically enhances their yield, and allows the exposure of any amino acid following cleavage of ubiquitin. The single exception is when proline is the amino acid immediately following ubiquitin; the ubiquitin-proline bond is poorly cleaved by presently studied ubiquitin-specific proteases. We show that the mouse ubiquitin-specific protease Unp, and its human homolog Unph, can efficiently cleave the ubiquitin-proline bond in ubiquitin fusion proteins of different sizes. N-terminal sequencing of the cleavage products reveals that cleavage occurs precisely at the ubiquitin-proline junction. The biological significance of this cleavage activity is unclear, as ubiquitin-proline fusions do not occur naturally. However, it may indicate a different catalytic mechanism for these ubiquitin-specific proteases and/or that they can cleave ubiquitin-like proteins. Unp and Unph thus represent versatile ubiquitin-specific proteases for cleaving ubiquitin-fusion proteins in biotechnology and basic research, regardless of both the amino acid immediately following ubiquitin, and the size of the fusion partner.

Amino Acid Sequence↗

PubMed1991

Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain.

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.

Animals↗