Ubiquitin: not just for proteasomes anymore

Ubiquitin is a small protein that can be covalently linked to itself or other proteins, either as single ubiquitin molecules or as chains of polyubiquitin. Addition of ubiquitin to a target protein requires a series of enzymatic activities (by ubiquitin-activating, -conjugating and -ligating enzymes). The first function attributed to ubiquitin was the covalent modification of misfolded cytoplasmic proteins, thereby directing proteasome-dependent proteolysis. More recently, additional functions have been ascribed to ubiquitin and ubiquitin-related proteins. Ubiquitin directs specific proteins through the endocytic pathway by modifying cargo proteins, and possibly also components of the cytoplasmic protein trafficking machinery.     

The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.     

Monoubiquitin carries a novel internalization signal that is appended to activated receptors

Ubiquitin modification of signal transducing receptors at the plasma membrane is necessary for rapid receptor internalization and downregulation. We have investigated whether ubiquitylation alters a receptor cytoplasmic tail to reveal a previously masked internalization signal, or whether ubiquitin itself carries an internalization signal. Using an alpha-factor receptor-ubiquitin chimeric protein, we demonstrate that monoubiquitin can mediate internalization of an activated receptor that lacks all cytoplasmic tail sequences. Furthermore, fusion of ubiquitin in-frame to the stable plasma membrane protein Pma1p stimulates endocytosis of this protein. Ubiquitin does not carry a functional tyrosine- or di-leucine-based internalization signal. Instead, the three-dimensional structure of the folded ubiquitin polypeptide carries an internalization signal that consists of two surface patches surrounding the critical residues Phe4 and Ile44. We conclude that ubiquitin functions as a novel regulated internalization signal that can be appended to a plasma membrane protein to trigger downregulation.     

Ubiquitin depletion as a key mediator of toxicity by translational inhibitors

Cycloheximide acts at the large subunit of the ribosome to inhibit translation. Here we report that ubiquitin levels are critical for the survival of Saccharomyces cerevisiae cells in the presence of cycloheximide: ubiquitin overexpression confers resistance to cycloheximide, while a reduced ubiquitin level confers sensitivity. Consistent with these findings, ubiquitin is unstable in yeast (t(1/2) = 2 h) and is rapidly depleted upon cycloheximide treatment. Cycloheximide does not noticeably enhance ubiquitin turnover, but serves principally to block ubiquitin synthesis. Cycloheximide also induces UBI4, the polyubiquitin gene. The cycloheximide-resistant phenotype of ubiquitin overexpressors is also characteristic of partial-loss-of-function proteasome mutants. Ubiquitin is stabilized in these mutants, which may account for their cycloheximide resistance. Previous studies have reported that ubiquitin is destabilized in the absence of Ubp6, a proteasome-associated deubiquitinating enzyme, and that ubp6 mutants are hypersensitive to cycloheximide. Consistent with the model that cycloheximide-treated cells are ubiquitin deficient, the cycloheximide sensitivity of ubp6 mutants can be rescued either by ubiquitin overexpression or by mutations in proteasome subunit genes. These results also show that ubiquitin wasting in ubp6 mutants is proteasome mediated. Ubiquitin overexpression rescued cells from additional translational inhibitors such as anisomycin and hygromycin B, suggesting that ubiquitin depletion may constitute a widespread mechanism for the toxicity of translational inhibitors.     

Simplified methods for isolation of ubiquitin from erythrocytes. Generation of ubiquitin polymers

1. Ubiquitin has been isolated from bovine erythrocytes by procedures in which the hemoglobin was removed by denaturation with either ethanol-chloroform mixtures or by heating. 2. The proteins soluble to the denaturation step were removed by 3% sodium trichloroacetate (TCA) at pH 2.0-2.5 or by 5% TCA. 3. Ubiquitin was isolated in relatively high yield from the TCA insoluble fraction by use of single ion-exchange chromatographic and gel permeation steps. 4. Ubiquitin shows relatively little cross-linking upon treatment with glutaraldehyde or with dimethyl suberimidate. Heating of the glutaraldehyde treated material in 4 M guanidine, however, leads to marked aggregation. 5. The polymers of ubiquitin react strongly with antibody in an immunoblot assay.     

Immunoelectron microscopic localization of ubiquitin in hepatoma cells.

Ubiquitin, a 76 amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Free (unconjugated) ubiquitin was localized in hepatoma cells using affinity purified anti-ubiquitin antibodies and colloidal gold immunoelectron microscopy. The anti-ubiquitin antibodies recognize only unconjugated ubiquitin. Ubiquitin is found within the cytoplasm, nucleus, the microvilli, autophagic vacuoles and lysosomes.     

Isolation, characterization, and esterase and CO2 hydration activities of ubiquitin from bovine erythrocytes

Ubiquitin was isolated from bovine erythrocytes by a relatively simple procedure involving extraction with chloroform and ethanol, chromatography on DEAE-cellulose, and gel filtration. Amino acid and partial sequence analyses showed it to be identical to previously isolated material. Ubiquitin released p-nitrophenolate from p-nitrophenyl acetate, but did not cleave other esterase substrates that were tested. It had a turnover number of 116 mmol for p-nitrophenyl acetate at pH 7.7 and 30 degrees C, and this activity was relatively stable to heat treatment. Electrophoretic studies indicated that the ubiquitin was sequentially acetylated by p-nitrophenyl acetate, as judged by the appearance of more anodically migrating components. The reactions of ubiquitin with p-nitrophenyl acetate at pH 7.0 were biphasic and consisted of (a) an initial phase, during which the release of p-nitrophenol resulted from monoacetylation of the ubiquitin and from ubiquitin-catalyzed hydrolysis of the ester; and (b) a second phase, during which the release of p-nitrophenol resulted only from the breakdown and reformation of the acetyl-enzyme complex. Ubiquitin also showed CO2 hydration activity and could be localized following gel electrophoresis by the CO2-bromthymol blue staining method. The strong inhibitor of carbonic anhydrase, acetazolamide, also inhibited the CO2 hydration activity and p-nitrophenyl acetate activity of ubiquitin. An antibody against this protein did not precipitate bovine carbonic anhydrase II. The esterase activity of ubiquitin was much higher than those previously reported for the carbonic anhydrases.     

The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences.

Ubiquitin coding sequences were isolated from a human genomic library and two cDNA libraries. One human ubiquitin gene consists of 2055 nucleotides and codes for a polyprotein consisting of 685 amino acid residues. The polyprotein contains nine direct repeats of the ubiquitin amino acid sequence and the last ubiquitin sequence is extended with an additional valyl residue at the C-terminal end. No spacer sequences separate the ubiquitin repeats and the coding regions are not interrupted by intervening sequences. This particular gene is transcribed since cDNAs corresponding to the genomic sequence have been isolated. At least two more types of ubiquitin genes are encoded in the human genome, one coding for an ubiquitin monomer while another presumably codes for three or four direct repeats of the ubiquitin sequence. Human DNA contains many copies of the ubiquitin sequence. Ubiquitin is therefore encoded in the human genome as a multigene family.     

Dynamic survey of mitochondria by ubiquitin

Ubiquitin is a post‐translational modifier with proteolytic and non‐proteolytic roles in many biological processes. At mitochondria, it performs regulatory homeostatic functions and contributes to mitochondrial quality control. Ubiquitin is essential for mitochondrial fusion, regulates mitochondria‐ER contacts, and participates in maternal mtDNA inheritance. Under stress, mitochondrial dysfunction induces ubiquitin‐dependent responses that involve mitochondrial proteome remodeling and culminate in organelle removal by mitophagy. In addition, many ubiquitin‐dependent mechanisms have been shown to regulate innate immune responses and xenophagy. Here, we review the emerging roles of ubiquitin at mitochondria.     

IAPS and ubiquitylation

The Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death, whose amplification has been correlated with tumor progression. Due to the presence of a RING domain, IAP proteins are classed as ubiquitin ligases and regulate cell survival by orchestrating a variety of ubiquitin modifications. Ubiquitin protein modification is fundamental in cell signaling and different ubiquitin modifications may label proteins for destruction, relocalization or provide a recruitment platform for ubiquitin binding proteins. Ubiquitin performs a myriad of different functions because it can be conjugated to a large range of target proteins through numerous different types of ubiquitin linkages. Despite the fact that ubiquitin is extremely versatile, the E3s such as the IAPs provide an important level of control due to their specificity for certain substrates. Several recent reviews have discussed the role of IAPs in regulating immune signaling so we have therefore focused our review on the interplay between IAPs and ubiquitin and discussed the importance of this relationship for the regulation of themselves, specific substrates and various cell death and survival signaling pathways.     

泛素连接酶的结构与功能研究进展

泛素化是体内蛋白质翻译后重要修饰之一,是蛋白质降解的信号.泛素连接酶E3是泛素化过程中的关键酶之一,介导活化的泛素从结合酶E2转移到底物,不同的泛素连接酶作用于不同的底物蛋白,决定了泛素化修饰的特异性.根据结构与功能机制的不同,可将泛素连接酶E3分为HECT (homologousto E6AP C terminus)家族和RING-finger家族,前者含有HECT结构域,可直接与泛素连接再将其传递给底物.RING-finger家族的E3发现较晚,庞大且功能复杂,是近年来研究的热点,此家族均包含相似的E2结合结构域和特异的底物结合部分,作为桥梁将活化的泛素从E2直接转移到靶蛋白,其本身并不与泛素发生作用.总结了这2种E3连接酶家族成员的三维结构及功能机制研究的最新进展.     

Ubiquitin in stressed chicken embryo fibroblasts

Ubiquitin, a small 76-amino acid protein which is highly conserved in eukaryotic cells, occurs in several forms other than the free polypeptide. Among these are protein conjugates in which ubiquitin is covalently linked in lysylpeptide bond to lysl residues of other proteins and fusion proteins in which the amino-terminal domain is the precise ubiquitin sequence. Ubiquitin plays a role in cellular proteolytic degradation and in chromatin structure and has been postulated to be involved in the induction of a set of proteins which function during the cellular response to various kinds of environmental stress. We have measured the various forms of ubiquitin in cultures of chicken embryo fibroblasts under normal growth conditions and after treatment with a thermal or chemical stress. Levels of free ubiquitin fell slightly, ubiquitin conjugate levels rose shortly after stress began, and both then increased substantially as one of the cell's ubiquitin-encoding genes was activated by stress. The level of a protein synthesized as the carboxyl-terminal domain of one ubiquitin fusion protein was unchanged by a heat stress. The most dramatic effect was seen in the rapid disappearance of the ubiquitinated form of histone H2A, one of the major ubiquitin conjugates in cells in the interphase portion of their growth cycle. A significant rise in protein turnover was detected as a result of the stress, but occurred only when cells were removed from the stress condition. These results suggest that ubiquitin plays an important role both during and after stress, but fails to support hypotheses for ubiquitin and proteolysis in the activation of stress genes.     

Evolution and function of ubiquitin-like protein-conjugation systems

Ubiquitin functions by covalently modifying other proteins. In the past few years, a surprising number of other proteins have been identified that, despite often being only slightly similar to ubiquitin, can also be attached to proteins. Newly discovered parallels between the activation of ubiquitin and the biosynthesis of certain enzyme cofactors now hint at the possible evolutionary origins of the ubiquitin system.     

Ubiquitin with a non-ATP-dependent slow intrinsic proteolytic activity: a mild and rapid purification procedure.

Ubiquitin has been purified to homogeneity, through a dialysis membrane having a NMW cutoff of 12 kDa, by taking advantage of its non-dialysable nature under these conditions. The dialysate was continuously recycled through a CM-52 cation exchange column at pH 4.5. The adsorbed fraction was eluted selectively at pH 7.2. Ubiquitin (25 mg) was obtained from 500 ml of packed RBCs. On SDS PAGE, ubiquitin showed varying mobility depending on the time of boiling in SDS. With 2 min of boiling, the molecular weight seemed to be 10.5 kDa, whereas 10 min of boiling resulted in a molecular weight of 8.5 kDa. Ubiquitin showed a slow intrinsic proteolytic activity against SDS-denatured beta-galactosidase in the absence of ATP. For the first 4 hr, there was no detectable degradation, but degradation was nearly complete after 8 hr. These data are not in agreement with those of Freid et al. [Proc. Natl Acad. Sci, USA, 84 (1987), 3685] who have reported a proteolytic activity comparable to that of other proteolytic enzymes.     

Regulation of the ubiquitin-mediated proteolytic pathway: role of the substrate alpha-NH2 group and of transfer RNA

Degradation of intracellular proteins via the ubiquitin pathway involves several steps. In the initial event, ubiquitin becomes covalently linked to the protein substrate in an ATP-requiring reaction. Following ubiquitin conjugation, the protein moiety of the adduct is selectively degraded with the release of free and reusable ubiquitin. Ubiquitin modification of a variety of protein targets in the cell plays a role in basic cellular functions. Modification of core nucleosomal histones is probably involved in regulation of gene expression at the level of chromatin structure. Ubiquitin attachment to cell surface proteins may play roles in processes of cell-cell interaction and adhesion, and conjugation of ubiquitin to other yet to be identified protein(s) could be involved in the progression of cells through the cell cycle. Despite the considerable progress that has been made in the elucidation of the mode of action and cellular roles of the ubiquitin pathway, many major problems remain unsolved. A problem f central importance is the specificity in the ubiquitin ligation system. Why are certain proteins conjugated and committed for degradation, whereas other proteins are not? A free α-NH₂ group is an important feature of the protein structure recognized by the ubiquitin conjugation system, and tRNA is required for the conjugation of ubiquitin to selective proteo-lytic substrates and for their subsequent degradation. These findings can shed light on some of the features of a substrate that render it susceptile to ubiquitin-mediated degradation.     

泛肽系统的组成和功能(一) 一 系统组成 底物识别与蛋白质泛肽化

泛肽(Ubiquitin 简称Ub)是一个由76个氨基酸残基组成的非常保守的小蛋白质。泛肽依赖性的蛋白质降解途径(Ubiquitin-dependebt proteiytic pathway)是目前已知的最重要的、有高度选择性的蛋白质降解途径。泛肽系统由Ub、Ub活比酶、Ub结合酶、Ub-蛋白质连接酶、Ub-C末端水解酶和26S蛋白酶体组成。本文详细地介绍了泛肽系统各个组成部分的种类、结构与功能,蛋白质泛肽化及其降解机制和底物识别模式。     

Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34

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

泛肽系统的组成和功能(一)—系统组成、底物识别与蛋白质泛肽化

泛肽（Ｕｂｉｑｕｉｔｉｎ,简称Ｕｂ）是一个由７６个氨基酸残基组成的非常保守的小蛋白质。泛肽依赖性的蛋白质降解途径（Ｕｂｉｑｕｉｔｉｎ＿ｄｅｐｅｎｄｅｎｔｐｒｏｔｅｏｌｙｔｉｃｐａｔｈｗａｙ）是目前已知的最重要的、有高度选择性的蛋白质降解途径。泛肽系统由Ｕｂ、Ｕｂ活化酶、Ｕｂ结合酶、Ｕｂ＿蛋白质连接酶、Ｕｂ＿Ｃ末端水解酶和２６Ｓ蛋白酶体组成。本文详细地介绍了泛肽系统各个组成部分的种类、结构与功能,蛋白质泛肽化及其降解机制和底物识别模式。