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GREAT EXPERIMENTS
Ubiquitin conjugation as a proteolytic signal: The first experiments

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Avram Hershko and Aaron Ciechanover

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The dynamic turnover of cellular proteins was discovered during the pioneering studies of Rudolf Schoenheimer in the 1930s, when he first used isotopically labeled compounds for biological studies (1815). The discovery in the 1960s that different enzymes in eukaryotic cells have widely varying half lives made it evident that protein degradation is a highly selective process that plays important roles in regulating metabolic processes (1809). However, the molecular mechanisms responsible for this process remained unknown. Up until this time, the lysosome was considered to be the sole site of protein degradation, but it was clear that the activity of lysosomal proteases was not selective. Still, certain studies attempted to attribute selectivity to degradation within lysosomes. According to one model, for example, all cellular proteins would be rapidly engulfed by the lysosome, but only short-lived proteins would be degraded, while long-lived proteins would escape back to the cytosol (1810).

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Using an assay for measuring the release of amino acids from proteins in liver slices, Simpson demonstrated that energy is required for proteolysis (1805). A similar energy requirement for the degradation of many other cellular proteins was observed in a variety of experimental systems (1811). While proteolysis per se is an exergonic process that does not require energy, lysosomal degradation requires metabolic energy for the activity of the proton pump that maintains the low acidic intralysosomal pH. Therefore, the arrest of degradation observed when energy production inhibitors were used could have been a secondary effect of inhibition of the lysosomal proton pump.

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Background

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Despite this possible explanation for a connection between lysosomes and the energy requirement for protein degradation, Hershko was convinced that lysosomal autophagy could not account for the selectivity and regulation of intracellular protein breakdown. Hershko was attracted to the field as a postdoctoral fellow in the laboratory of Gordon Tomkins 30 years ago (1969-71). At that time, the main subject of research in the laboratory was the mechanism by which corticosteroid hormones induce synthesis of tyrosine aminotransferase (TAT). Hershko decided to study degradation, a process that also regulates TAT levels. He found that the degradation of TAT in cultured hepatoma cells is completely arrested by inhibitors of cellular energy production, such as fluoride or azide (1789). These results confirmed and extended the previous observations of Simpson. From this point, Hershko considered the argument of whether selective protein degradation could take place in the lysosome. He concluded that it could not, since once proteins are engulfed by the lysosome, they are degraded at a similar rate, and this degradation is not substrate-specific. He assumed that the cell must be equipped with a yet unknown proteolytic system that utilizes energy for the high selectivity and specificity of protein degradation.

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Experiments of Brian Poole and colleagues lent support to this hypothesis when they demonstrated that degradation of metabolically labeled intracellular proteins is not affected by lysosomotropic agents such as chloroquine. These agents are weak bases that dissipate the low intralysosomal pH and thus inhibit the activity of lysosomal proteases. In striking contrast, the same agents strongly inhibited degradation of pinocytosed extracellular proteins, such as bovine serum albumin (BSA) (4105). These experiments strongly suggested the existence of a proteolytic system(s) that is distinct from the lysosome and that is involved in degradation of intracellular proteins.

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The experiment

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An ATP-dependent proteolytic system from reticulocytes was first described by Etlinger and Goldberg (1806) and a bit later by Hershko and Ciechanover (3768). This was the stage when Aaron Ciechanover joined the Hershko's laboratory as a graduate student. We were convinced that the best way to characterize this novel proteolytic system was to use classical biochemistry to fractionate and reconstitute the system in order to identify, characterize and purify the different components and determine their individual modes of action. Important support and advice were provided by Irwin Rose, who hosted Hershko in his laboratory for a sabbatical year in 1977-78, and also hosted us many times afterwards.

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Figure 1  
Anion exchange chromatography of reticulocyte lysate into two complementing proteolytic activities. (Adapted from 1812.)

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Because of its abundance, the first aim was to remove hemoglobin from the reticulocyte extracts. This could be achieved in a single ion exchange chromatography step over DEAE-cellulose (an anion exchange resin). We resolved the lysate into two fractions: Fraction 1, which contained nonadsorbed proteins (including hemoglobin), and Fraction 2, which contained all the proteins that were adsorbed to the resin and eluted with high salt. We expected that the proteolytic activity would be found in Fraction 2, but the Fraction contained only very low ATP-dependent proteolytic activity towards the radiolabeled denatured globin that we used as a proteolytic substrate. Fraction 1 did not have any activity. The proteolytic activity could be recovered, however, following reconstitution of Fractions 1 and 2, as shown in Figure 1 (1812). This was a surprising finding, as until then proteolytic activities were confined to single proteases, and researchers in the field had been trying to characterize and purify them as such. This critically important experiment taught us that we had at hand a complex system that contained at least two (and more, as it turned out later) complementing factors, rather than a single ATP-dependent protease.

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From then, we used the power of "classical" biochemistry and established a "complementing add and subtract" experimental approach. Using this approach, each novel factor was identified, purified and characterized by assaying its activity (stimulation of ATP-dependent degradation of a labeled protein) against the remaining crude fraction from which it was resolved/removed during the previous purification step. For example, the active component in Fraction 1 was purified to homogeneity via several purification steps and characterized by assaying its stimulatory activity using crude Fraction 2 in the assay (1790). This component in Fraction 1 was designated APF-1 (ATP-dependent Proteolytic Factor-1) to denote that it was the first factor characterized. At the same time we started to identify additional complementing factors from Fraction 2. APF-1 showed some unusual characteristics. It was a small (~8.5 kDa) protein that was purified by taking advantage of its remarkable heat stability: a single step, heating Fraction 1 to 90°C (which causes most proteins to precipitate) resulted in a 210-fold purification of the protein (1812; 1790). The protein we called APF-1 was later identified by Wilkinson and colleagues as ubiquitin (1791), a known protein of hitherto unknown function.

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Ubiquitin was first thought to be a thymic hormone, but subsequently was found to be universally present in many tissues and organisms — hence its name (1807). It was found to be conjugated to a small proportion of histone 2A molecules (1814), but did not target the protein for degradation, and its function in this context has remained largely unknown. Though we did not know at that time that APF-1 is ubiquitin, from the time we discovered the identity between the two we adopted its original name, ubiquitin, coined by the researcher who discovered it. For the purposes of this text, we shall use the term APF-1/ubiquitin to describe experiments carried out prior to the discovery of its common identity.

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Purification of APF-1/ubiquitin from Fraction 1 was the key to the elucidation of its mode of action in the proteolytic system. Initially, we thought that it could be either an activator or a regulatory subunit of a protease, or it could be a component of a multienzyme cascade system, with the remaining components of the pathway being contained in Fraction 2. To examine these alternatives, purified APF-1/ubiquitin was radio-iodinated and incubated with crude Fraction 2 in the presence or absence of ATP. Gel filtration chromatography revealed a dramatic ATP-dependent shift of 125I-APF-1/ubiquitin to the high-molecular mass region (490). Chemical analysis revealed that APF-1/ubiquitin generated a covalent amide linkage with protein(s) in Fraction 2, as the high molecular mass "complex" was stable to treatments with acid, alkali, hydroxylamine and boiling with SDS and mercaptoethanol (490). Analysis of the reaction products by SDS-polyacrylamide gel electrophoresis showed that APF-1/ubiquitin was covalently ligated to numerous proteins. Since crude Fraction 2 from reticulocytes contains many endogenous substrates that are degraded by the system, in addition to enzymes of the proteolytic system itself, we suspected that APF-1/ubiquitin might be linked to protein substrates rather than to enzymes of the system. In support of this interpretation, efficient substrates for ATP-dependent proteolysis, such as lysozyme, were found to form multiple conjugates with APF-1/ubiquitin (though we realized that the substrates we used were artificial, as they were all extracellular rather than intracellular proteins). These multiple adducts represented a ladder where an increasing number of APF-1/ubiquitin moieties are attached to a single substrate molecule (1792).

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Figure 2  
Multiple molecules of APF-1/ubiquitin are covalently attached to the protein substrate targeted for degradation. Complete reaction mixtures including 125I-APF-1/ubiquitin and increasing concentrations of lysozyme were resolved on SDS-PAGE (lanes 3-5), resulting in the appearance of several new bands (C1-C6). These bands were not seen in the absence of ATP (lane 1) or added lysozyme (lane 2; in this case, 125I-APF-1/ubiquitin became ligated to endogenous substrates present in Fraction 2). 125I-labeled lysozyme and unlabeled APF-1/ubiquitin were used in reactions in the absence (lane 6) or presence (lane 7) of ATP. Whether complete reactions were incubated in the presence of labeled lysozyme (lane 7) or labeled APF-1/ubiquitin (lanes 3-5), the same bands (C1-C6) appeared. "Cont" = contaminating protein in lysosomal preparation. (Reproduced from 1792.)

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Figure 2 shows the original experiment that convinced us that APF-1/ubiquitin is ligated to the protein substrate. As can be clearly seen, similar high-molecular weight derivatives (see C3 through C6) were formed when 125I-APF-1/ubiquitin was incubated in the presence of unlabeled lysozyme (lanes 3-5), and when 125I-lysozyme was incubated with unlabeled APF-1/ubiquitin (lane 7). The ~8.0 kDa difference in the molecular mass of the bands corresponds to the size of APF-1/ubiquitin, and analysis of the ratio of radioactivity in APF-1/ubiquitin as compared to lysozyme indicated that the various derivatives consisted of increasing numbers of APF-1/ubiquitin moieties linked to a single molecule of lysozyme.

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Figure 3  
The predicted APF-1/ubiquitin proteolytic pathway, 1980. The following enzymatic steps were proposed: 1. "APF-1-protein amide synthetase" ligates APF-1/ubiquitin to the protein substrate. 2. a "correction" amidase/­isopeptidase that acts upon "erroneously" ligated proteins. 3. a protease that cleaves peptide bonds, liberating peptides, amino acids, and APF-1 linked to a short peptide ("APF-1•X"). 4. an amidase/­isopeptidase that cleaves the bond between APF-1 and lysine residues. (Adapted from 1792.)

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Based on these findings, in 1980 we proposed the model shown in Figure 3 that explains the role of APF-1/ubiquitin in marking intracellular proteins for degradation, rendering them susceptible to recognition by a downstream protease. We suggested that multiple molecules of APF-1/ubiquitin are linked to ε-NH2 groups of internal Lys residues in the protein substrate by an "APF-1-protein amide synthetase" (Step 1). Proteins ligated to multiple APF-1/ubiquitin moieties were proposed to be broken down by a specific protease that recognizes such conjugates (Step 3). The products are free amino acids, peptides, and APF-1/ubiquitin still linked by isopeptide linkage to lysine or a small peptide ("APF-1•X"). Finally, free APF-1/ubiquitin is released for reutilization by the action of a specific amidase/isopeptidase (Step 4). According to a suggestion of Irwin Rose, a hypothetical "correcting" isopeptidase was added to this scheme, that would release free ubiquitin and substrate protein from adducts of erroneously ligated proteins (Step 2). An isopeptidase that may have such correction function has been described recently (1793). We hypothesized that the conjugation step, Step 1 (catalyzed by the putative "APF-1-protein amide synthetase") endows the system with its high degree of specificity towards its many substrates (1811).

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Subsequent work

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Figure 4  
The ubiquitin-proteasome pathway for intracellular protein degradation, 2001. See text and explanations in the figure for description of the pathway. Steps 1, 2, and 3 in this model correspond to Step 1 of the original 1980 model (shown in Figure 3); the light blue arrows at right correspond to Step 2 of the original model; Step 5 corresponds to Step 3 of the original model; and Step 6 corresponds to Step 4 of the original model.

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Comparison of the original model with our current knowledge of the reactions of the ubiquitin pathway (shown in Figure 4) (1819; for reviews see 1813; 996; 1818) (and see Ubiquitination targets proteins for degradation), shows that the original model was essentially correct, but more components were discovered and details added that provide explanations for the high specificity of ubiquitin-mediated proteolysis. Thus, we have found that the predicted "APF-1-protein amide synthetase" is composed of three types of enzymes: a ubiquitin-activating enzyme E1, a ubiquitin-carrier protein, E2, and a ubiquitin-protein ligases, E3 (1794).

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E1 activates ubiquitin, in an ATP-dependent reaction, in which the C-terminal residue of ubiquitin binds to an internal residue in E1 (Step 1). The activated ubiquitin is then transferred through any one of several ubiquitin-carrier proteins, E2s, to the substrate, which is complexed specifically (1795) with a member of one of the two known ubiquitin-protein ligase families, or E3s. In the case of a RING-H2 domain E3, the transfer is direct, from E2 to the E3-bound substrate (Step 2, to the left). In the case of HECT (Homologous to the E6-AP C-T terminal) domain-E3, the transfer is mediated by an E3-ubiquitin intermediate (Step 2, to the right). The two families of E3s, the RING finger- and HECT domain-containing proteins, are composed of many members that recognize specific structural features in the target substrates, and thus account for substrate selectivity. These proteins catalyze the last step in the conjugation process, covalent attachment of ubiquitin to the substrate. The first ubiquitin moiety is transferred to an ε-NH2 group of an internal Lys residue of the protein substrate (although in some cases it can be conjugated linearly to the free α-NH2) to generate an isopeptide bond. This is followed by successive transfer of additional activated ubiquitin moieties to the previously conjugated ubiquitin molecule to generate a polyubiquitin chain (Step 3).

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The chain probably serves as a recognition marker for the protease (Step 4). Additional work led to corroboration of a key step of the model, in which only ubiquitin-tagged proteins are targeted by the protease and then degraded to short peptides (Step 5) (1796). The protease was purified and characterized initially by Rechsteiner and colleagues (1797) and later by Goldberg and colleagues (1798), and was identified as the 26S proteasome complex. It was found that ATP is required not only to catalyze ubiquitin-protein ligation, as originally proposed, but also for the action of the 26S proteasome (1796; 1797; 1798).Finally, free and reusable ubiquitin is released by the action of a large variety of ubiquitin-C-terminal hydrolases (Step 6, reviewed in 1813).

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Because we carried out all of our studies in a cell-free reconstituted system from terminally differentiating reticulocytes and used secretory model proteins as substrates, the problem of the physiological relevance of our initial findings had remained open. The first evidence that the system plays a role in degradation of proteins in vivo came from immunochemical analysis of ubiquitin adducts in nucleated eukaryotic cells (1799). Cells were incubated in the presence of amino acid analogs to induce synthesis of abnormal, rapidly degrading proteins. Antibodies we raised against ubiquitin were then used to monitor the initial level and kinetics of decay of ubiquitin-protein adducts. We found that immediately following incubation of cells with the amino acid analog, the level of the conjugates was significantly higher compared with cells that were incubated without the analog. This reflected the rapid degradation of this pool of abnormal proteins even as their synthesis had just begun. Blocking protein synthesis resulted in the rapid disappearance of the abnormal proteins, along with a concomitant disappearance of the adducts. Thus, the rate of formation and degradation of the short-lived, abnormal proteins was in tight and direct correlation with that of their respective adducts. These findings strongly suggested that the adducts indeed serve as essential intermediates in the proteolytic process.

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A stronger and more direct proof for the involvement of the ubiquitin system in the degradation of cellular proteins came from observations of Alexander Varshavsky, Daniel Finley and Aaron Ciechanover. They found that a cell cycle arrest mutant that harbors a thermolabile E1 is also defective in degradation of abnormal, amino acid analog-containing short-lived proteins at the nonpermissive temperature (1800; 1801). In parallel, the first targeting signal in the substrate (out of many that were later discovered) — the N-terminal residue — was also identified using both a biochemical approach (1802; 1803), and a more thorough and systematic genetic approach (1804). This mode of recognition, which is dependent on the identity of the free N-terminal residue, was designated by Varshavsky and colleagues the "N-end rule" pathway.

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The legacy

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In the two decades that have followed, it has become clear that the proteolysis of ubiquitinated proteins plays a major role in a broad array of basic cellular processes. Among these are regulation of the cell cycle, differentiation and development, the cellular response to stress, modulation of cell surface receptors and ion channels, DNA repair, regulation of the immune and inflammatory responses, biogenesis of organelles, and quality control in the cytosol and the secretory pathway (see Protein degradation is important in mitosis and The translocon forms a pore). The list of cellular proteins targeted by the ubiquitin system has grown exponentially and includes (to mention a few): cell cycle regulators, tumor suppressors and growth modulators, transcriptional activators and their inhibitors, cell surface receptors and endoplasmic reticulum proteins. Mutated or otherwise damaged/misfolded proteins are also recognized specifically and removed rapidly. With the many substrates targeted and processes involved, it is not surprising that aberrations in the system have been implicated in the pathogenesis of many diseases, including malignancies, neurodegeneration, and disorders of the immune and inflammatory responses. Drug companies are attempting to develop drugs based on these mechanisms to modulate the degradation of normal and abnormal proteins in different disease states.

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As pointed out in (1808), the main lesson from our story is the continued importance of the use of biochemistry in modern biomedical research. Without biochemistry, it is doubtful whether an entirely novel system could have been discovered. On the other hand, molecular genetics was essential to uncovering the myriad roles of the system in vivo.

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The authors

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Avram Hershko (left) was born in Karcag, Hungary in 1937. He immigrated to Israel in 1950. He obtained his M.D. degree in 1964 and his Ph.D. degree in 1969 from "Hadassah" and the Hebrew University School of Medicine in Jerusalem, Israel. During the years 1967-69, Avram served in the Israel Defense Forces (I.D.F.) as a military physician. Following his graduate studies, Avram joined the laboratory of Gordon Tomkins in the University of California in San Francisco in the U.S. where he initiated his studies on intracellular protein degradation. He returned to Israel in 1972 and joined the newly established Faculty of Medicine in the Technion-Israel Institute of Technology, in Haifa, where he continued his studies on protein degradation. In 1976, Aaron Ciechanover joined his laboratory as a graduate student, and together they discovered the ubiquitin proteolytic system. Avram is currently a Professor of Biochemistry in the Faculty of Medicine of the Technion. Along with Aaron Ciechanover and Alexander Varshavsky, he shared the 2000 Lasker Award for Basic Medical Research.

Aaron Ciechanover (right) was born in Haifa, Israel in 1947. He obtained his M.Sc. Degree in 1970 and his M.D. degree in 1972 from "Hadassah" and the Hebrew University School of Medicine in Jerusalem, Israel. Following national service in the Israel Defense Forces (I.D.F.) as a military physician, Aaron started his graduate studies in the laboratory of Avram Hershko at the Faculty of Medicine of the Technion-Israel Institute of Technology, in Haifa, Israel. During his studies (1976-81), they discovered the ubiquitin proteolytic system. After completing his Ph.D. thesis, Aaron joined Harvey Lodish's laboratory at the Massachusetts Institute of Technology in the U.S. While there, he studied receptor-mediated endocytosis and also different aspects of the ubiquitin system, some in collaboration with Alexander Varshavsky and Daniel Finley, and some independently. Following his postdoctoral training, Aaron returned to the Faculty of Medicine of the Technion in Haifa, Israel, where he is currently a Professor. He shared the 2000 Lasker Award for Basic Medical Research with Avram Hershkoand Alexander Varshavsky.


Avram Hershko
Aaron Ciechanover
Department of Biochemistry
The Bruce Rappaport Faculty of Medicine
and the Rappaport Family Institute for Research in the Medical Sciences
Technion-Israel Institute of Technology
Haifa 31096, Israel
Phone: 972 4 829 5356
Fax: 972 4 851 3922
E-mail: hershko@tx.technion.ac.il
E-mail: mdaaron@tx.technion.ac.il

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Last Revised on October 22, 2004

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reviews
  • 996 Voges, D., Zwickl, P., and Baumeister, W. (1999).  The 26S proteasome: a molecular machine designed for controlled proteolysis.  Annu. Rev. Biochem. 68, 1015-1068.  PubMed   Journal
  • 1808 Hershko, A. (1996).  Lessons from the discovery of the ubiquitin system.  Trends Biochem. Sci. 21, 445-449.  PubMed   Journal
  • 1809 Schimke, R.T., and Doyle, D. (1970).  Control of enzyme levels in animal tissues.  Annu. Rev. Biochem. 39, 929-976.  PubMed   Journal
  • 1811 Hershko, A. and Ciechanover, A. (1982).  Mechanisms of intracellular protein breakdown.  Annu. Rev. Biochem. 51, 335-364.  PubMed   Journal
  • 1813 Hershko, A., and Ciechanover, A. (1998).  The ubiquitin system.  Annu. Rev. Biochem. 67, 425-479.  PubMed   Journal
  • 1815 Schoenheimer, R. (1942). The Dynamic State of Body Constituents (Cambridge, MA: Harvard University Press).
  • 1818 Weissman, A. M. (2001).  Themes and variations on ubiquitylation.  Nat. Rev. Mol. Cell Biol. 2, 169-178.  PubMed   Journal
  • 1819 Hilt, W., and Wolf, D.H (2000). Proteasomes: The World of Regulatory Proteolysis (Austin, TX: Landes Bioscience), pp. 1-139.
  • 3768 Hershko, A., Heller, H., Ganoth, D., and Ciechanover, A. (1978).  Mode of degradation of abnormal globin chains in rabbit reticulocytes. In Protein Turnover and Lysosome Function Segal, H L and Doyle, D J, eds. (New York: Academic Press), pp. 149-169.
reviews
  • 490 Ciechanover, A. et al. (1980).  ATP-dependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation.  Proc. Natl. Acad. Sci. USA 77, 1365-1368.  PubMed  
  • 1789 Hershko, A. and Tomkins, G. M. (1971).  Studies on the degradation of tyrosine aminotransferase in hepatoma cells in culture. Influence of the composition of the medium and adenosine triphosphate dependence.  J. Biol. Chem. 246, 710-714.  PubMed  
  • 1790 Ciechanover, A., Elias, S., Heller, H., Ferber, S., and Hershko, A. (1980).  Characterization of the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes.  J. Biol. Chem. 255, 7525-7528.  PubMed  
  • 1791 Wilkinson, K. D., Urban, M. K., and Haas, A. L. (1980).  Ubiquitin is the ATP-dependent proteolysis factor I of rabbit reticulocytes.  J. Biol. Chem. 255, 7529-7532.  PubMed  
  • 1792 Hershko, A., Ciechanover, A., Heller, H., Haas, A. L., and Rose, I. A. (1980).  Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.  Proc. Natl. Acad. Sci. USA 77, 1783-1786.  PubMed  
  • 1793 Lam, Y. A., Xu, W., DeMartino, G. N., and Cohen, R. E. (1997).  Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome.  Nature 385, 737-740.  PubMed  
  • 1794 Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983).  Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown.  J. Biol. Chem. 258, 8206-8214.  PubMed  
  • 1795 Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986).  The protein substrate binding site of the ubiquitin-protein ligase system.  J. Biol. Chem. 261, 11992-11999.  PubMed  
  • 1796 Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984).  ATP-dependent degradation of ubiquitin-protein conjugates.  Proc. Natl. Acad. Sci. USA 81, 1619-1623.  PubMed  
  • 1797 Hough, R., Pratt, G., and Rechsteiner, M. (1986).  Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates.  J. Biol. Chem. 261, 2400-2408.  PubMed   Journal
  • 1798 Waxman, L., Fagan, J. M., and Goldberg, A. L. (1987).  Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes, one of which degrades ubiquitin conjugates.  J. Biol. Chem. 262, 2451-2457.  PubMed  
  • 1799 Hershko, A., Eytan, E., Ciechanover, A., and Haas, A. L. (1982).  Immunochemical analysis of the turnover of ubiquitin-protein conjugates in intact cells. Relationship to the breakdown of abnormal proteins.  J. Biol. Chem. 257, 13964-13970.  PubMed  
  • 1800 Finley, D., Ciechanover, A., and Varshavsky, A. (1984).  Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85.  Cell 37, 43-55.  PubMed   Journal
  • 1801 Ciechanover, A., Finley, D., and Varshavsky, A. (1984).  Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85.  Cell 37, 57-66.  PubMed   Journal
  • 1802 Hershko, A., Heller, H., Eytan, E., Kaklij, G., and Rose, I. A. (1984).  Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown.  Proc. Natl. Acad. Sci. USA 81, 7021-7025.  PubMed  
  • 1803 Ferber, S. and Ciechanover, A. (1987).  Role of arginine-tRNA in protein degradation by the ubiquitin pathway.  Nature 326, 808-811.  PubMed   Journal
  • 1804 Bachmair, A., Finley, D., and Varshavsky, A. (1986).  In vivo half-life of a protein is a function of its amino-terminal residue.  Science 234, 179-186.  PubMed  
  • 1805 Simpson, M.V. (1953).  The release of labeled amino acids from proteins in liver slices.  J. Biol. Chem. 201, 143-154.  PubMed  
  • 1806 Etlinger, J. D. and Goldberg, A. L. (1977).  A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes.  Proc. Natl. Acad. Sci. USA 74, 54-58.  PubMed  
  • 1807 Goldstein, G., Scheid, M., Hammerling, U., Schlesinger, D. H., Niall, H. D., and Boyse, E. A. (1975).  Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells.  Proc. Natl. Acad. Sci. USA 72, 11-15.  PubMed  
  • 1810 Haider, M. and Segal, H. L. (1972).  Some characteristics of the alanine aminotransferase- and arginase-inactivating system of lysosomes.  Arch. Biochem. Biophys. 148, 228-237.  PubMed  
  • 1812 Ciehanover, A., Hod, Y., and Hershko, A. (1978).  A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes.  Biochem. Biophys. Res. Commun. 81, 1100-1105.  PubMed  
  • 1814 Goldknopf, I. L. and Busch, H. (1977).  Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A24.  Proc. Natl. Acad. Sci. USA 74, 864-868.  PubMed  
  • 4105 Poole, B., Ohkuma, S., and Warburton, M. (1978).  Some aspects of the intracellular breakdown of exogenous and endogenous proteins. In Protein Turnover and Lysosome Function Segal, H. L. and Doyle, D. J. , eds. (New York: Academic Press), pp. 43-58.

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