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GREAT EXPERIMENTS
The physiological functions of the ubiquitin system

Alex Varshavsky

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Background

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Proteolysis, or protein degradation, is a set of processes that result in the hydrolysis of one or more of the peptide bonds in a protein. That most protein molecules do not last as long as the cell in which they reside was suggested more than 60 years ago, by studies that utilized stable isotopes to label proteins in vivo (1815). The prevailing view, until the 1980s, was that intracellular protein degradation was a simple and even mundane process, serving largely to dispose of "aged" or otherwise damaged proteins. Cellular regulation was believed to be a separate affair, mediated primarily by repressors and activators of gene expression, which were assumed to be long-lived. Thus, most people studying gene expression in the 1960s and 1970s assumed that the regulatory circuits they cared about did not involve short-lived proteins. As we know now, just the opposite proved true, especially in eukaryotic cells, where many transcriptional regulators are conditionally short-lived proteins whose levels are determined as much by the rates of their ubiquitin-dependent proteolysis as by their rates of synthesis.

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Ubiquitin is a 76-residue protein that was first identified by G. Goldstein and colleagues, who detected it in many different organisms. The first covalent ubiquitin conjugate, to histone H2A, was described by H. Busch and colleagues (1814). In 1978, A. Hershko and his graduate students A. Ciechanover and Y. Hod reported that a small heat-stable protein is required for the degradation of denatured globin added to rabbit reticulocyte extracts (4358). In 1980, Hershko and colleagues demonstrated that this heat-stable protein, ATP-dependent proteolytic factor 1 (APF-1), is conjugated to proteins in an ATP-dependent reaction, and proposed that APF-1 conjugation marked proteins for selective degradation (1792). Also in 1980, K. Wilkinson, M. Urban, and A. Haas (1791) showed that APF-1 and ubiquitin are the same protein.

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Figure 1  
The ubiquitin system as known in 1984. The ~ designates ubiquitin (u) conjugated to enzymes by high-energy (thioester) bonds. The subscript n denotes the number of ubiquitin moieties covalently bound to the acceptor protein. The steps from un+1-protein to "peptides" involve the 26S proteasome, an ATP-dependent protease that had not yet been identified directly.

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In 1980-83, the Hershko laboratory dissected the enzymatic cascade of ubiquitin conjugation (see Ubiquitin conjugation as a proteolytic signal: The first experiments). As shown in Figure 1, the initial step involves ubiquitin-activating enzyme E1, which catalyzes an ATP-dependent reaction in which the C-terminal Gly residue of ubiquitin is covalently linked, via a thioester bond, to a specific Cys residue of E1. The activated, E1-linked ubiquitin moiety is transesterified to specific Cys residues of the ubiquitin-conjugating enzymes (E2s), which thereafter mediate the conjugation of ubiquitin to ε-amino groups of Lys residues in substrate proteins (4359). Yet another set of proteins, E3s, is also required for the conjugation reaction; E3 proteins play a role in substrate recognition (4359). Proteins conjugated to ubiquitin are targeted for degradation by a large ATP-dependent protease that became known later as the 26S proteasome (reviewed in 4360). (For a historical account of the early biochemical work, see 4361.) These studies defined the enzymology of ubiquitin conjugation, but did not address the nature of its specificity and particularly its physiological functions. Consequently, there was, at the time, widespread skepticism concerning the physiological significance of this protein modification.

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Introduction

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I emigrated from Russia to the U.S. in 1977, joined the faculty of MIT's Biology Department, and resumed research on the structure of chromosomes, the subject of my studies in Moscow. In 1978, I came across the paper by the Busch laboratory that described the ubiquitin-H2A conjugate (Ub-H2A) (1814) and decided to determine the distribution of this ubiquitin conjugate amongst the chromatin's nucleosomes. Back in Russia, I had begun to develop a method for high-resolution analysis of nucleosomes. These DNA-protein complexes were subjected to electrophoresis in a low-ionic-strength polyacrylamide gel (a forerunner of the gel shift assay), followed by second-dimension electrophoresis of either DNA or proteins. L. Levinger, then a postdoctoral student, and I located Ub-H2A in a subset of the nucleosomes, separated these nucleosomes from those lacking Ub-H2A, and showed that nucleosomes containing Ub-H2A are enriched in transcribed genes and excluded from the centromeric heterochromatin (4362).

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In 1980, I saw the paper showing the identity of ubiquitin and APF-1 (1791). Two seemingly independent realms, protein degradation and chromosomes, came together for me, suggesting a regulatory pathway of immense complexity and a broad, still to be discovered, range of functions. Because a system of such complexity was unlikely to be understood through biochemistry alone, I decided to find genetic approaches to the functions of ubiquitin conjugation and ubiquitin-dependent proteolysis. In 1980, reverse-genetic techniques were about to become feasible with the yeast Saccharomyces cerevisiae, but were still a decade away for mammalian genetics. Near the end of 1980, I came across a paper by M. Yamada and colleagues that described a conditionally lethal, temperature-sensitive mouse cell line called ts85. They showed that a specific nuclear protein disappears from ts85 cells at increased temperatures and suggested that this protein might be Ub-H2A (4363). From the paper's data, I concluded that the protein was virtually certain to be Ub-H2A, because in the preceding two years we had learned much about the properties of this ubiquitin conjugate. On the hunch that mouse ts85 cells might be mutated in a component of the ubiquitin system, I wrote to Yamada, and received from him, in 1981, both ts85 and the parental ("wild-type") cell line, FM3A. Using these cell lines, my laboratory established two main results (1800; 1801):

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  • Mouse ts85 cells have a temperature-sensitive ubiquitin-activating (E1) enzyme.

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  • In contrast to the wild-type parental cells, ts85 cells stop degrading the bulk of their short-lived proteins at nonpermissive temperature.

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The path that led to these conclusions is described below.

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The experiments

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Figure 2  
In vitro ubiquitin-protein conjugation in crude extracts from FM3A and ts85 cells. Extracts were prepared from cells cultured at 32°C or 39°C. The extracts were preincubated at the indicated temperature, then assayed by adding 125I-ubiquitin and denatured lysozyme. The samples were analyzed by SDS-PAGE and fluorography. α, β, and γ denote bands of lysozyme containing increasing amounts of covalently linked 125I-ubiquitin moieties. (Results were originally published in 1800.)

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D. Finley, who spearheaded the ts85 project, joined my laboratory as a graduate student soon after the arrival of ts85 cells. Finley intended to work on regulation of gene expression, the lab's major subject at the time, but soon switched to a systematic study of ts85 and FM3A cells. A few months into the project, Finley and I made the crucial observation that ubiquitin conjugation in an extract from ts85 cells is temperature-sensitive, in contrast to an extract from the parental cells (Figure 2). Soon afterward, I invited Ciechanover (who had been working on unrelated projects in the MIT laboratory of H. Lodish) to join me and Finley in the ongoing study of ts85 cells, which he did.

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To determine whether the E1, E2, and E3 enzymes from the ts85 and parental cell lines differed in their ability to mediate ubiquitin conjugation, we first used affinity chromatography on ubiquitin-Sepharose (4359) to copurify these enzymes. This preparation was subjected to heat treatments, and assayed for ubiquitin conjugation. We found that ubiquitin conjugation mediated by the enzymes from ts85 cells was hypersensitive to heating, in comparison to an identical preparation from wild-type (parental) cells. In addition, we found that, in contrast to the wild-type enzyme preparation, the ts85 enzymes strongly conjugated 125I-ubiquitin to a specific endogenous substrate. It seemed possible that ubiquitin was conjugated to the mutated (conformationally perturbed) enzyme of the ubiquitin system that we wanted to identify. The apparent molecular mass (~113 kDa) of the substrate suggested that it might be monoubiquitylated ubiquitin-activating enzyme (E1).

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Figure 3  
Heat inactivation of purified ubiquitin-activating enzymes (E1s) from ts85 and FM3A cells. After preincubation at 40°C for the indicated times, 125I-ubiquitin was added for 10 minutes. Samples were electrophoresed and radiolabeled proteins detected by fluorography. 2ME is 2-mercaptoethanol. (Results were originally published in 1800.)

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We therefore sought to show directly that the E1 enzyme from ts85 cells was temperature-sensitive. To do this, we purified E1 to more than 90% homogeneity and assayed the formation of the ubiquitin-E1 thioester, E1-S~u, an intermediate in the transfer of activated ubiquitin to acceptor proteins (see Figure 1) (1800). The purified E1 enzymes from ts85 and FM3A cells were incubated in the presence of 125I-ubiquitin and ATP. Figure 3 shows that at the permissive temperature (30°C), E1-S~u is observed with both enzyme preparations. However, at the restrictive temperature (40°C), E1-S~u is not detected in the ts85 samples. As a control, we incubated replicate samples with 2-mercaptoethanol to cleave the thioester bond between E1 and ubiquitin. These and related biochemical tests supported the main conclusion of our first ts85 paper that the E1 enzyme from ts85 cells is thermolabile, in comparison to E1 from the parental FM3A cells (1800).

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Figure 4  
The ts85 and FM3A cells were grown, incubated with amino acid analogs, and labeled with 35S-methionine at 30.5°C. The chase with unlabeled methionine was done in the absence of amino acid analogs, at either 30.5°C or 39.5°C for the indicated times. The percentages of radioactivity precipitable by trichloroacetic acid (TCA) represent the amounts of nondegraded protein remaining after the chase. (Results were originally published in 1801.)

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Concurrently with these experiments, we compared protein degradation in the ts85 and FM3A cells. The results of these studies were described in the second ts85 paper (1801). To assay protein degradation in vivo, we incubated cells with chemical analogs of certain amino acids. These analogs can substitute for cognate amino acids in translation, producing "abnormal proteins" that were known to be rapidly degraded in vivo. We incubated ts85 and FM3A cells with the amino acid analogs and pulse-labeled newly synthesized proteins with 35S-methionine. The degradation of 35S-labeled proteins was followed during a chase period in the absence of 35S-methionine. As shown in Figure 4, at the permissive temperature (30.5°C) both cell lines degrade abnormal intracellular proteins at approximately equal rates. In striking contrast, at the nonpermissive temperature (39.5°C) the ts85 cells degrade less than 15% of the 35S-labeled proteins during the same time period.

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Figure 5  
The FM3A and ts85 cells were incubated with amino acid analogs and pulse-labeled with 35S-methionine. The chase incubations were at the indicated temperatures and times, in the presence of unlabeled methionine and the absence of amino acid analogs. Cell extracts were electrophoresed and radiolabeled proteins visualized by fluorography. (Results were originally published in 1801.)

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In Figure 5, the degradation of more than 20 discrete protein species could be inspected individually over time. At the nonpermissive temperature, ts85 cells showed not only a general defect in protein degradation, but also specific alterations in protein synthesis. Particularly striking was the induction of an ~70 kDa protein, which was later shown to be a member of the HSP70 family of heat-stress proteins. The induction of HSP70 synthesis at the nonpermissive temperature was observed specifically in ts85 cells and was therefore likely to be caused by thermoinactivation of ubiquitin-activating enzyme (E1) in these cells (1800).

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Figure 6  
The FM3A and ts85 cells were incubated with amino acid analogs, and pulse-labeled with 35S-methionine at 30.5°C or 39.5°C. The chase incubation was in the presence of unlabeled methionine and the absence of amino acid analogs, for the times indicated. Cell extracts were incubated with antibodies to ubiquitin, and immunoprecipitates were analyzed by scintillation counting or by gel electrophoresis and fluorography. (Results were originally published in 1801.)

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In our first ts85 paper (1800), we showed that the conjugation of ubiquitin to histone H2A in vivo is strongly suppressed at the nonpermissive temperature in ts85 but not in FM3A cells. In our second ts85 paper (1801), we showed that at the nonpermissive temperature ts85 cells fail to ubiquitylate efficiently not only histone H2A but also the abnormal proteins generated with amino acid analogs. We detected these proteins by immunoprecipitation with antibodies to ubiquitin. As shown in Figure 6, at the permissive temperature (30.5°C) the level of ubiquitin-protein conjugates in ts85 and FM3A cells treated with amino acid analogs is approximately 4% of the total label in 35S-methionine pulse-labeled proteins. During the chase with cold methionine at the permissive temperature, the kinetics of disappearance of 35S-labeled ubiquitin-protein conjugates is similar in both ts85 and FM3A cells. However, incubation at the nonpermissive temperature (39.5°C) strongly suppresses the formation of ubiquitin-protein conjugates in ts85 cells but not in FM3A cells. Analysis of the immunoprecipitates by SDS-PAGE detected many immunoreactive 35S-labeled proteins conjugated to ubiquitin (Figure 6). These and other data, including tests with inhibitors of lysosomal proteolysis (1801), indicated that the ts85 defect in selective protein degradation is directly dependent on the ts85 defect in ubiquitin-protein conjugation.

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Figure 7  
The FM3A and ts85 cells were incubated at 30.5°C or 39.5°C and pulse-labeled with 35S-methionine. The chase incubation was in the presence of unlabeled methionine, for the times indicated. Cell extracts were incubated with trichloroacetic acid (TCA) to precipitate nondegraded proteins. The TCA soluble counts represent the amounts of degradation products. (Results were originally published in 1801.)

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We also tested whether physiological translation products (produced in the absence of abnormal amino acids) are substrates of the ubiquitin system (1801). FM3A and ts85 cells were incubated at 30.5°C or shifted to the nonpermissive temperature (39.5°C), and the cells were labeled briefly with 35S-methionine and chased in the presence of cold methionine. To follow the degradation of the labeled normal proteins, we measured the presence of 35S in trichloroacetic acid-soluble degradation products at different times of the chase. As shown in Figure 7, the degradation of prelabeled proteins at nonpermissive temperature is strongly (though incompletely) suppressed in ts85 cells, but not in FM3A cells.

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Other experiments with ts85 cells indicated that ubiquitin conjugation is essential for cell viability (1800). In addition, ts85 cells are preferentially arrested at the G2 phase of the cell cycle, and the synthesis of heat-stress proteins is strongly induced in these cells at nonpermissive temperature. These observations indicated that ubiquitin conjugation is involved in the cell cycle progression and stress response (1800). In 1983, T. Hunt and colleagues discovered unusual proteins in clam embryos (825). These proteins, called cyclins, are degraded during exit from mitosis. We suggested that cyclins were destroyed by the ubiquitin system (1801), a hypothesis shown to be correct in 1991, by M. Glotzer, A. Murray, and M. Kirschner (845), and independently by Hershko and colleagues (4364).

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In one of our ts85 papers, a specific (and at the time entirely original) hypothesis was suggested about the mechanism of action of heat-stress proteins. It was proposed that "the ubiquitin-dependent proteolytic pathway and the heat shock response are complementary systems designed (among other things) to prevent cellular damage that abnormal proteins could inflict. ... It is possible that at least some of the heat shock proteins recognize the same binding sites by which abnormal proteins recognize each other in precipitate formation, but bind monovalently to abnormal proteins so as to prevent precipitate formation" (1800). At the time of this hypothesis, which proved correct, published speculations on the mechanism of heat-stress proteins were largely about RNA and chromatin regulation; the era of modern understanding commenced soon after the ts85 work.

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Our other 1984 proposal was that specific heat-stress proteins (such as HSP70) and ubiquitin-dependent proteolysis play complementary roles in containing and reversing stress damage through the recognition of unfolded proteins via exposed hydrophobic surfaces (1800). This has also proven correct, as illustrated by numerous papers in the 1990s.

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Subsequent work

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In addition to having been a breakthrough that indicated the importance, indeed the requirement, of the ubiquitin system for intracellular proteolysis, cell viability and cell cycle progression, the ts85 papers were the first instance of a study that addressed the in vivo workings of this system. The ts85 papers underlay the subsequent expansion of the ubiquitin field, as well as the later scientific strategy of my laboratory. Although the ts85 discoveries left little doubt, among the optimists, about the importance of the ubiquitin system in cellular physiology, it was difficult to extend these findings in mammalian cells due to the impossibility, at the time, of altering genes at will. In addition, the advances with ts85 cells did not address the existence and nature of degradation signals, that is, features of proteins that make them targets for ubiquitin conjugation.

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Therefore in 1983, even before the completion of ts85 work, Finley and I, together with other colleagues in my lab, began systematic analysis of the ubiquitin system in the genetically tractable S. cerevisiae. Between 1983 and 1990, this work yielded key discoveries and established the physiological fundamentals of the ubiquitin field. In 1984, Finley, E. Özkaynak, and I cloned the first ubiquitin gene, UBI4, which encodes a polyubiquitin precursor protein (4365). By 1987, we showed that UBI4 is strongly induced by different stresses, and that a deletion of UBI4 results in stress-hypersensitive cells (4366). These advances established a broad and essential function of the ubiquitin system.

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In 1986, A. Bachmair, Finley, and I discovered the first degradation signals (degrons) that target proteins for ubiquitin conjugation and proteolysis (1804). One set of these degradation signals (1804) gives rise to the N-end rule, which relates the in vivo half-life of a protein to the identity of its N-terminal residue.

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In 1987, S. Jentsch, J. McGrath, and I discovered that RAD6, a protein known to yeast geneticists as an essential component of DNA repair pathways, is a ubiquitin-conjugating (E2) enzyme, the first such enzyme shown to mediate a specific physiological function (4367). A subsequent collaboration between B. Byers' and my laboratories demonstrated that CDC34, an essential cell cycle regulator, is also a ubiquitin-conjugating enzyme (4368). This discovery extended the ubiquitin-cell cycle connection of our ts85 work to the first demonstration of a specific function of ubiquitin conjugation in cell cycle control.

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Soon thereafter, Finley, B. Bartel, and I discovered the functions of the other yeast ubiquitin genes, UBI1-UBI3, which encode fusions of ubiquitin to one protein of the large ribosomal subunit and one protein of the small ribosomal subunit (4369). The association of ubiquitin with a ribosomal protein moiety is transient, yet essential for efficient ribosome biogenesis. Here, ubiquitin acts not as a degradation signal but as a cotranslational chaperone (4369).

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In 1989, V. Chau and colleagues in my laboratory demonstrated the existence and unique topology of polyubiquitin chains (491). We also showed that a substrate-linked polyubiquitin chain is essential for the substrate's degradation by the proteasome. In 1990, E. Johnson, D. Gonda, and I discovered that ubiquitin-dependent proteolysis of oligomeric proteins is subunit-selective, a fundamental feature of the ubiquitin system that allows protein degradation to function as an instrument of either negative or positive control (4370). Among many examples are activation of the transcription factor NF-κB, through selective degradation of the inhibitory subunit IκB, and inactivation of cyclin-dependent kinases, through selective degradation of their cyclin subunits.

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The ubiquitin-dependent N-end rule pathway gradually became the major focus of my laboratory's work (4372; 4371). The ubiquitin fusion technique, which made possible the pathway's discovery (1804), led us to other methods that utilize the unique properties of ubiquitin (4373).

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By the early 1990s, several physiological substrates of the ubiquitin system were identified, including MATα2 (4375; 4374), p53 (4376), and cyclins (4364; 845). More and more laboratories began to study the functions and mechanisms of the ubiquitin system. The trickle became a flood, and today research on ubiquitin is going from strength to strength in hundreds of laboratories throughout the world. Within a decade from now (this is being written in 2003), we will probably see useful drugs that target specific components of the ubiquitin system, and also, with luck, drugs that direct this system to destroy, selectively, any specific protein.

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Since the early 1990s, some of the major discoveries in the ubiquitin field revealed additional, nonproteolytic functions of ubiquitin, as well as several ubiquitin-like conjugation systems. One example is the ubiquitin-related SUMO protein, whose conjugation to other proteins (catalyzed by distinct enzymes that are similar to ubiquitin-specific enzymes) mediates a range of functions that include protein localization and regulation of the "canonical" ubiquitin system (for review see 4377). Ubiquitin was also found to play an important role in intracellular vesicle-mediated protein trafficking. In particular, monoubiquitylation of some cell surface receptors marks them for endocytosis and eventual degradation in the lysosomes (for review see 4379; 4378). These more recent discoveries are but a small subset of new advances that have been made in this field over the last decade.

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Ubiquitin studies are now one of the largest and most important arenas of modern biology, the point of convergence of many disparate disciplines. The two 1984 papers about mouse ts85 cells (1801; 1800) played a key role in bringing this about, in that they established the ubiquitin system as the dominant pathway for selective protein degradation and at the same time revealed its broad significance.

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The author

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Alexander Varshavsky was born in Moscow, Russia in 1946. In 1970, he received a B.S. degree in Chemistry, at the Department of Chemistry, Moscow University. Varshavsky did his graduate work in 1970-73 at the Institute of Molecular Biology in Moscow, receiving a Ph.D. in Biochemistry. In 1977, Varshavsky moved to the United States and joined the faculty at the Massachusetts Institute of Technology (MIT) in Cambridge. He remained at MIT until 1992. Since 1992, his laboratory is at the California Institute of Technology (Caltech) in Pasadena, where he is the Howard and Gwen Laurie Smits Professor of Cell Biology. Varshavsky is a member of the National Academy of Sciences, the American Academy of Arts and Sciences, the American Philosophical Society, and the European Molecular Biology Organization. He received the 1998 Novartis-Drew Award, the 1999 Gairdner International Award, the 2000 Lasker Award in Basic Medical Research, the 2000 General Motors Sloan Prize in Cancer Research, the 2000 Hoppe-Seyler Award, the 2000 Shubitz Prize in Cancer Research, the 2001 Merck Award, the 2001 Wolf Prize in Medicine, the 2001 Horwitz Prize, the 2001 Max Planck Research Prize, the 2001 Pasarow Award in Cancer Research, the 2001 Massry Prize, and the 2002 Wilson Medal.


Alexander Varshavsky
Division of Biology, 147-75
California Institute of Technology
1200 East California Blvd.
Pasadena, CA 91125, USA
E-mail: avarsh@caltech.edu

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reviews
  • 1815 Schoenheimer, R. (1942). The Dynamic State of Body Constituents (Cambridge, MA: Harvard University Press).
  • 4360 Zwickl, P., Baumeister, W., and Steven, A. (2000).  Dis-assembly lines: the proteasome and related ATPase-assisted proteases.  Curr. Opin. Struct. Biol. 10, 242-250.  PubMed   Journal
  • 4361 Hershko, A., Ciechanover, A., and Varshavsky, A. (2000).  The ubiquitin system.  Nat. Med. 10, 1073-1081.  PubMed   Journal
  • 4371 Varshavsky, A. (1996).  The N-end rule: functions, mysteries, uses.  Proc. Natl. Acad. Sci. USA 93, 12142-12149.  PubMed   Journal
  • 4372 Varshavsky, A. (2003).  The N-end rule and regulation of apoptosis.  Nat. Cell Biol. 5, 373-376.  PubMed  
  • 4377 Schwartz, D. C. and Hochstrasser, M. (2003).  A superfamily of protein tags: ubiquitin, SUMO and related modifiers.  Trends Biochem. Sci. 28, 321-328.  PubMed   Journal
  • 4378 Katzman, D.J., Odorizzi, G., and Emr, S. (2002).  Nat. Rev. Mol. Cell Biol. 3, 893-905.  PubMed   Journal
reviews
  • 491 Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989).  A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.  Science 243, 1576-1583.  PubMed  
  • 825 Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D., and Hunt, T. (1983).  Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division.  Cell 33, 389-396.  PubMed   Journal
  • 845 Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991).  Cyclin is degraded by the ubiquitin pathway.  Nature 349, 132-138.  PubMed   Journal
  • 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   Journal
  • 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  
  • 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
  • 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  
  • 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  
  • 4358 Ciechanover, A., Hod, Y., and Hershko, A. (1978).  A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes.  Biochem. Biophys. Res. Comm. 81, 1100-1105.
  • 4359 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   Journal
  • 4362 Levinger, L. and Varshavsky, A. (1982).  Selective arrangement of ubiquitinated and D1 protein-containing nucleosomes within the Drosophila genome.  Cell 28, 375-385.  PubMed  
  • 4363 Marunouchi, T., Mita, S., Matsumoto, Y., and Yamada, M. (1980).  Disappearance of a chromosomal basic protein from cells of a mouse temperature-sensitive mutant defective in histone phosphorylation.  Biochem. Biophys. Res. Commun. 95, 126-131.  PubMed  
  • 4364 Hershko, A., Ganoth, D., Pehrson, J., Palazzo, R. E., and Cohen, L. H. (1991).  Methylated ubiquitin inhibits cyclin degradation in clam embryo extracts.  J. Biol. Chem. 266, 16376-16379.  PubMed   Journal
  • 4365 Ozkaynak, E., Finley, D., and Varshavsky, A. (1984).  The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein.  Nature 312, 663-666.  PubMed  
  • 4366 Finley, D., Ozkaynak, E., and Varshavsky, A. (1987).  The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses.  Cell 48, 1035-1046.  PubMed  
  • 4367 Jentsch, S., McGrath, J. P., and Varshavsky, A. (1987).  The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme.  Nature 329, 131-134.  PubMed  
  • 4368 Goebl, M. G., Yochem, J., Jentsch, S., McGrath, J. P., Varshavsky, A., and Byers, B. (1988).  The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme.  Science 241, 1331-1335.  PubMed  
  • 4369 Finley, D., Bartel, B., and Varshavsky, A. (1989).  The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis.  Nature 338, 394-401.  PubMed  
  • 4370 Johnson, E. S., Gonda, D. K., and Varshavsky, A. (1990).  cis-trans recognition and subunit-specific degradation of short-lived proteins.  Nature 346, 287-291.  PubMed  
  • 4373 Varshavsky, A. (2000).  Ubiquitin fusion technique and its descendants.  Methods Enzymol. 327, 578-593.  PubMed  
  • 4374 Hochstrasser, M. and Varshavsky, A. (1990).  In vivo degradation of a transcriptional regulator: the yeast alpha 2 repressor.  Cell 61, 697-708.  PubMed  
  • 4375 Hochstrasser, M., Ellison, M. J., Chau, V., and Varshavsky, A. (1991).  The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo.  Proc. Natl. Acad. Sci. USA 88, 4606-4610.  PubMed   Journal
  • 4375 Hochstrasser, M., Ellison, M. J., Chau, V., and Varshavsky, A. (1991).  The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo.  Proc. Natl. Acad. Sci. USA 88, 4606-4610.  PubMed   Journal
  • 4376 Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J., and Howley, P. M. (1990).  The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53.  Cell 63, 1129-1136.  PubMed  
  • 4379 Schnell, J. D. and Hicke, L. (2003).  Non-traditional functions of ubiquitin and ubiquitin-binding proteins.  J. Biol. Chem. 278, 35857-35860.  PubMed   Journal

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