The discovery of ubiquitin-dependent proteolysis Keith D. Wilkinson* Department of Biochemistry, Emory University School of Medicine, 4017 Rollins Research Building, 1510 Clifton Road, Atlanta, GA 30322 Edited by Marc W. Kirschner, Harvard Medical School, Boston, MA, and approved August 29, 2005 (received for review June 9, 2005) In early 1980, Irwin A. Rose, Avram Hershko, and Aaron Ciechanover published two papers in PNAS that reported the astounding observation that energy-dependent intracellular proteolysis was far more complicated than the previously accepted models of lyso- somal proteolysis or the action of ATP-dependent proteases such as bacterial lon. In fact, it has turned out to be even more compli- cated than they could have suspected. The general model of covalently attaching a small protein as a targeting signal has proved to be every bit as important to eukaryotic cells as the better understood modifications such as phosphorylation or acetylation. The key player in this modification, a small protein called ubiquitin (APF-1 in these papers), is the founding member of a large family of pro- teins containing the ␤-grasp fold and is used as a posttranslational targeting signal to modify the structure, function, and͞or local- ization of other proteins. The story of this discovery is a textbook example of the confluence of intellectual curiosity, unselfish col- laboration, chance, luck, and preparation. t is a truism in science that the first example of any biological phenom- enon is the hardest to prove. We rely so much on precedent to for- Imulate our hypothesis that something truly unique and novel is often over- looked for many years. The covalent modification of proteins by the attach- ment of other proteins is one such ex- ample (1–6). As we now know, this modification is a targeting mechanism used to move proteins around in the cell. The ubiquitin family of modifiers (ubiquitin, Nedd8, SUMO, ISG15, etc.) has been implicated in the regulation of proteolysis, nuclear localization, chroma- tin structure, genetic integrity, protein quality control, and signaling (6). The prototypical example of this modifica- tion is the covalent attachment of ubiq- uitin to proteins to target them for Fig. 1. The laureates at the Karolinska Institute after their Nobel addresses. Shown are (left to right) degradation by the proteasome and was Aaron Ciechanover, Irwin Rose, and Avram Hershko. first reported in two PNAS papers pub- lished early in 1980 by the 2004 Nobel laureates in chemistry, Avram Hershko, Goldberg’s group showed that damaged transferase and on the rates of bulk pro- Aaron Ciechanover, and Irwin A. Rose or abnormal proteins were rapidly tein turnover in bacteria and mamma- (Fig. 1), and their collaborators (1, 7). cleared from the cell (9–11). He and lian cells. Hershko then established his These two papers outlined the essentials Schimke (11) pointed out that enzymes own laboratory at the Technion-Israel of the system and were amazingly pre- that catalyzed rate-limiting steps in Institute of Technology in Haifa and scient in their interpretations and pre- metabolic pathways were generally continued to collaborate with Tompkins dictions based on simple biochemical short-lived and that their amounts were until his untimely death in 1975. Aaron analysis. responsive to metabolic conditions. Ciechanover (M.D. 1974 and Ph.D. 1981 The authors set out to explain a sim- Thus, by the late 1970s, we began to from Hebrew University-Hadassah Med- ple biological curiosity: the fact that in- suspect that the energy dependence of ical School) completed his military tracellular proteolysis in mammalian intracellular proteolysis reflected some service before joining Hershko as a cells requires energy. Melvin Simpson energy-dependent regulation of proteo- graduate student at the Technion-Israel first showed this in his 1953 studies with lytic systems. Institute of Technology. Irwin A. isotopic labeling of cellular proteins (8), The collaboration of Ciechanover, ‘‘Ernie’’ Rose (Ph.D. in 1952 from the and for the next 25 years there were few Hershko, and Rose was uniquely posi- University of Chicago) did postdoctoral insights into the mechanisms or meta- tioned and qualified to define this regu- studies with Charles Carter at Case bolic logic of this observation. The lation. Avram Hershko (M.D. 1965 and Western Reserve University and Severo hydrolysis of the peptide bond is exer- Ph.D. 1969 from Hebrew University- Ochoa at New York University. He gonic, and there is no thermodynamic Hadassah Medical School) did postdoc- joined the Department of Pharmacology reason to use energy. The apparent re- toral work in the laboratory of Gordon at Yale University in 1954 and moved to quirement for energy could mean only Tompkins at the University of Califor- that there was something we didn’t un- nia at San Francisco where he first be- derstand. Part of the answer began to came interested in protein degradation. *E-mail: [email protected]. become apparent in the mid 1970s when His early studies were on tyrosine amino © 2005 by The National Academy of Sciences of the USA 15280–15282 ͉ PNAS ͉ October 25, 2005 ͉ vol. 102 ͉ no. 43 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504842102 Downloaded by guest on September 23, 2021 PERSPECTIVE the Institute for Cancer Research at the association of APF-1 with other compo- Fox Chase Cancer Center in 1963. As a nents of the system. mechanistic enzymologist, he gained In the first paper (1), Ciechanover et fame for his studies on proton transfer al. showed that 125I-labeled APF-1 was reactions and the use of isotopic label- promoted to a high molecular weight ing to examine the chemical mechanisms form upon incubation with fraction II used by enzymes. Rose’s interest in pro- and ATP. This association required low tein degradation dated back to the ob- concentrations of ATP and was reversed servations of Simpson, his colleague at upon removal of the ATP. At this time, Yale, who had demonstrated the ATP a postdoctoral fellow in Rose’s labora- dependence of proteolysis in 1953. They tory, Art Haas, began to characterize talked often about this biochemical curi- this association, and he found that the osity, and Rose would come back to this complex survived high pH. To every- question periodically, but made little one’s surprise, the association of 125I- progress. Hershko and Rose first met at labeled APF-1 with proteins in fraction a Fogarty Foundation meeting in 1977 II was covalent! They went on to show where they discovered their mutual in- that the bond was stable to NaOH treat- terests in ATP-dependent proteolysis. ment and that APF-1 was bound to Rose invited Hershko to do a sabbatical many different proteins as judged by in his laboratory at the Institute for SDS͞PAGE. The authors concluded Cancer Research in Philadelphia. This that it was likely that conjugation was began a 10-year collaboration that saw required for proteolysis; the nucleotide Rose hosting the Israeli group every and metal ion requirements were similar summer. Rose was a patron and intellec- for conjugation and proteolysis, as were tual contributor far beyond what might the amounts of ATP and fraction II Fig. 2. Formation of covalent compounds be- be indicated by his authorship on the necessary to maximally stimulate. The tween APF-1 and lysozyme in an ATP-dependent papers of that era. Thus, these two tal- covalent attachment of APF-1 to cellu- reaction. Tracks 1–5, compound formation with 125 ented investigators entered into the col- lar proteins also explained why some I-APF-1; track 1, without ATP; track 2, with ATP; laboration that would define one of the investigators had so much difficulty tracks 3–5, with ATP and 5, 10, or 25 mg of unla- beled lysozyme, respectively; and tracks 6 and 7, several mechanisms of ATP-dependent demonstrating a requirement for APF-1 compound formation with 125I-lysozyme. Condi- protein degradation and frame a new in ATP dependent proteolysis. When tions were as detailed in Methods of ref. 7, except means of viewing cellular regulation. fraction II was prepared from reticulo- that 5 ␮gof125I-lysozyme (40,000 cpm) and 3 ␮gof [See Ciechanover (12) and Goldberg cytes without first depleting the ATP, unlabeled APF-1 were used. Track 6, ATP omitted; (13) for a more complete discussion of most of the APF-1 was initially present track 7, with 2 mM ATP. Contamination in 125I- the various ATP-dependent processes.] in high molecular weight conjugates that labeled lysozyme is indicated. [Reproduced with By 1979, Hershko and Ciechanover were subsequently found in fraction II. permission from ref. 7 (Copyright 1980).] had exploited the seminal observations These conjugates were rapidly disassem- of Etlinger and Goldberg that reticulo- bled by amidases in fraction II, thereby cyte lysates (which lack lysosomes) ex- liberating free APF-1. Thus, there was H2a was covalently modified by the at- hibited ATP-dependent proteolysis of sufficient APF-1 in fraction II to sup- tachment of a small protein called ubiq- denatured proteins (14) and would be port proteolysis. If one first depleted the uitin. Gideon Goldstein first discovered amenable to biochemical fractionation. ATP, APF-1 was liberated before the ubiquitin in his search for thymopoietin Hershko and Ciechanover first showed chromatographic preparation of fraction (17), and he generously shared authentic the system could be separated into two II and APF-1 had to be added back to samples with me. Intrigued by this simi- fractions (I and II) that had to be re- obtain maximal rates of proteolysis. This larity, Urban, Haas, and I went on to combined to generate ATP-dependent series of rather simple experiments con- show that APF-1 was the previously proteolysis (15). Fraction I contained a vincingly demonstrated that APF-1 was known protein called ubiquitin (18).