Update Ubiquitin-mediated Protein Modification and Degradation Alan L. Schwartz and Aaron Ciechanover Departments of Pediatrics and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri, and The Rappaport Institute for Research in the Medical Sciences and the Department of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
Ubiquitin is a small, 8 leD protein found in all eukaryotic cells. It is involved in a wide variety of regulatory roles within the cell, including gene expression, ribosome biosynthesis, receptor expression, and the stress response. The best understood of these is that of ubiquitin-mediated proteolysis, in which ubiquitin is covalently attached to specific protein target substrates that are then recognized and degraded by a high molecular weight protease.
General Functions
Cellular proteins are in a constant state of turnover. Their half-lives range over more than three orders of magnitude (1). For example, normal hemoglobin is extremely stable whereas many abnormal variants are degraded within minutes. In general, shott-lived proteins (t1/2 of minutes) are predominantly degraded through the cytoplasmic ubiquitinmediated pathway (2-4), whereas the majority of long-lived proteins (t1/ 2 of hours-days) are degraded within the vacuolar system. Recent evidence suggests that these two major proteolytic pathways within the cell interact (5). Only in the past few years have studies begun to sort out the biology and underlying molecular mechanisms responsible for the selective degradation of cellular proteins: What determines the specificity whereby two proteins of similar structure within the same cellular compartment are recognized and specifically degraded at widely different rates? This selectivity for protein turnover accounts for the rapid degradation of regulatory proteins (e.g., the mitotic cyclins, see below); the rapid and specific degradation of proteins following a critical regulatory signal (e.g., the effect of light on degradation of plant phytochromes); and the removal of abnormal cellular proteins (e.g., thalassemic hemoglobins). In addition to the role of the ubiquitin system in the rapid and specific turnover of cellular proteins, ubiquitin modification of a variety of protein targets within the cell appears to be important in a number of basic cellular functions, such as regulation of gene expression, regulation of the cell cycle, modification of cell surface receptors, biogenesis of ribosomes, DNA repair, (Received for publication July 10, 1992) Address correspondence to: Alan L. Schwartz, M.D., Ph.D., Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110. Am. J. Respir. CeU Mol. BioI. Vol. 7. pp. 463-468, 1992
and antigen processing and presentation. Despite the considerable progress that has been made in elucidating the mode of action and roles of the ubiquitin pathway (for recent reviews, see references 3, 4, 6, 7, and below), many major issues remain unresolved. The best understood of the cellular biologic processes involving the ubiquitin system is that of ubiquitin-mediated proteolysis. Degradation of intracellular protein via this system involves several discrete steps (see Figure 1). Initially, ubiquitin is covalently linked to the protein substrate in an ATP-dependent reaction. Following ubiquitin conjugation, the protein is selectively degraded and ubiquitin is released for further use. The use of in vitro reconstituted biochemical systems, purification of many of the individual components, molecular genetic studies in yeast, and the use of mammalian cell mutants of the ubiquitin system have shed considerable light on the details of this pathway (4). Overall, the first step in the ubiquitin conjugation system is the activation of ubiquitin to a high-energy thiolester intermediate. This reaction is catalyzed by El, the ubiquitinactivating enzyme. The activated ubiquitin moiety is then transferred to one of the ubiquitin carrier proteins (E2s) to generate a similar thiolester linkage. At this point, ubiquitin can either be linked to a target protein directly (to generate monoubiquitin adducts) or conjugated to proteins via the ubiquitin protein ligases (E3s) (to generate polyubiquitin adducts). A 26S protease carries out the proteolytic degradation of the polyubiquitin protein conjugates in an ATPdependent manner. A group of ubiquitin C-terminal isopeptidases catalyze the cleavage of ubiquitin from conjugated proteins or from short peptides and lysine residues that are released during the proteolytic process. This process plays an important role in "correction" of mistakenly conjugated proteins and in the recycling of free and reutilizable ubiquitin.
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A
I Substrate I
Ubiquitin Ubiquitin Activation
CD
1' E2 ATP
comp,e0 E3 Formation (Ubiquitin Protein Ligase)
®
I Activated Ubiquitin Iy I Substrate ± E31 ®•
Ubiquitin Ligation
I
Substrate (Ubiquitin}n
I
Figure 1. Overall pathway of ubiquitin-mediated conjugation and degradation. (j) Ubiquitin is activated via El, E2, and ATP. (2, 3) Protein substrates are then either recognized for ubiquitin ligation directly via E2 or via E2 and E3. (4) The resultant ubiquitin-protein conjugates are subject to degradation via a 26S protease and ATP.
(Ubiquitin - Protein Conjugate) 268 Protease ATP
I
t
0
I Degradation I Ubiquitin Ubiquitin is an abundant, highly conserved 76 amino acid protein with sequence identity among mammals and only three amino acid differences between mammals and plants (8). The three-dimensional structure of ubiquitin has been resolved at 1.8 Aand reveals a highly compact, near spherical molecule with a critical Lys-48 residue located on the surface (9). In addition, the 3 amino acid carboxyl-terminal tail (Arg-Gly-Gly-COOH) protrudes outward. Ubiquitin is highly resistant to cellular proteases and is not denatured by boiling. Ubiquitin has been localized to the cytoplasm, nucleus, and several subcellular organelles (10, 11). The covalent attachment of ubiquitin to the substrate protein is mediated by an isopeptide bond between the carboxylterminal group of ubiquitin and the e-amino group of lysine residues within the acceptor protein. These ubiquitin-protein adducts exist either as monoubiquitin conjugates (e.g., histones H2A, H2B) or as polyubiquitin conjugates (e.g., intermediates in degradation). Ubiquitin-protein conjugates are found widely dispersed in cells, including within the nucleus, cytoplasm, lysosomes, and so forth (10-12). Ubiquitin-activating Enzyme, El The ubiquitin-activating enzyme, E1, carries out the first reaction of the ubiquitin pathway via a two-step reaction involving the formation of a high-energy ubiquitin adenylate intermediate and the subsequent transfer of this activated ubiquitin moiety to a thiol site within the E1 molecule. The native form of the enzyme is a dimer that consists of two identical 115 kD subunits (13). A single ubiquitous E1 mRNA of 3.5 kb has been cloned and sequenced from humans and yeast (14-17). In humans, the El gene is found at Xpl1.22. Multiple El genes have been found in wheat and arabidopsis, although the functional significance of these multiple genes is unclear. Disruption of the E1 gene in yeast (URAl) is lethal and clearly indicates a single El gene in this
organism (15). Four mammalian cell lines have been described that appear to have a single temperature-sensitive defect in E1; at the nonpermissive temperature, AlS9, ts20, ts85, and tsBN85 cells arrest at the S/G2 transition (4, 7). Transvection of AIS9 with E1 cDNA abolishes the temperature-sensitive growth arrest and reconstitutes cellular proliferation (17). Localization studies have shown that the EI enzyme is found in both the cytoplasm and nucleus, though its role in each of these cellular compartments is not yet fully defined (18). Ubiquitin-conjugating Enzymes, E2s The family of ubiquitin-conjugating enzymes, E2s, are responsible for the transfer of the activated ubiquitin from the thiol site of El to a similar thiol site on E2. Thereafter, the activated ubiquitin is either ligated to a target protein to yield a monoubiquitin conjugate or to other target proteins to yield multiubiquitin adducts that require the presence of ubiquitin
TABLE 1
Ubiquitin-conjugating enzymes, £2s* Protein Size Yeast Gene UBCl UBC2/RAD6 UBC31CDC34 UBC4 UBCS UBC6 UBC7 UBC8 UBC9 UBCIOIPAS2
Functions (Yeast) Sporulation DNA repair, protein degradation Gl-S cell cycle Protein degradation Protein degradation, stress Protein secretion (Stress) (?) (Viability) (Peroxisome function)
(kD)
24
20 34 16 16
28 18 23
* Summary of yeast family of ubiquitin-conjugating enzymes (VBC) (E2s); adapted from references 7 and 19.
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protein ligases (E3s; see below). E2s exist as a multigene family in all species examined, including yeast and humans (7, 19). At present, at least 10 E2 coding genes have been described in yeast (UBC1-10) (Table 1). Each E2 contains a short region of similarity surrounding the cysteine residue at the active site. One member of the E2 family denoted in yeast UBC2 is identical to the RAD6 gene (20), which is involved in DNA repair. A second E2, UBC3, is encoded by the cell cycle progression CDC34 gene. Some members of the E2 gene family code for proteins with polyacidic C-terminal extensions, which are important as determinants of substrate specificity. In general, the molecular size of the E2 enzymes fall within the 16 to 34 kD range. Several of the E2 enzymes catalyze monoubiquitination, while current evidence suggests that only E2 14ill• is specific for the formation of the multiply ubiquitinated adducts generated in proteins destined for ubiquitin-mediated proteolysis. Addition of multiple ubiquitin moieties to a single protein substrate molecule can be mediated via two independent mechanisms. In most cases, ubiquitination of a single and defined lysine residue occurs initially. This is followed by ubiquitin branch chain elongation (21) (ubiquitin linked to ubiquitin via Lys-48). Formation of the resulting "branched tree" substrate target is dependent upon the presence of ubiquitin protein ligases, E3s. It is also possible that single ubiquitin residues will be conjugated each to various lysine moieties of the protein substrate (22).
Asp Glu + tRNAArg Cys
aa=
L [
aa= aa= aa -
protein
aa= aa=
protein
Reiss and Hershko have purified E3 from rabbit reticulocytes and found that it is composed of two subunits, each with a molecular mass of 180 kD (23). Two independent lines of evidence indicate that the E3 molecules contain the protein substrate binding site of the ligase system (24). One set of observations is based on the use of chemical cross-linking reagents. The second series of studies made use of short competitive peptides to define the specificity of the recognition sites in the E3 molecules. Based on these observations, differential recognition of several distinct substrate classes by E3 has been defined (Figure 2, see below). Based on these different substrate specificities, two E3 molecules, termed E3a and E3{3, are currently defined. E3a recognizes substrates with both basic and bulky-hydrophobic N-termini. E3a can also complex with specific E2s. The generation of multiubiquitin chains (see above) takes place while the target substrate is bound to the E3a molecule. The second E3, E3{3, has also been purified from reticulocyte lysates and demonstrates substrate specificity for target proteins with Ser, Ala, or Thr residues at the amino terminus (25). Like E3a, E3{3 is able to ligate multiple ubiquitin molecules to a single target substrate. E3a and E3{3 share several physical characteristics including apparent molecular mass of approximately 350 kD. A yeast E3 gene, designated UBRi, encoding a 225 kD protein, has been isolated and appears to be functionally homologous to the mammalian E3a. Deletion of UBRi gene
_
LArS-ASP Arg-Glu
Arg-Cys
Asp + tRNAArg _ Glu
[
Recognition by
E3a
Arg-Asp Arg-Glu
(basic "head" site)
Lys Arg His
L
[
Leu Trp Phe Tyr Ser Ala Thr
L
aa= [
acetyl -
Asn _ Gin
Ubiquitin-Protein Ligases, E3s
.[
Met lie Pro Gly Val
~ ~
Recognition by
E3a (bulky - hydrophobic "head" site) Recognition by
E3fJ rhead- site)
Recognition by E3a(?fJ) (-body- sites)
acetyl -
Figure 2. Recognition of the proteolytic substrates by ubiquitin-protein ligases (E3s). Distinct sites of E3s recognize a spec.ific group.of substrates that are characterized by the nature of their N-terminal amino acid residue as described in the text. Some of these residues require post-translational modification prior to recognition. Other proteins are probably recognized by signals that reside downstream from the N-terminal residue ("body" sites).
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from yeast reveals a nonlethal phenotype with a partial defect in sporulation and growth rate (26). Recently, a second E3coding yeast gene with substrate specificity similar to both E3a and E3J3 has been described in VERI null mutant. The function of this gene is not presently clear (27). Target Substrate Recognition While activation of ubiquitin by El is protein substrate-independent, catalysis of ubiquitin-protein isopeptide bond formation requires E2, or E2 and E3 (see Figure 1). Thus, it appears that both enzymes (E2, E3) have specific recognition sites for a subset of potential protein substrates. Recently, several lines of evidence have begun to shed light on the specificity of substrate recognition (28). Hershko and associates noted that protein substrates with blocked (i.e., acetylated) N-termini were not degraded in an in vitro reconstituted degradation system (29). They then went on to show that specific binding sites resident on E3 molecules conferred specificity for substrate recognition, whereby a functional complex is formed between E3 and the protein substrate. Using a variety of model substrates as well as competing dipeptides, it was shown that a single E3a molecule contains two distinct and independent sites (see Figure 2): one for basic (Arg, His, Lys), the other for bulky-hydrophobic (Leu, Phe, Trp, and Tyr) N-terminal residues. It appears that the association between protein substrate and E3 involves binding of the N-terminal residue of the substrate to a specific site of the ligase. A second ligase, E3J3, has recently been identified with recognition for Ala, Ser, or Thr at their amino termini. However, the signal recognized by this ligase (E3J3) is apparently complex and involves amino acids distal to the amino terminus (25). An additional recognition system has been elucidated for proteins that have acidic amino termini (30). Arginyl modification of the terminal Asp or Glu via arginyl-tRNA-protein transferase is required prior to recognition by E3a for ubiquitin conjugation. Furthermore, protein substrates with Asn or GIn amino termini are deamidated to Asp or Glu prior to the transferase modification and ligase recognition (31). In vivo, the amino terminal residue also functions as a key recognition signal for ubiquitin conjugation/degradation. Bachmair and associates examined yeast expressing the 20 mutants of J3-galactosidase each with a different amino acid residue at the amino terminus (32). The in vivo half-lives of the expressed J3-galactosidase proteins varied from more than 20 h to less than 2 min, depending on the nature of the amino-terminal residue: molecules with Met, Ser, Ala, Val, Thr, Gly,Pro, and Cys had relatively long half-lives ("'20 h), whereas Ile, Glu, Tyr, GIn, His, Arg, Lys, Phe, Trp, Leu, Asn, or Asp conferred extremely short « 30 min) half-lives. However, this behavior of "stabilizing" and "destabilizing" amino-terminal residues is not sufficient to signal ubiquitindependent degradation, since a similar series of studies using 20 dihydrofolate reductase amino terminal mutants expressed in yeast demonstrated no difference in the in vivo half-lives (33). Further characterization has revealed "downstream" features, including the location of acceptor lysine residues which were critical for ubiquitin conjugation. While the amino terminal residue is one major deter-
minant of recognition, approximately 80% of cellular proteins have N-acetyl-blocked amino termini. Some of these proteins are also recognized and degraded by the ubiquitin system in a process that seems to be distinct from that described above for free amino termini proteins (34). A factor that is partially responsible for the degradation of this group of proteins has been recently purified by Gonen and coworkers and shown to be a protein composed of two identical 46 kD subunits (35). However, its function is not in substrate recognition but in conjugate degradation. Thus, the recognition step for N-acetyl-blocked protein substrates is still unclear. 26S Protease/Proteasome Multiply ubiquitinated protein substrates are rapidly and efficiently degraded in an ATP-dependent manner by a large (26S) multi-subunit proteolytic particle (36-38). The complex is composed of three multi-subunit protein factors with molecular masses of 250, 600 and 700 kD. One of these (the 700 kD factor) had been described earlier as a 20S protease (termed proteasome, macropain, prosome, or multicatalytic protease) (39, 40). The proteasome is a cylindrical particle as observed in the electron microscope and is composed of at least 15 distinct polypeptides (in the 15 to 32 kD range). Many of the individual subunits show striking homology both within and between species. Disruption of individual proteasome subunits in yeast is lethal. The proteasome is an ATP-independent protease with a wide range of substrate specificities. Association of the three factors confers both ATP dependence and specificity towards ubiquitinated proteins. Cellular Systems of Ubiquitin-mediated Control Ubiquitin is involved in a multitude of cellular functions (see reference 6). These include protein degradation, regulation of cell cycle, gene expression, ribosome biogenesis, cell surface receptor expression, response to stress, regulation of autophagy, and protein import into mitochondria. A few selected examples are provided below. As noted above, one of the E2 enzymes is the RAD6 gene product, mutations in which cause hypersensitivity to chemical mutagens and irradiation. In vitro, E2/RAD6 efficiently catalyzes the addition of multiple ubiquitin molecules to purified histones. The disruption of the single active enzyme site cysteine residue (Cys-88) completely abrogates the E2 activity of RAD6 when assayed in vitro or in vivo (41). Taken together, these studies suggest the DNA repair function of RAD6 may involve the ubiquitination of chromosomal regulatory proteins (including histones). Ubiquitin-mediated degradation is essential for cell cycle progression from metaphase. In this case, degradation of both cyclin-A and cyclin-B appears to be mediated by the ubiquitin system (42,43). The mechanisms whereby the mitotic cyclins are specifically degraded in metaphase are poorly understood. Both cyclin-A and cyclin-B are known to form complexes with the highly conserved phosphoserine/threonine kinase, p34cdc2. The cyclin-B/cdc2 complex is thought to activate a cyclin destruction pathway presumably by phosphorylation/dephosphorylation of an unknown
Update
substrate, driving the cells into interphase of the next cell cycle. It is clear that a motif residing within the first 90 amino acid residues of the cyclin molecule serves as a proteolysis recognition marker (42). Given that the timing of cyclin destruction is determined by the cell cycle stage of the cytoplasm rather than the cell cycle stage of the cyclins themselves, it is most likely that one or more of the components of the ubiquitin system or kinases are modified in such a way that degradation of a specific protein is initiated. In addition, studies of the yeast cell cycle gene CDC341E2 provide strong evidence for an additional role of the ubiquitin system in the control of cell cycle progression during Gl. Conditional mutants of CDC341E2 fail to undergo separation of the duplicated spindle pole bodies required for spindle formation and essential for Gl/S transition. However, the physiologic substrates for CDC341E2 are not currently known. As noted above, several mammalian cell lines have been described that have temperature-sensitive defects in El (4, 7). Following inactivation of the El enzyme, the cells arrest at the S/G2 transition. Since the removal of ubiquitin from histone H2A is a prerequisite for chromosome condensation, one major role of the ubiquitin cycle in cells most likely relates to its ability to regulate ubiquitin attachment to histones H2A and H2B during interphase (44). Indeed, the capacity of these mutant cells to convert newly replicated small DNA strands to chromosomal DNA is markedly impaired at the nonpermissive temperature. Most recently, a role for ubiquitin-mediated proteolysis and turnover of several short-lived oncoproteins has been demonstrated (45, 46). In cells, the degradation of a broad class of growth control-associated oncoproteins (e.g., fos, myc, p53) is rapid (tl/2 < 10 min). In vitro reconstituted systems have demonstrated the capacity of the ubiquitin-mediated system to perform this rapid, ATP-dependent degradation. Thus, since the discovery of the ubiquitin-mediated conjugation and proteolytic system less than 15 years ago, our understanding of the multiple critical cellular events under its control has expanded rapidly. The underlying cellular and molecular mechanisms are just now being dissected and certainly will provide us with more insight into the fundamental functions of this complex and intricate biologic system. Acknowledgments: We thank Susan Starbuck for secretarial assistance. Alan L. Schwartz has been supported by NIH and Monsanto. Aaron Ciechanover has been supported by US-Israel Binational Science Foundation, German-Israeli Foundation for Research and Scientific Development, the Israel Academy of Sciences, Council for Tobacco Research, Israel Cancer Research Fund, and the Israel Cancer Society.
References 1. Goldberg, A. L., and A. C. St.-John. 1976. Intracellular protein degradation in mammalian and bacterial cells. Annu. Rev. Biochem. 45:747-803. 2. Rechsteiner, M. 1987. Ubiquitin-mediated pathways for intracellular proteolysis. Annu. Rev. Cell BioI. 3:1-30. 3. Hershko, A. 1991. The ubiquitin pathway for protein degradation. Trends Biochem. Sci. 16:265-268. 4. Hershko, A., and A. Ciechanover. 1992. Ubiquitin-mediated protein degradation. Annu. Rev. Biochem. In press. 5. Gropper, R., R. A. Brandt, S. Elias et al. 1991. The ubiquitin-activating
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enzyme, EI, is required for stress-induced lysosomal degradation of cellular proteins. J. BioI. Chem. 266:3602-3610. 6. Finley, D., and V. Chau. 1991. Ubiquitination. Annu. Rev. Cell BioI. 7:25-69. 7. Jentsch, S. 1992. Ubiquitin dependent protein degradation: a cellular perspective. Trends Cell BioI. 2:98-103. 8. Ozkaynak, E., D. Finley, M. J. Solomon, and A. Varshavsky. 1987. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 6: 14291439. 9. Vijay-Kumar, S./ C. E. Bugg, and W. J. Cook. 1987. Structure ofubiquit in refined at 1.8 A resolution. J. Mol. BioI. 194:531-544. 10. Haas, A. L., and P. M. Bright. 1985. The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. J. BioI. Chem. 260: 12424-12473. 11. Schwartz, A. L., A. Ciechanover, R. Brandt, and H. Geuze. 1988. Immunoelectron microscopic localization of ubiquitin in hepatoma cells. EMBO J. 7:2961-2966. 12. Doherty, F. J., N. U. Osborn, J. A. Wassell, P. E. Heggit, L. Laszlo, and R. J. Mayer. 1989. Ubiquitin-protein conjugates accumulate in the lysosomal system of fibroblasts treated with cysteine proteinase inhibitors. Biochem. J. 263:47-55. 13. Ciechanover, A., S. Elias, H. Heller, and A. Hershko. 1982. "Covalent affinity" purification of ubiquit in-activating enzyme. J. BioI. Chem. 257: 2537-2542. 14. Handley, P. M., M. MueckJer, N. R. Siegel, A. Ciechanover, and A. L. Schwartz. 1991. Molecular cloning, sequence, and tissue distribution of the human ubiquitin-activating enzyme EI. Proc. Natl. Acad. Sci. USA 88:258-262. 15. McGrath, J. P., S. Jentsch, and A. Varshavsky. 1991. UBAI: an essential yeast gene encoding ubiquitin-activating enzyme. EMBO 1. 10:227-236. 16. Hatfield, P., J. Callis, and R. Vierstra. 1990. Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in E. coli. J. BioI. Chem. 265:15813-15817. 17. Zachsenhaus, E., and R. Scheinen. 1990. Molecular cloning, primary structure and expression of the human X-linked A1S9 gene eDNA which complements the tsAIS9 mouse L cell defect in DNA replication. EMBO J. 9:2923-2929. 18. Schwartz, A. L., J. S. Trausch, A. Ciechanover, J. W. Slot, and J. W. Geuze. 1992. Immunoelectron microscopic localization of the ubiquitinactivating enzyme, EI, in HepG2 cells. Proc. Natl. Acad. Sci. USA 89:5542-5546. 19. Jentsch, S., W. Seufert, T. Sommer, and H. Reins. 1990. Ubiquitinconjugating enzymes: novel regulators of eukaryotic cells. Trends Biochem. Sci. 15:195-198. 20. Jentsch, S., J. McGrath, and A. Varshavsky. 1987. The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature 329:131134. 21. Chau, V., J. W. Tobias, A. Bachmair et al. 1989. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243:1576-1583. 22. Hershko, A., andH. Heller. 1985. Occurrence ofa polyubiquitin structure in ubiquitin-protein conjugates. Biochem. Biophys. Res. Commun. 128: 1079-1086. 23. Reiss, Y., and A. Hershko. 1990. Affinity purification of ubiquitin-protein ligase on immobilized protein substrates. J. Bioi. Chem. 265:3685-3690. 24. Hershko, A., H. Heller, E. Eytan, and Y. Reiss. 1986. The protein substrate binding site of the ubiquitin-protein ligase system. J. BioI. Chem. 261: 11992-11999. 25. Heller, H., and A. Hershko. 1990. A ubiquitin protein ligase specific for type III protein substrates. J. Bioi. Chem. 265:6532-6535. 26. Bartel, B., I. Wunning, and A. Varshavsky. 1990. The recognition component of the N-end rule pathway. EMBO J. 9:3179-3189. 27. Sharon, G., B. Raboy, H. A. Parag, D. Dimitrovsky, and R. G. Kulka. 1991. RAD6 gene product of Saccharomyces cerevisiae requires a putative ubiquitin protein ligase (E3) for the ubiquitination of certain proteins. J. BioI. Chem. 266:15890-15894. 28. Ciechanover, A., and A. L. Schwartz. 1989. How are substrates recognized by the ubiquitin-mediated proteolytic system. Trends Biochem. Sci. 14:483-488. 29. Hershko, A., H. Heller, E. Eytan, G. KakJij, and I. A. Rose. 1984. Role ofthe a-amino group of protein in ubiquitin-mediated protein breakdown. Proc. Natl. Acad. Sci. USA 81:7021-7025. 30. Ferber, S., and A. Ciechanover. 1987. Role of arginine-tRNA in protein degradation by the ubiquitin pathway. Nature 326:808-811. 31. Gonda, D., A. Bachmair, I. Wunning, J. Tobias, W. Lane, and A. Varshavsky. 1989. Universality and structure of the N-end rule. J. BioI. Chem. 264:16700-16712. 32. Bachmair, A., D. Finley, and A. Varshavsky. 1986. In vivo half-life of a protein is a function of its amino terminal residue. Science 234: 179-186. 33. Bachmair, A., and A. Varshavsky. 1989. The degradation signal in a shortlived protein. Cell 56:1019-1032.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 71992
34. Mayer, A., N. R. Siegel, A. L. Schwartz, and A. Ciechanover. 1988. Degradation of proteins with acetylated amino termini by the ubiquitin system. Science 244:1480-1483. 35. Gonen, H., A. L. Schwartz, and A. Ciechanover. 1991. Purification and characterization of a novel protein that is required for degradation of N-aacetylated proteins by the ubiquitin system. J. Biol. Chem. 266: 1922119231. 36. Hough, R., G. Pratt, and M. Rechsteiner. 1986. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte extracts. J. BioI. Chem. 261:2400-2408. 37. Eytan, E., D. Ganoth, T. Armon, and A. Hershko. 1989. ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin. Proc. Nat!. Acad. Sci. USA 86:7751-7755. 38. Driscoll, J., and A. L. Goldberg. 1990. The proteasome (multicatalytic protease) is a component of the 1500 kDa proteolytic complex which degrades ubiquitin-conjugated proteins. J. Biol. Chem. 265:4789-4792. 39. Arrigo, A.-P., K. Tanaka, A. L. Goldberg, and W. J. Welch. 1988. Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome). Nature 331:192-194. 40. Falkenburg, P. E., C. Haass, P. M. Kloetzel eta!. 1988. Drosophila small
41.
42. 43. 44. 45. 46.
cytoplasmic 19S ribonucleoprotein is homologous to the rat multicatalytic proteinase. Nature 331:190-192. Sung, P., S. Prakash, and L. Prakash. 1990. Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitinconjugating activity and its various biological functions. Proc. Natl. Acad. Sci. USA 87:2695-2699. Glotzer, M., A. W. Murray, and M. W. Kirschner. 1991. Cyclin is degraded by the ubiquitin pathway. Nature 349: 132-138. Hershko, A., D. Ganoth, J. Pehrson, R. Palazzo, and L. Cohen. 1991. Methylated ubiquitin inhibits cyclin degradation in clam embryo extracts. J. BioI. Chem. 266:16376-16379. Bonner, W. M., C. L. Hatch, and R. S. Wu. 1988. Ubiquinated histones and chromatin. In Ubiquitin. M. Rechsteiner, editor. Plenum Publishing Corp., New York. 157-172. Ciechanover, A., J. A. DiGiuseppe, B. Bercovich et al. 1991. Degradation of nuclear oncoproteins by the ubiquitin system in vitro. Proc. Nat!. Acad. Sci. USA 88:139-143. Scheffner, M., B. A. Werness, J. M. Huibrgste, A. J. Levine, and P. M. Howley. 1990. The E6 oncoprotein encoded by human papillomavirus type 16 and 18 promotes the degradation ofp53. Cell 63:1129-1136.
Ubiquitin is a small, 8 leD protein found in all eukaryotic cells. It is involved in a wide variety of regulatory roles within the cell, including gene expression, ribosome biosynthesis, receptor expression, and the stress response. The best understood of these is that of ubiquitin-mediated proteolysis, in which ubiquitin is covalently attached to specific protein target substrates that are then recognized and degraded by a high molecular weight protease.
General Functions
Cellular proteins are in a constant state of turnover. Their half-lives range over more than three orders of magnitude (1). For example, normal hemoglobin is extremely stable whereas many abnormal variants are degraded within minutes. In general, shott-lived proteins (t1/2 of minutes) are predominantly degraded through the cytoplasmic ubiquitinmediated pathway (2-4), whereas the majority of long-lived proteins (t1/ 2 of hours-days) are degraded within the vacuolar system. Recent evidence suggests that these two major proteolytic pathways within the cell interact (5). Only in the past few years have studies begun to sort out the biology and underlying molecular mechanisms responsible for the selective degradation of cellular proteins: What determines the specificity whereby two proteins of similar structure within the same cellular compartment are recognized and specifically degraded at widely different rates? This selectivity for protein turnover accounts for the rapid degradation of regulatory proteins (e.g., the mitotic cyclins, see below); the rapid and specific degradation of proteins following a critical regulatory signal (e.g., the effect of light on degradation of plant phytochromes); and the removal of abnormal cellular proteins (e.g., thalassemic hemoglobins). In addition to the role of the ubiquitin system in the rapid and specific turnover of cellular proteins, ubiquitin modification of a variety of protein targets within the cell appears to be important in a number of basic cellular functions, such as regulation of gene expression, regulation of the cell cycle, modification of cell surface receptors, biogenesis of ribosomes, DNA repair, (Received for publication July 10, 1992) Address correspondence to: Alan L. Schwartz, M.D., Ph.D., Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110. Am. J. Respir. CeU Mol. BioI. Vol. 7. pp. 463-468, 1992
and antigen processing and presentation. Despite the considerable progress that has been made in elucidating the mode of action and roles of the ubiquitin pathway (for recent reviews, see references 3, 4, 6, 7, and below), many major issues remain unresolved. The best understood of the cellular biologic processes involving the ubiquitin system is that of ubiquitin-mediated proteolysis. Degradation of intracellular protein via this system involves several discrete steps (see Figure 1). Initially, ubiquitin is covalently linked to the protein substrate in an ATP-dependent reaction. Following ubiquitin conjugation, the protein is selectively degraded and ubiquitin is released for further use. The use of in vitro reconstituted biochemical systems, purification of many of the individual components, molecular genetic studies in yeast, and the use of mammalian cell mutants of the ubiquitin system have shed considerable light on the details of this pathway (4). Overall, the first step in the ubiquitin conjugation system is the activation of ubiquitin to a high-energy thiolester intermediate. This reaction is catalyzed by El, the ubiquitinactivating enzyme. The activated ubiquitin moiety is then transferred to one of the ubiquitin carrier proteins (E2s) to generate a similar thiolester linkage. At this point, ubiquitin can either be linked to a target protein directly (to generate monoubiquitin adducts) or conjugated to proteins via the ubiquitin protein ligases (E3s) (to generate polyubiquitin adducts). A 26S protease carries out the proteolytic degradation of the polyubiquitin protein conjugates in an ATPdependent manner. A group of ubiquitin C-terminal isopeptidases catalyze the cleavage of ubiquitin from conjugated proteins or from short peptides and lysine residues that are released during the proteolytic process. This process plays an important role in "correction" of mistakenly conjugated proteins and in the recycling of free and reutilizable ubiquitin.
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A
I Substrate I
Ubiquitin Ubiquitin Activation
CD
1' E2 ATP
comp,e0 E3 Formation (Ubiquitin Protein Ligase)
®
I Activated Ubiquitin Iy I Substrate ± E31 ®•
Ubiquitin Ligation
I
Substrate (Ubiquitin}n
I
Figure 1. Overall pathway of ubiquitin-mediated conjugation and degradation. (j) Ubiquitin is activated via El, E2, and ATP. (2, 3) Protein substrates are then either recognized for ubiquitin ligation directly via E2 or via E2 and E3. (4) The resultant ubiquitin-protein conjugates are subject to degradation via a 26S protease and ATP.
(Ubiquitin - Protein Conjugate) 268 Protease ATP
I
t
0
I Degradation I Ubiquitin Ubiquitin is an abundant, highly conserved 76 amino acid protein with sequence identity among mammals and only three amino acid differences between mammals and plants (8). The three-dimensional structure of ubiquitin has been resolved at 1.8 Aand reveals a highly compact, near spherical molecule with a critical Lys-48 residue located on the surface (9). In addition, the 3 amino acid carboxyl-terminal tail (Arg-Gly-Gly-COOH) protrudes outward. Ubiquitin is highly resistant to cellular proteases and is not denatured by boiling. Ubiquitin has been localized to the cytoplasm, nucleus, and several subcellular organelles (10, 11). The covalent attachment of ubiquitin to the substrate protein is mediated by an isopeptide bond between the carboxylterminal group of ubiquitin and the e-amino group of lysine residues within the acceptor protein. These ubiquitin-protein adducts exist either as monoubiquitin conjugates (e.g., histones H2A, H2B) or as polyubiquitin conjugates (e.g., intermediates in degradation). Ubiquitin-protein conjugates are found widely dispersed in cells, including within the nucleus, cytoplasm, lysosomes, and so forth (10-12). Ubiquitin-activating Enzyme, El The ubiquitin-activating enzyme, E1, carries out the first reaction of the ubiquitin pathway via a two-step reaction involving the formation of a high-energy ubiquitin adenylate intermediate and the subsequent transfer of this activated ubiquitin moiety to a thiol site within the E1 molecule. The native form of the enzyme is a dimer that consists of two identical 115 kD subunits (13). A single ubiquitous E1 mRNA of 3.5 kb has been cloned and sequenced from humans and yeast (14-17). In humans, the El gene is found at Xpl1.22. Multiple El genes have been found in wheat and arabidopsis, although the functional significance of these multiple genes is unclear. Disruption of the E1 gene in yeast (URAl) is lethal and clearly indicates a single El gene in this
organism (15). Four mammalian cell lines have been described that appear to have a single temperature-sensitive defect in E1; at the nonpermissive temperature, AlS9, ts20, ts85, and tsBN85 cells arrest at the S/G2 transition (4, 7). Transvection of AIS9 with E1 cDNA abolishes the temperature-sensitive growth arrest and reconstitutes cellular proliferation (17). Localization studies have shown that the EI enzyme is found in both the cytoplasm and nucleus, though its role in each of these cellular compartments is not yet fully defined (18). Ubiquitin-conjugating Enzymes, E2s The family of ubiquitin-conjugating enzymes, E2s, are responsible for the transfer of the activated ubiquitin from the thiol site of El to a similar thiol site on E2. Thereafter, the activated ubiquitin is either ligated to a target protein to yield a monoubiquitin conjugate or to other target proteins to yield multiubiquitin adducts that require the presence of ubiquitin
TABLE 1
Ubiquitin-conjugating enzymes, £2s* Protein Size Yeast Gene UBCl UBC2/RAD6 UBC31CDC34 UBC4 UBCS UBC6 UBC7 UBC8 UBC9 UBCIOIPAS2
Functions (Yeast) Sporulation DNA repair, protein degradation Gl-S cell cycle Protein degradation Protein degradation, stress Protein secretion (Stress) (?) (Viability) (Peroxisome function)
(kD)
24
20 34 16 16
28 18 23
* Summary of yeast family of ubiquitin-conjugating enzymes (VBC) (E2s); adapted from references 7 and 19.
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Update
protein ligases (E3s; see below). E2s exist as a multigene family in all species examined, including yeast and humans (7, 19). At present, at least 10 E2 coding genes have been described in yeast (UBC1-10) (Table 1). Each E2 contains a short region of similarity surrounding the cysteine residue at the active site. One member of the E2 family denoted in yeast UBC2 is identical to the RAD6 gene (20), which is involved in DNA repair. A second E2, UBC3, is encoded by the cell cycle progression CDC34 gene. Some members of the E2 gene family code for proteins with polyacidic C-terminal extensions, which are important as determinants of substrate specificity. In general, the molecular size of the E2 enzymes fall within the 16 to 34 kD range. Several of the E2 enzymes catalyze monoubiquitination, while current evidence suggests that only E2 14ill• is specific for the formation of the multiply ubiquitinated adducts generated in proteins destined for ubiquitin-mediated proteolysis. Addition of multiple ubiquitin moieties to a single protein substrate molecule can be mediated via two independent mechanisms. In most cases, ubiquitination of a single and defined lysine residue occurs initially. This is followed by ubiquitin branch chain elongation (21) (ubiquitin linked to ubiquitin via Lys-48). Formation of the resulting "branched tree" substrate target is dependent upon the presence of ubiquitin protein ligases, E3s. It is also possible that single ubiquitin residues will be conjugated each to various lysine moieties of the protein substrate (22).
Asp Glu + tRNAArg Cys
aa=
L [
aa= aa= aa -
protein
aa= aa=
protein
Reiss and Hershko have purified E3 from rabbit reticulocytes and found that it is composed of two subunits, each with a molecular mass of 180 kD (23). Two independent lines of evidence indicate that the E3 molecules contain the protein substrate binding site of the ligase system (24). One set of observations is based on the use of chemical cross-linking reagents. The second series of studies made use of short competitive peptides to define the specificity of the recognition sites in the E3 molecules. Based on these observations, differential recognition of several distinct substrate classes by E3 has been defined (Figure 2, see below). Based on these different substrate specificities, two E3 molecules, termed E3a and E3{3, are currently defined. E3a recognizes substrates with both basic and bulky-hydrophobic N-termini. E3a can also complex with specific E2s. The generation of multiubiquitin chains (see above) takes place while the target substrate is bound to the E3a molecule. The second E3, E3{3, has also been purified from reticulocyte lysates and demonstrates substrate specificity for target proteins with Ser, Ala, or Thr residues at the amino terminus (25). Like E3a, E3{3 is able to ligate multiple ubiquitin molecules to a single target substrate. E3a and E3{3 share several physical characteristics including apparent molecular mass of approximately 350 kD. A yeast E3 gene, designated UBRi, encoding a 225 kD protein, has been isolated and appears to be functionally homologous to the mammalian E3a. Deletion of UBRi gene
_
LArS-ASP Arg-Glu
Arg-Cys
Asp + tRNAArg _ Glu
[
Recognition by
E3a
Arg-Asp Arg-Glu
(basic "head" site)
Lys Arg His
L
[
Leu Trp Phe Tyr Ser Ala Thr
L
aa= [
acetyl -
Asn _ Gin
Ubiquitin-Protein Ligases, E3s
.[
Met lie Pro Gly Val
~ ~
Recognition by
E3a (bulky - hydrophobic "head" site) Recognition by
E3fJ rhead- site)
Recognition by E3a(?fJ) (-body- sites)
acetyl -
Figure 2. Recognition of the proteolytic substrates by ubiquitin-protein ligases (E3s). Distinct sites of E3s recognize a spec.ific group.of substrates that are characterized by the nature of their N-terminal amino acid residue as described in the text. Some of these residues require post-translational modification prior to recognition. Other proteins are probably recognized by signals that reside downstream from the N-terminal residue ("body" sites).
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from yeast reveals a nonlethal phenotype with a partial defect in sporulation and growth rate (26). Recently, a second E3coding yeast gene with substrate specificity similar to both E3a and E3J3 has been described in VERI null mutant. The function of this gene is not presently clear (27). Target Substrate Recognition While activation of ubiquitin by El is protein substrate-independent, catalysis of ubiquitin-protein isopeptide bond formation requires E2, or E2 and E3 (see Figure 1). Thus, it appears that both enzymes (E2, E3) have specific recognition sites for a subset of potential protein substrates. Recently, several lines of evidence have begun to shed light on the specificity of substrate recognition (28). Hershko and associates noted that protein substrates with blocked (i.e., acetylated) N-termini were not degraded in an in vitro reconstituted degradation system (29). They then went on to show that specific binding sites resident on E3 molecules conferred specificity for substrate recognition, whereby a functional complex is formed between E3 and the protein substrate. Using a variety of model substrates as well as competing dipeptides, it was shown that a single E3a molecule contains two distinct and independent sites (see Figure 2): one for basic (Arg, His, Lys), the other for bulky-hydrophobic (Leu, Phe, Trp, and Tyr) N-terminal residues. It appears that the association between protein substrate and E3 involves binding of the N-terminal residue of the substrate to a specific site of the ligase. A second ligase, E3J3, has recently been identified with recognition for Ala, Ser, or Thr at their amino termini. However, the signal recognized by this ligase (E3J3) is apparently complex and involves amino acids distal to the amino terminus (25). An additional recognition system has been elucidated for proteins that have acidic amino termini (30). Arginyl modification of the terminal Asp or Glu via arginyl-tRNA-protein transferase is required prior to recognition by E3a for ubiquitin conjugation. Furthermore, protein substrates with Asn or GIn amino termini are deamidated to Asp or Glu prior to the transferase modification and ligase recognition (31). In vivo, the amino terminal residue also functions as a key recognition signal for ubiquitin conjugation/degradation. Bachmair and associates examined yeast expressing the 20 mutants of J3-galactosidase each with a different amino acid residue at the amino terminus (32). The in vivo half-lives of the expressed J3-galactosidase proteins varied from more than 20 h to less than 2 min, depending on the nature of the amino-terminal residue: molecules with Met, Ser, Ala, Val, Thr, Gly,Pro, and Cys had relatively long half-lives ("'20 h), whereas Ile, Glu, Tyr, GIn, His, Arg, Lys, Phe, Trp, Leu, Asn, or Asp conferred extremely short « 30 min) half-lives. However, this behavior of "stabilizing" and "destabilizing" amino-terminal residues is not sufficient to signal ubiquitindependent degradation, since a similar series of studies using 20 dihydrofolate reductase amino terminal mutants expressed in yeast demonstrated no difference in the in vivo half-lives (33). Further characterization has revealed "downstream" features, including the location of acceptor lysine residues which were critical for ubiquitin conjugation. While the amino terminal residue is one major deter-
minant of recognition, approximately 80% of cellular proteins have N-acetyl-blocked amino termini. Some of these proteins are also recognized and degraded by the ubiquitin system in a process that seems to be distinct from that described above for free amino termini proteins (34). A factor that is partially responsible for the degradation of this group of proteins has been recently purified by Gonen and coworkers and shown to be a protein composed of two identical 46 kD subunits (35). However, its function is not in substrate recognition but in conjugate degradation. Thus, the recognition step for N-acetyl-blocked protein substrates is still unclear. 26S Protease/Proteasome Multiply ubiquitinated protein substrates are rapidly and efficiently degraded in an ATP-dependent manner by a large (26S) multi-subunit proteolytic particle (36-38). The complex is composed of three multi-subunit protein factors with molecular masses of 250, 600 and 700 kD. One of these (the 700 kD factor) had been described earlier as a 20S protease (termed proteasome, macropain, prosome, or multicatalytic protease) (39, 40). The proteasome is a cylindrical particle as observed in the electron microscope and is composed of at least 15 distinct polypeptides (in the 15 to 32 kD range). Many of the individual subunits show striking homology both within and between species. Disruption of individual proteasome subunits in yeast is lethal. The proteasome is an ATP-independent protease with a wide range of substrate specificities. Association of the three factors confers both ATP dependence and specificity towards ubiquitinated proteins. Cellular Systems of Ubiquitin-mediated Control Ubiquitin is involved in a multitude of cellular functions (see reference 6). These include protein degradation, regulation of cell cycle, gene expression, ribosome biogenesis, cell surface receptor expression, response to stress, regulation of autophagy, and protein import into mitochondria. A few selected examples are provided below. As noted above, one of the E2 enzymes is the RAD6 gene product, mutations in which cause hypersensitivity to chemical mutagens and irradiation. In vitro, E2/RAD6 efficiently catalyzes the addition of multiple ubiquitin molecules to purified histones. The disruption of the single active enzyme site cysteine residue (Cys-88) completely abrogates the E2 activity of RAD6 when assayed in vitro or in vivo (41). Taken together, these studies suggest the DNA repair function of RAD6 may involve the ubiquitination of chromosomal regulatory proteins (including histones). Ubiquitin-mediated degradation is essential for cell cycle progression from metaphase. In this case, degradation of both cyclin-A and cyclin-B appears to be mediated by the ubiquitin system (42,43). The mechanisms whereby the mitotic cyclins are specifically degraded in metaphase are poorly understood. Both cyclin-A and cyclin-B are known to form complexes with the highly conserved phosphoserine/threonine kinase, p34cdc2. The cyclin-B/cdc2 complex is thought to activate a cyclin destruction pathway presumably by phosphorylation/dephosphorylation of an unknown
Update
substrate, driving the cells into interphase of the next cell cycle. It is clear that a motif residing within the first 90 amino acid residues of the cyclin molecule serves as a proteolysis recognition marker (42). Given that the timing of cyclin destruction is determined by the cell cycle stage of the cytoplasm rather than the cell cycle stage of the cyclins themselves, it is most likely that one or more of the components of the ubiquitin system or kinases are modified in such a way that degradation of a specific protein is initiated. In addition, studies of the yeast cell cycle gene CDC341E2 provide strong evidence for an additional role of the ubiquitin system in the control of cell cycle progression during Gl. Conditional mutants of CDC341E2 fail to undergo separation of the duplicated spindle pole bodies required for spindle formation and essential for Gl/S transition. However, the physiologic substrates for CDC341E2 are not currently known. As noted above, several mammalian cell lines have been described that have temperature-sensitive defects in El (4, 7). Following inactivation of the El enzyme, the cells arrest at the S/G2 transition. Since the removal of ubiquitin from histone H2A is a prerequisite for chromosome condensation, one major role of the ubiquitin cycle in cells most likely relates to its ability to regulate ubiquitin attachment to histones H2A and H2B during interphase (44). Indeed, the capacity of these mutant cells to convert newly replicated small DNA strands to chromosomal DNA is markedly impaired at the nonpermissive temperature. Most recently, a role for ubiquitin-mediated proteolysis and turnover of several short-lived oncoproteins has been demonstrated (45, 46). In cells, the degradation of a broad class of growth control-associated oncoproteins (e.g., fos, myc, p53) is rapid (tl/2 < 10 min). In vitro reconstituted systems have demonstrated the capacity of the ubiquitin-mediated system to perform this rapid, ATP-dependent degradation. Thus, since the discovery of the ubiquitin-mediated conjugation and proteolytic system less than 15 years ago, our understanding of the multiple critical cellular events under its control has expanded rapidly. The underlying cellular and molecular mechanisms are just now being dissected and certainly will provide us with more insight into the fundamental functions of this complex and intricate biologic system. Acknowledgments: We thank Susan Starbuck for secretarial assistance. Alan L. Schwartz has been supported by NIH and Monsanto. Aaron Ciechanover has been supported by US-Israel Binational Science Foundation, German-Israeli Foundation for Research and Scientific Development, the Israel Academy of Sciences, Council for Tobacco Research, Israel Cancer Research Fund, and the Israel Cancer Society.
References 1. Goldberg, A. L., and A. C. St.-John. 1976. Intracellular protein degradation in mammalian and bacterial cells. Annu. Rev. Biochem. 45:747-803. 2. Rechsteiner, M. 1987. Ubiquitin-mediated pathways for intracellular proteolysis. Annu. Rev. Cell BioI. 3:1-30. 3. Hershko, A. 1991. The ubiquitin pathway for protein degradation. Trends Biochem. Sci. 16:265-268. 4. Hershko, A., and A. Ciechanover. 1992. Ubiquitin-mediated protein degradation. Annu. Rev. Biochem. In press. 5. Gropper, R., R. A. Brandt, S. Elias et al. 1991. The ubiquitin-activating
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enzyme, EI, is required for stress-induced lysosomal degradation of cellular proteins. J. BioI. Chem. 266:3602-3610. 6. Finley, D., and V. Chau. 1991. Ubiquitination. Annu. Rev. Cell BioI. 7:25-69. 7. Jentsch, S. 1992. Ubiquitin dependent protein degradation: a cellular perspective. Trends Cell BioI. 2:98-103. 8. Ozkaynak, E., D. Finley, M. J. Solomon, and A. Varshavsky. 1987. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 6: 14291439. 9. Vijay-Kumar, S./ C. E. Bugg, and W. J. Cook. 1987. Structure ofubiquit in refined at 1.8 A resolution. J. Mol. BioI. 194:531-544. 10. Haas, A. L., and P. M. Bright. 1985. The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. J. BioI. Chem. 260: 12424-12473. 11. Schwartz, A. L., A. Ciechanover, R. Brandt, and H. Geuze. 1988. Immunoelectron microscopic localization of ubiquitin in hepatoma cells. EMBO J. 7:2961-2966. 12. Doherty, F. J., N. U. Osborn, J. A. Wassell, P. E. Heggit, L. Laszlo, and R. J. Mayer. 1989. Ubiquitin-protein conjugates accumulate in the lysosomal system of fibroblasts treated with cysteine proteinase inhibitors. Biochem. J. 263:47-55. 13. Ciechanover, A., S. Elias, H. Heller, and A. Hershko. 1982. "Covalent affinity" purification of ubiquit in-activating enzyme. J. BioI. Chem. 257: 2537-2542. 14. Handley, P. M., M. MueckJer, N. R. Siegel, A. Ciechanover, and A. L. Schwartz. 1991. Molecular cloning, sequence, and tissue distribution of the human ubiquitin-activating enzyme EI. Proc. Natl. Acad. Sci. USA 88:258-262. 15. McGrath, J. P., S. Jentsch, and A. Varshavsky. 1991. UBAI: an essential yeast gene encoding ubiquitin-activating enzyme. EMBO 1. 10:227-236. 16. Hatfield, P., J. Callis, and R. Vierstra. 1990. Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in E. coli. J. BioI. Chem. 265:15813-15817. 17. Zachsenhaus, E., and R. Scheinen. 1990. Molecular cloning, primary structure and expression of the human X-linked A1S9 gene eDNA which complements the tsAIS9 mouse L cell defect in DNA replication. EMBO J. 9:2923-2929. 18. Schwartz, A. L., J. S. Trausch, A. Ciechanover, J. W. Slot, and J. W. Geuze. 1992. Immunoelectron microscopic localization of the ubiquitinactivating enzyme, EI, in HepG2 cells. Proc. Natl. Acad. Sci. USA 89:5542-5546. 19. Jentsch, S., W. Seufert, T. Sommer, and H. Reins. 1990. Ubiquitinconjugating enzymes: novel regulators of eukaryotic cells. Trends Biochem. Sci. 15:195-198. 20. Jentsch, S., J. McGrath, and A. Varshavsky. 1987. The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature 329:131134. 21. Chau, V., J. W. Tobias, A. Bachmair et al. 1989. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243:1576-1583. 22. Hershko, A., andH. Heller. 1985. Occurrence ofa polyubiquitin structure in ubiquitin-protein conjugates. Biochem. Biophys. Res. Commun. 128: 1079-1086. 23. Reiss, Y., and A. Hershko. 1990. Affinity purification of ubiquitin-protein ligase on immobilized protein substrates. J. Bioi. Chem. 265:3685-3690. 24. Hershko, A., H. Heller, E. Eytan, and Y. Reiss. 1986. The protein substrate binding site of the ubiquitin-protein ligase system. J. BioI. Chem. 261: 11992-11999. 25. Heller, H., and A. Hershko. 1990. A ubiquitin protein ligase specific for type III protein substrates. J. Bioi. Chem. 265:6532-6535. 26. Bartel, B., I. Wunning, and A. Varshavsky. 1990. The recognition component of the N-end rule pathway. EMBO J. 9:3179-3189. 27. Sharon, G., B. Raboy, H. A. Parag, D. Dimitrovsky, and R. G. Kulka. 1991. RAD6 gene product of Saccharomyces cerevisiae requires a putative ubiquitin protein ligase (E3) for the ubiquitination of certain proteins. J. BioI. Chem. 266:15890-15894. 28. Ciechanover, A., and A. L. Schwartz. 1989. How are substrates recognized by the ubiquitin-mediated proteolytic system. Trends Biochem. Sci. 14:483-488. 29. Hershko, A., H. Heller, E. Eytan, G. KakJij, and I. A. Rose. 1984. Role ofthe a-amino group of protein in ubiquitin-mediated protein breakdown. Proc. Natl. Acad. Sci. USA 81:7021-7025. 30. Ferber, S., and A. Ciechanover. 1987. Role of arginine-tRNA in protein degradation by the ubiquitin pathway. Nature 326:808-811. 31. Gonda, D., A. Bachmair, I. Wunning, J. Tobias, W. Lane, and A. Varshavsky. 1989. Universality and structure of the N-end rule. J. BioI. Chem. 264:16700-16712. 32. Bachmair, A., D. Finley, and A. Varshavsky. 1986. In vivo half-life of a protein is a function of its amino terminal residue. Science 234: 179-186. 33. Bachmair, A., and A. Varshavsky. 1989. The degradation signal in a shortlived protein. Cell 56:1019-1032.
468
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 71992
34. Mayer, A., N. R. Siegel, A. L. Schwartz, and A. Ciechanover. 1988. Degradation of proteins with acetylated amino termini by the ubiquitin system. Science 244:1480-1483. 35. Gonen, H., A. L. Schwartz, and A. Ciechanover. 1991. Purification and characterization of a novel protein that is required for degradation of N-aacetylated proteins by the ubiquitin system. J. Biol. Chem. 266: 1922119231. 36. Hough, R., G. Pratt, and M. Rechsteiner. 1986. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte extracts. J. BioI. Chem. 261:2400-2408. 37. Eytan, E., D. Ganoth, T. Armon, and A. Hershko. 1989. ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin. Proc. Nat!. Acad. Sci. USA 86:7751-7755. 38. Driscoll, J., and A. L. Goldberg. 1990. The proteasome (multicatalytic protease) is a component of the 1500 kDa proteolytic complex which degrades ubiquitin-conjugated proteins. J. Biol. Chem. 265:4789-4792. 39. Arrigo, A.-P., K. Tanaka, A. L. Goldberg, and W. J. Welch. 1988. Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome). Nature 331:192-194. 40. Falkenburg, P. E., C. Haass, P. M. Kloetzel eta!. 1988. Drosophila small
41.
42. 43. 44. 45. 46.
cytoplasmic 19S ribonucleoprotein is homologous to the rat multicatalytic proteinase. Nature 331:190-192. Sung, P., S. Prakash, and L. Prakash. 1990. Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitinconjugating activity and its various biological functions. Proc. Natl. Acad. Sci. USA 87:2695-2699. Glotzer, M., A. W. Murray, and M. W. Kirschner. 1991. Cyclin is degraded by the ubiquitin pathway. Nature 349: 132-138. Hershko, A., D. Ganoth, J. Pehrson, R. Palazzo, and L. Cohen. 1991. Methylated ubiquitin inhibits cyclin degradation in clam embryo extracts. J. BioI. Chem. 266:16376-16379. Bonner, W. M., C. L. Hatch, and R. S. Wu. 1988. Ubiquinated histones and chromatin. In Ubiquitin. M. Rechsteiner, editor. Plenum Publishing Corp., New York. 157-172. Ciechanover, A., J. A. DiGiuseppe, B. Bercovich et al. 1991. Degradation of nuclear oncoproteins by the ubiquitin system in vitro. Proc. Nat!. Acad. Sci. USA 88:139-143. Scheffner, M., B. A. Werness, J. M. Huibrgste, A. J. Levine, and P. M. Howley. 1990. The E6 oncoprotein encoded by human papillomavirus type 16 and 18 promotes the degradation ofp53. Cell 63:1129-1136.
