Ubiquitin, proteasomes, and the regulation of intracellular protein degradation

Ubiquitin, proteasomes, and the regulation of intracellular protein degradation Mark Hochstrasser University

Rapid

degradation

pathways work

of specific

is a component

has shown

of Chicago, Chicago, USA

proteins

by ubiquitin/proteasome-dependent

of many cellular

that protein

ubiquitination

regulatory

mechanisms.

and deubiquitination

mediated by large families of enzymes and that proteolysis by alterations

of the proteasome itself. The complexity

Recent are both

can be modulated

of the ubiquitin

system

is reflected in the broad range of processes it regulates; these include key steps in cell cycle progression, class

processing

of foreign

proteins

for presentation

by

I MHC molecules, and the control of cell proliferation.

Current

Opinion

in Cell Biology

Introduction Cells often switch from one cellular state to another, either in response to environmental cues or as part of regulated developmental pathways. Such switches generally require rapid dismantlement of an existing regulatory network, a process that is frequently dependent on protein degradation [l]. Selective protein turnover offers several advantages over other kinds of regulatory controls (and frequently functions in concert with other types of regulation). One advantage is speed: a protein’s half-life determines the time it takes to reach a new steady-state level following a change in the rate of its synthesis. A second advantage is irreversibility: elimination of a protein removes any chance of its being reactivated inappropriately. These features help explain why selective protein degradation is almost always a component of regulatory mechanisms that involve timing controls. Examples abound, ranging horn cell cycle progression, circadian rhythms, and various signal transduction pathways to cell lineage specification, metabolic control, and embryogenesis [ 1,2]. The specificity of intracellular protein degradation must be extremely high. Mistargeting of essential proteins or degradation of proteins at inappropriate times would wreak havoc in the cell. Important phenotypic alterations may also result from small changes in the rates of protein turnover. Many proto-oncogene products, for instance, are very short-lived in ho, and relatively small increases in their intracellular concentrations can be tumorigenic (see, for example, [3]). For many short-lived eukaryotic proteins, conjugation to the polypeptide ubiquitin is an obligatory step in their

1995,

7915-223

degradation [4]. Ubiquitin is joined reversibly to other proteins via linkage between the a-carboxyl group of ubiquitin and lysine E-amino groups of the acceptor proteins (isopeptide bonds). A simplified view of the ubiquitin pathway, which is highly conserved among diverse eukaryotes, is depicted in Fig. la. In an energyis first activated by an dependent reaction, ubiquitin enzyme called El, to which it becomes linked by a high energy thiolester bond. Ubiquitin then forms a thiolester bond with a second protein, ubiquitin-conjugating enzyme, or E2. This enzyme, ofien with the additional Victor E3, catalyzes isopeptide bond formation between ubiquitin and the substrate. For substrates destined for proteolysis, additional ubiquitin molecules are usually added to the substrate by this same enzyme cascade to form a chain or chains of ubiquitin molecules in isopeptide linkage to one another. Degradation of such multi-ubiquitinated proteins occurs on a large proteinase called the 26s proteasome complex (Fig. 1). In this review, I highlight advances from the past -two years in our understanding of ubiquitin-dependent proteolytic targeting, proteasome-mediated degradation, and the utilization of selective proteolysis in specific cellular regulatory circuits, placing emphasis on the complexity of the ubiquitin system. Such complexity is likely to be a consequence both of the need for high substrate specificity and regulatory flexibility and of the centrality of the ubiquitin system in the cell’s regulatory arsenal. As this is an Opinion journal, I include quite a number of speculations horn what may at times be a fairly idiosyncratic viewpoint. A sampling of alternative perspectives can be obtained by reference to other recent reviews [1,4-6].

Abbreviations EC-AP-E6-associated

protein; PA-proteasome

activator.

0 Current Biology Ltd ISSN 0955-0674

215

216

Cell regulation

Ubiquitination

(a)

Degradation

0

Ub

26s Proteasome

fi

Peptides

PA700

PA28

n

ATP

PAZELProteasome

20s Proteasome

26s Proteasome complex Q 1995

Current Ophon

in Cell Biology

Fig. 1. (a) Simplified view of protein ubiquitination and degradation (see text for details). Isopeptide-linked ubiquitin chains that form on proteolytic substrates are dynamic structures, with ubiquitinating (El, E2, E3) and deubiquitinating (Ubp) enzymes rapidly modifying these adducts. Ubiquitinated substrates are degraded by a large -2,000 kDa protease called the 26s proteasome complex. (b) Interaction of the 20s proteasome with alternative regulatory complexes. A number of activators and inhibitors have been shown to regulate the 20s proteasome in vitro. A -700 kDa complex called proteasome activator 700 (PA700; also know as the ATPase complex or the p-particle) can bind to both ends of the CZ-symmetric proteasome, thereby conferring ubiquitin-dependence on the complex, which is now called the 26s proteasome complex (for structural details, see [50*,51*]). The 20s proteasome activator 28 fPA28) consists of a hexamer or heptamer of 28 kDa subunits and is known to stimulate peptidase (but not protease) activity in vitro [52,53]. The physiological relevance of the PA28-proteasome complex has not yet been established, nor is it clear whether opposite sides of the proteasomal cylinder can be simultaneously occupied by a PA28 complex and a PA700 complex.

Naturally

short-lived

intracellular

proteins

Substrates of the ubiquitin system must be structurally distinguishable from the many stable proteins in the same cellular compartment. Transplantable sequence elements recognized by a proteolytic targeting apparatus (degradation signals) have been identified in a number of short-lived proteins (see [7]). Although many new substrates of the ubiquitin system have been identified in the past few years (Table l), the structural features that comprise their degradation signals are still largely unknown. Interestingly, the ATP-dependent degradation of one protein, ornithine decarboxylase, occurs via the ubiquitin conjugate degrading 26s proteasome complex, but ornithine decarboxylase is not ubiquitinated [8]. Instead, ornithine decarboxylase targeting to the 26s enzyme depends on its association with another small protein, called antizyme. How this interaction leads to the destruction of ornithine decarboxylase, while sparing antizyme, is not known. Discussion of many of the

substrates listed in Table below.

1 can be found in the sections

Ubiquitination A primary determinant of substrate specificity in the ubiquitin pathway is the E2-E3 ubiquitin-protein ligase complex. In yeast, twelve E2 enzymes have been identified ([9]; D Finley, personal communication), and mutations in many of them lead to distinct phenotypes, suggesting that E2 proteins have different substrate specificities. Degradation of several short-lived proteins has now been shown to depend on particular E2 enzymes. Ubiquitination of the yeast MATa transcription factor, the first natural substrate for which a specific E2 enzyme was identified, turned out to be unexpectedly complex [lO”]. Four different E2 enzymes are necessary for wildtype rates of MATa degradation in viva. These enzymes

Ubiquitin, proteasomes, and the regulation of intracellular protein degradation Hochstrasser 217 Table 1. Currently known proteolytic substrates of the ubiquitin-proteasome pathway (in approximate order of discovery). Substrate

Function

Ubiquitination

Regulation of turnover

References

Phytochrome

Plant light-responsive regulator

Yes

Red light stimulates turnover

[54]

MAT0~2

Transcription factor (yeast)

Yes

Unknown

[55]

Mitotic cyclins

Cell cycle regulation

Yes

Turnover is cell-cycle dependent

[56]

p53

Tumor suppressor

Yes

HPV E6 protein stimulates turnover

[57]

Mos

Oocyte Ser/Thr protein kinase

Yes

Phosphorylation inhibits turnover

[58]

Ornithine decarboxylase

Synthesis of polyamines

No

Polyamines stimulate turnover

[8]

IgE, T cell receptors

Immune system surface receptors

Yes

Ubiquitination regulated by ligand

Ste6

Export of pheromone (yeast)

Yes

Unknown

[61 "]

Get

Signal transduction (yeast)

Yes

Unknown

[62"]

NFKB, IKB

Transcriptional regulators

Yes

Modulated by a variety of factors

[63"]

c-Jun

Transcription factor

Yes

Turnover depends on 6 element

[64"]

Fructose bisphosphatase

Gluconeogenesis (yeast)

ND

Turnover induced by glucose

[65]

Sicl

Inhibitor of Cln-Cdc28 (yeast)

ND

Turnover is cell-cycle dependent

Itrl

Inositol permease (yeast)

ND

Inositol stimulates turnover

Gcn4

Transcription factor (yeast)

Yes

Stabilized by amino acid starvation

Cln2,3

G I cyclins (yeast)

Yes

Turnover depends on phosphorylation

[59,60]

[47"] (a) [66°] [67",68 °]

ND, not determined; (a) P McGraw, personal communication. HPV, human papillomavirus.

participate in two distinct ubiquitination pathways, one involving a complex between the Ubc6 and Ubc7 E2 proteins. Interestingly, mutants that lack Ubc6 or Ubc7 have disparate phenotypes, indicating that these enzymes work independently in some cellular processes. These observations lead to the idea that the ubiquitin conjugation system could expand its repertoire of substrate specificities by association of a limited set of' E2 proteins into multiple hetero-oligomeric complexes. This combinatorial expansion of specificities might be extended by interactions with E3 proteins. The yeast Rad6 E2 enzyme, which is important for DNA repair, combines with the Ubrl E3 protein to form a ubiquitin-protein ligase complex that targets substrates bearing particular 'destabilizing' amino-terminal residues (N-end rule substrates) [5]. Ubrl plays no role in DNA repair, however, wherein Rad6 appears to form a distinct complex with the Rad18 protein [11"]. R.ad18, unlike Rad6, is a DNA-binding protein. It is therefore possible that Rad18 functions by an E3-like mechanism, bringing Rad6 to chromatin sites bearing DNA lesions. The Rad6-Rad18 ubiquitin-protein ligase complex could target either chromatin-associated proteins that block

DNA repair enzymes or components of the DNA repair machinery. There may be very different kinds of E3 proteins; for example, Rad18 bears no similarity to Ubrl. Another protein, E6-associated protein (E6-AP), has E3-like properties, and it is unrelated to either Rad18 or Ubrl [12"]. E6-AP was identified as a protein that binds the oncogenic human papillomavirus E6 protein in vitro and, together with E6, binds to and helps to trigger the degradation of the p53 tumor suppressor. E6-AP contains a carboxy-terminal segment required for E6-dependent ubiquitination of p53. This segment bears similarity to carboxy-terminal elements in over a half dozen known proteins, including the essential Drosophila protein hyperplastic discs, which has properties akin to those of a tumor suppressor [13]. Unexpectedly, E6-AP can itself form a thiolester bond with ubiquitin; formation of this bond is abolished by mutation of a specific cysteine residue in the conserved carboxy-terminal domain, as is E6-dependent ubiquitination of p53 [14°']. Ubrl can also form a thiolester bond with ubiquitin (V Chau, personal communication). These data suggest that E3 proteins may not simply be adaptor molecules that bring

218

Cell regulation

E2 proteins to their correct targets (as was previously thought) but may participate as intermediates in ubiquitin transfer reactions. The biochemical logic of such a ubiquitin thiolester ‘bucket brigade’ mechanism (El+ E2+ E3+ substrate) remains uncertain. Perhaps the efficiency/processivity of substrate multi-ubiquitination is increased by allowing the El and E2 enzymes to ‘reload’ with ubiquitin even while the substrate-ubiquitin bond is being forged.

Deubiauitination Attempts to understand the regulation of intracellular protein turnover have naturally focused on the enzymes that attach ubiquitin to proteins, but recent results strongly suggest that regulatory events will also center on rates of substrate deubiquitination. Ubiquitin chains that assemble on various proteins are highly dynamic, with rapid addition and removal of ubiquitin units. The enzymes responsible for removing ubiquitin from substrates, known as ubiquitin carboxy-terminal hydrolases, ubiquitin isopeptidases, or deubiquitinating enzymes, fall into two distinct families: a set of relatively small proteins that preferentially cleave ubiquitin from small molecules, such as peptides, lysine or the abundant intracellular nucleophile glutathione, and a group of larger proteins that can generally cleave ubiquitin from a range of protein substrates in vitro [15]. This latter family of enzymes, the so-called Ubp (ubiquitin-specific processing protease) class, is extremely diverse, but all members contain several short conserved sequences that probably form the active site of the enzyme [16,17”]. The explosion of sequence information from the various organism-based genome projects now indicates that the Ubp enzyme family is remarkably large. In yeast alone the number of likely or confirmed Ubp enzymes now stands at fifteen (my unpublished data), which exceeds the number of known E2 ubiquitinconjugating enzymes. Changing the rate of ubiquitin removal from a substrate will alter the probability of the multi-ubiquitinated intermediate being recognized by the 26s proteasome complex. The unexpectedly high number of deubiquitinating enzymes now being uncovered therefore raises the possibility that specific protein turnover rates can be differentially regulated by these enzymes. Such regulation would imply that Ubps possess a considerable degree of substrate specificity. A striking example of specificity comes from recent work on isopeptidase T, a Ubp-type enzyme that acts largely if not exclusively on unanchored ubiquitin chains, that is, ubiquitin polymers with a free ubiquitin a-carboxyl group ([lg]; KD Wilkinson, personal communication). A number of E2 enzymes can synthesize unanchored chains de novo, and such chains may also be generated by the action of the 26s proteasome. Because the 26s proteasome avidly binds ubiquitin chains

[19**], competitive binding by an excess of unanchored chains in vivo could inhibit 26s enzyme action. Thus, isopeptidase T may have a broad effect on proteolysis by preventing inhibition of the 26s proteasome complex by unanchored ubiquitin chains. Evidence for such an effect may be provided by a yeast mutant lacking Ubp14, a deubiquitinating enzyme which may be a homolog of isopeptidase T (KD Wilkinson, M Hochstrasser, unpublished data). The mutant shows general defects in proteolysis and accumulates ubiquitinated species of low molecular mass that appear to be unanchored ubiquitin polymers (A Amerik, S Swaminathan, M Hochstrasser, unpublished data). Another yeast ubiquitin isopeptidase, Doa4, has been shown to have a very broad role in ubiquitin-dependent degradation in vivo [17”]. Molecular and genetic analyses strongly suggest that Doa works in conjunction with the 26s proteasome complex, and purified preparations of the apparent yeast 26s complex contain Doa (FR Papa, M Hochstrasser, unpublished data). In doa mutants, small ubiquitinated species accumulate; these species are all slightly larger than unanchored ubiquitin chains, leading to the suggestion that they may be the ubiquitinated proteolytic remnants of 26s proteasome action. Such remnants may collect on the protease in dou4 cells, preventing its recycling for further rounds of protein degradation. Intracellular levels of Doa are at least partially rate-limiting for the turnover of certain substrates [17**]. Conceivably then, the Doa isopeptidase may function as a proteolytic ‘governor,’ analogous to the governors placed on steam engines, controlling rates of proteolysis by controlling the rate at which the end products of protease action are cleared from the 26s enzyme. A negative feedback loop regulating protein degradation rates could have several important effects. First, by restraining the action of the 26s proteasome complex, it could confer on the protease a degree of substrate discrimination that it would not have if rates of proteolysis greatly exceeded rates of substrate ubiquitination. In fact, elimination of Doa from yeast cells stabilizes various proteins to strikingly different degrees, as might be predicted if substrates were competing for limiting amounts of active protease in the mutant. A second consequence of an isopeptidase-based 26s proteasome governor is that broad changes in relative protein-degradation rates could be engendered by varying the level or activity of Doa or Doa4-like isopeptidases. For instance, as yeast cells enter stationary phase or are starved, rates of general proteolysis rise [20], which may at least partly reflect an increase in 26s proteasome activity required for amino acid mobilization and protein remodeling. DOA gene expression is known to rise as cells approach stationary phase [17”]. In mammalian cells, several proteins implicated in tumorigenesis [21,22] have recently been shown to be deubiquitinating enzymes [17**]. Interestingly, in the case of the human tre-2 oncogene product, it appears that it is an inactive form of the deubiquitinating enzyme

Ubiquitin, proteasomes, and the regulation of intracellular protein degradation Hochstrasser 219 that is tumorigenic. The inactive protein may act in a dominant-negative fashion, interfering with tre-2-mediated degradation of one or more positive regulators of cell prohferation, such as the G 1 cychns. Alternatively, the tre-2 enzyme may normally hmit the degradation of a negative regulator(s) of growth, such as p53, by rapidly disassembhng ubiquitinated intermediates. A Drosophila protein important for embryogenesis and eye development, fat facets, has sequence elements that are characteristic of Ubp proteins as well [17°°]. Collectively, these findings suggest that deubiquitinating enzymes play central but as yet largely unexplored regulatory roles in the growth and development of eukaryotic organisms.

Degradation As depicted in Fig. Ib, the 26S proteasome complex is a large supramolecular complex, composed of a core proteinase known as the 20S proteasome and a pair of regulatory complexes that are probably equivalent to a separable multisubunit protein known as PA700 (for 700 kDa proteasome activator) [23°°,24°-26°]. I will use the name PA700 here, but the exact composition and functions of the protein complexes attached to either end of the 20S proteasome cylinder in the 26S complex have yet to be fully defined [6]. The 26S enzyme may exist in several dynamic and interconvertible forms in vivo. Studies on antigen presentation in mammals have provided an example of different proteasomal subpopulations in the same cell. Interferon-y is an antiviral factor that induces components of the M H C class I antigenpresentation pathway. The antigens for this pathway include peptides derived from viral proteins found in the cytoplasm or nucleus of infected cells. Two 20S proteasomal subunit genes, LMP2 and LMP7, map to the M H C region, and expression of these genes is induced by interferon-y. Several groups have now demonstrated that LMP2 and LMP7 can specifically replace certain 'constitutive' proteasome subunits [27,28]. Biogenesis of the proteasome involves an ordered assembly pathway, requiring proteolytic processing of certain subunits (including LMP2 and LMP7) at specific stages of the pathway [29°]. These processing steps are conserved between yeast and mammals (P Chen, M Hochstrasser, unpubhshed data). In mammalian cells, proteasome maturation occurs over a period of hours, so subunit replacement by LMP2 and LMP7 must also be fairly slow. The mechanistic consequences of these subunit replacements remain unclear. Both mutant and inhibitor studies suggest that the 20S proteasome or 26S proteasome complex helps to generate the peptides presented by class I M H C molecules [30°,31,32°]. Evidence has also been presented that favors the notion that the range of possible protein cleavages can be modulated by altering the composition of these enzymes, perhaps

tailoring cleavage specificity to match the peptide-binding specificity of M H C molecules. However, 'the reported interferon-induced changes in peptide cleavage preferences were discrepant between various laboratories [33°,34,35], and several important limitations of the experiments performed comphcate their interpretation. Despite the mechanistic uncertainties, a deficit in presentation of certain antigens has now been shown with cells from mice carrying a deletion of either LMP2 or LMP7 [32°,36°]. The PA700 component of the 26S proteasome complex appears to confer both ubiquitin-dependence and ATPdependence on protein degradation by the 20S proteasome. The 20S proteasome itself is unable to degrade stably folded proteins, but it can break these proteins down completely (in the absence of ATP) if they are first denatured [37°]. Thus, one anticipated function of PA700 is as a protein unfoldase. The requirement for an unfoldase activity brings to mind the action of molecular chaperones, which can catalyze protein conformational changes [38]. Recent experiments with the plasmid P1 repA rephcation protein demonstrate that repA is degraded by the Escherichia coli clpA--clpP protease complex in vitro [39°°]. ClpA is an ATP-dependent activator of the clpP protease, a property which has some similarity to the effect of PA700 on the 20S proteasome. Surprisingly, in the absence of clpP, clpA functions as a chaperone, dissociating repA dimers into active monomers by a mechanism identical to that of the hsp70 chaperone system [39°°]. Hence, an ATP-dependent chaperone can conformationally alter a substrate protein in such a way that it either changes the protein's activity or, if the chaperone is coupled to a proteolytic enzyme, causes the protein to be destroyed. At least four PA700 subunits turn out to be members of a closely related family of ATPases ([24°,6,40°°], and PA700 has ATPase activity [24"]. Another anticipated function of the PA700 complex, the binding of multi-ubiquitinated proteins, was recently traced to a specific PA700 subunit, with binding affinity increasing dramatically at ubiquitin chain lengths of four or more monomers [19°°]. PA700 is clearly relevant physiologically to ubiquitin-dependent proteolysis inasmuch as mutations in yeast genes encoding several PA700 subunits result in defects in the degradation of ubiquitin system substrates ([40°°]; my unpubhshed data). On the basis of the above data, a reasonable but largely hypothetical scheme for proteolysis by the 26S proteasome complex would include the following steps: binding of the multi-ubiquitinated protein by its ubiquitin chain(s) to the chain-binding subunits of PA700; a series of ATP-dependent unfolding and translocation steps that feed the unfolded polypeptide into the central channel of the 20S proteasome, perhaps with concomitant movement or partial disassembly of the ubiquitin chain within PA700; cleavage of substrate to small peptides, which are released through fenestrations in the proteasome wall (by analogy to the related GroEL chaperonin structure;

220

Cell regulation

ubiquitin-protein ligase complex [45*], and/or in a cyclin-specific deubiquitinating enzyme.

[41]); and, in a final step, disassembly of ubiquitin chains on the remaining proteolytic remnants, which facilitates their release horn the protease. In this view, the ubiquitin chain functions as both tag and tether, targeting the substrate to the protease and then ensuring the processivity of the ensuing proteolytic cleavages by holding the substrate in place as it is being destroyed.

Two recent papers [46**,47”] provide insight into how selective protein degradation underlies the transition between cell cycle stages in yeast. In the first [46”], experiments with synchronized cultures revealed, unexpectedly, that degradation of Clb2 occurs not just in a brief time window at the end of mitosis but continues into early G1 phase. It is only upon activation of Cdc28 by the newly synthesized Gl cyclins (Clns) that Clb2 degradation is halted. The important consequence of the degradation of mitotic cyclins early in G1 is that they are prevented from reaccumulating before the formation of Cm-Cdc28 complexes. The Cln-Cdc28 complex stops Clb2 degradation, a prerequisite for the subsequent accumulation of Clb2, which in turn shuts off synthesis of the Cln proteins. The combination of feedback controls on both cyclin proteolysis and synthesis leads to a cyclical alternation between Cln-Cdc28 and Clb-Cdc28 complexes, which helps ensure the strictly ordered passage of cells through stages of DNA synthesis and mitotic division.

Ubiauitin and cell cycle progression Regulated ubiquitin-mediated protein turnover is emerging as a primary mechanism governing passage through the cell cycle (reviewed in [42]). The central cell cycle regulator is the cyclin-dependent protein kinase, called Cdc28 in budding yeast (multiple cyclin-dependent kinases exist in most organisms). Genetic experiments in yeast are beginning to reveal the logic of cell cycle traversal. From a regulatory perspective, the cell cycle can now be recast as a sequence of timed regulatory-subunit eliminations and replacements in the Cdc28 kinase complex; these regulatory subunits are the cyclins, which are positive regulatory subunits, and the cyclin-dependent kinase inhibitors. In all cases examined, ubiquitin-dependent proteolysis turns out to be the mechanism of subunit elimination. The cell cycle timings of these eliminations are outlined in Fig. 2.

The second paper [47**] describes how the G1 +S transition is controlled by the destruction of a specific cyclindependent kinase inhibitor, the p4OSICl (p40) protein. In yeast, two B-type cyclins, Clb5 and Clb6, appear in Gl and form complexes with Cdc28; these Cdc28 complexes are important for entry into S phase but are inactive through earlier parts of G1 because they are tightly bound to ~40, which accumulates shortly after mitosis. Destruction of p40 triggers entry into S phase and is mediated by the Cdc34 ubiquitin-conjugating enzyme. It is not known what signals the timed turnover of ~40, but initial data suggest that its phosphorylation by the Cln-Cdc28 kinase is an important component of this signal.

The precipitous, cell cycle stage specific degradation of cyclins was the physiological property that led to their discovery [42]. Rapid turnover of B-type mitotic cyclins in vitro was later shown to correlate with their multi-ubiquitination. In viva degradation of two yeast B-type cyclins, Clb2 and Clb5, has now been found to be strongly inhibited by conditional mutations in Ubc9, an essential E2 enzyme [43*]. Interestingly, Clb2, a mitotic cyclin, is degraded in a cell stage specific manner, whereas Clb5, which is involved in entry into S phase, is degraded throughout the cell cycle. This suggests that the stage specificity of Clb2 instability is due to something other than Ubc9 activation. The molecular mechanism governing the regulated switch between the stable and unstable states of cyclin proteins remains the most intriguing aspect of cyclin degradation; it may involve alteration of the substrate [44], modification of the

In summary, rapid ubiquitin-dependent proteolysis of Cdc28 kinase regulatory subunits is a key mechanism controlling both the M+Gl transition and the Gl+S transition. On the basis of work on other cell cycle regulators (Table l), it is likely that ubiquitin-dependent turnover is critical in other aspects of cell cycle progression. Consistent with this, ubiquitin mutants that are unable to assemble ubiquitin chains on proteolytic substrates are inviable, with most cells arresting near the G+M boundary of the cell cycle [48*]. Mutants in the

Start 1

G, cyclins (Cln2, Cln3)

t

I

I

S

Gl

I

I

c,

I

,

M

1 -1

,H

Sic1 B-type cyclins: Clb2 H Clb5

I

changes

I

0 1995 Current Opinion in Cell Biology

Fig. 2. Periods of the budding yeast cell cycle during which degradation of the indicated Cdc28 kinase regulators occurs. For those proteins degraded throughout the cell cycle, such as Clb5, it is still possible that proteolytic rates can vary between stages. ‘Start’ refers to the point when the cell becomes committed to completing the mitotic cycle regardless of nutritional status. Budding yeast do not have a cytologically well defined Cz phase.

Ubiquitin, proteasomes, and the regulation of intracellular protein degradation Hochstrasser 26s proteasome [40”,49].

complex

also exhibit

cell cycle defects

is degraded by the 265 proteasome Nature 1992, 360:597-599. 9.

Jentsch 5: The ubiquitin conjugation 1992, 26:177-205.

without

ubiquitination.

system. Annu Rev Genet

10. ..

Conclusions The ubiquitin/proteasome field is entering what should be a very exciting period. We have begun to sense the breadth of processes in which ubiquitin-dependent proteolysis plays a key role, and we have assembled a he@ catalog of proteins that function in the ubiquitin pathway. We are therefore in a position to answer some of the fundamental questions in the field, to wit: what comprises a protein degradation signal? How are substrate ubiquitination and degradation regulated, for example, in cell cycle control or in response to changes in nutritional status? How are the dynamics of intracellular ubiquitin and ubiquitin-protein conjugate pools regulated? What are the mechanistic roles of ubiquitin chains? What kinds of supramolecular complexes are formed by the proteasome in vivoand what are their functions? How is a protein broken down into peptides by the proteasome and how is this regulated? Many of these problems, one may hope, will soon yield to experimental attack.

Chen P, Johnson P, 5ommer T, Jentsch 5, Hochstrasser M: Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MATa repressor. Cell 1993, 74:357-369. The first time a natural substrate of the ubiquitin system was linked to a specific E2 enzyme. Surprisingly, two separate ubiquitination pathways were uncovered, one involving a complex of different E2 enzymes. The data suggest a combinatorial mechanism of substrate selection wherein different E2 enzymes partition into multiple ubiquitination complexes. 11. .

Bailly V, Lamb J, Sung P, Prakash 5, Prakash L: Specific comples fprmation between yeast RAD6 and Radl8 proteim: a potential mechanism for targeting RADC ubiquitin-conjugating activity to DNA damage sites. Genes Dev 1994, 8:811-820. Demonstrates that RadlB is a DNA-binding protein that can form a tight complex with the Rad6 E2 enzyme. This complex was shown to be important for DNA repair.

12. ..

Scheffner M, Huibregste JM, Vierstra RD. Howley PM: The HPV E6 and ECAP complex functions as a ubiquitin-protein ligase in the ubiquitination of ~53. Cell 1993, 75:495-505. The authors used biochemical fractionation of an in vitro ubiquitination system to show that the role of the E6-associated protein (E6-AP)/E6 complex was most likely to be that of an E3 ubiquitirr-protein ligase. They were able to reconstitute p53 ubiquitination with isolated El, E2, ubiquitin, and Eb-AP/E6 proteins. 13.

Acknowledgements 1 would

like to thank J Shapiro, K Wilkinson, and members of my laboratory, especially L Stillman, for comments on the manuscript and V Chau, D Finley, P McGraw, and K Wilkinson for providing data prior to publication. Work from my laboratory was funded by grants from the National lnstitutes of Health (GM46904). the Cancer Research Foundation, and the Searle Scholars Program of the Chicago Community Trust.

References and recommended Papers of particular interest, published review, have been highlighted as: . of special inter&t .. of outstanding interest

Scheffner M, Nuber U, Huibregtse JM: Protein ubiquitination involving an El-EZ-E3 enzyme ubiquitin thioester cascade. Nature 1995, 373:81-83. Reports the surprising finding that E6-associated protein (E6-AP), an E3 ligase, can form a thiolester bond with ubiquitin. This E3-ubiquitin thiolester appears to be a required intermediate in the ubiquitination of p53 and other cellular proteins. A cysteine residue in E&AP was identified as being necessary for ubiquitin thiolester formation. 14. ..

15.

Mayer AN, Wilkinson KD: Detection, resolution, and nomenclature of multiple ubiquitin carboxyl-terminal esterases from bovine calf thymus. Biochemistry 1989, 28:166-l 72.

16.

Baker RT, Tobias JW, Varshavsky A: Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and U8P3, and functional analysis of the U8P gene family. / Biol Chem 1992, 267:23364-23375.

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3.

Okazaki K, Nishizawa M, Furuno N, Yasuda H, Sagata N: Differential occurrence of CSF-like activity and transforming activity of Mos during the cell cycle in fibroblasts. EM60 / 1992, 11:2447-2456.

4.

Hershko A, Ciechanover A: The ubiquitin system for protein degradation. Annu Rev B&hem 1992, 61:761-807.

5.

Varshavsky A: The N-end rule. Cell 1992, 69:725-735.

6.

Rechsteiner M: The multicatalytic Chem 1993, 268:6065-6068.

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Hochstrasser M: Ubiquitin and intracellular protein tion. Curr Opin Cell Biol 1992, 4:1024-1031.

a.

Murakami Y, Matsufuji Tamura T, Tanaka K,

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5, Kameji T, Hayashi lchihara A: Omithine

1 Biol

degrada-

5, lgarashi K, decarboxylase

Mansfield E, Hersperger E, Biggs J, Shearn A: Genetic and molecular analysis of hyperplastic discs, a gene whose product is required for regulation of cell proliferation in Drosophila melanogaster imaginal discs and germ cells. Dev Biol 1994, 165:507-526.

17. ..

Papa FR, Hochstrasser M: The yeast DOA gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene. Nature 1993, 366:313-319. Demonstrates that Doa is a ubiquitin isopeptidase that is required for normal rates of ubiquitin-dependent proteolysis in vivo. The data suggest that Doa works with or is pan of the 265 proteasome complex. The authors also show that the Doa4-related rre-2 proto-oncogene product is a ubiquitin isopeptidase and that an oncogenic rre-2 derivative is enzymatically inactive.

ia.

Hadari T, Warms JVB, Rose IA, Hershko A: A ubiquitin C-terminal isopeptidase that acts on polyubiquitin chains role in protein degradation. / Biol Chem 1992, 267:719-727.

Deveraux Q, Ustrell V, Pickart C, Rechsteiner M: A 26s protease subunit that binds ubiquitin conjugates. 1 Bio/ Chem 1994, 269:7059-7061. Far-western analysis of purified preparations of 265 proteasome complex identifies a single protein component that binds tightly to either unanchored ubiquitin chains or ubiquitin-lysozyme conjugates. Ubiquitin chains of four or more monomers bind much more tightly than do smaller chains. 19. ..

20.

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Wickner S, Gottesman S, Skowyra D, Hoskins J, McKenney K, Maurizi MR: A molecular chaperone, ClpA, functions like DnaK and DnaJ. Proc Nat/ Acad Sci USA 1994, 91:12218-12222. Reports the remarkable finding that a single protein, ClpA, can either function as a chaperone, being able to alter the conformation of the substrate protein, or, if bound to a proteinase, CIpP, can lead to the ATP-dependent degradation of that same substrate.

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Ghislain M, Udvardy A, Mann C: S. cerevisiae 26s protease mutants arrest cell division in CZ/metaphase. Nature 1993, 366:358-362. The first evidence for the physiological importance to ubiquitin-dependent protein degradation of the 265 proteasome complex. Interestingly, the mutations in two different yeast PA700 subunits were identified by their enhancement of the cell cycle defect of a cdc28 mutant at the GZ-+M stage, which may be related to the finding that Cdc28 is required for activation of mitotic cyclin degradation.

Michalek M, Grant E, Gramm C, Goldberg AL, Rock K: A role for the ubiquitin-dependent proteolytic pathway in MHC class I-restricled antigen presentation. Nature 1993, 363:552-554. A mouse cell with a thermolabile El ubiquitin-activating enzyme is deficient in class I restricted antigen presentation at restrictive temperatures, suggesting the involvement of the ubiquitin system in the generation of peptides for presentation to cytotoxic T lymphocytes.

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Palombella VJ, Rando OJ, Goldberg AL, Maniatis T: The ubiquitin-proteasome pathway is required for processing the NF-KBI precursor protein and the activation of NF-~8. Cell 1994, 78~773-785. This paper provides the first example of a natural substrate of the ubiquitin system that is not broken down completely into peptides by the proteasome, but, rather, is released as a proteolytically truncated protein, the transcriptionally active form of the NF-KB transcription factor. 64. .

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Kornitzer D, Raboy B, Kulka RG, Fink CR: Regulated degradation of the transcription factor Ccn4. EM60 I 1994, 13:60214030. The yeast Gcn4 bZlP protein, which controls expression of many amino acid biosynthetic genes, is shown here to be ubiquitinated and rapidly degraded in viva. Degradation is inhibited by amino acid starvation, which could provide a homeostatic mechanism for controlling amino acid levels different from the well established translational control of Gcn4. An internal element of Gcn4 containing PEST sequences (single letter code for amino acids), which were previously postulated to contribute to protein turnover, was important for Gcn4 degradation. 67. .

Deshaies RJ, Chau V, Kirschner MW: Ubiquitination of the Gl cyclin Cln2p by a CdcMp-dependent pathway. EM60 / 1995, . . 14:303-31 i. Yeast extracts were made that could support multi-ubiquitination of in vitro translated Cln2 protein. Extracts from mutants lacking the Cdc34 E2 enzyme were deficient in this activity, and cdc34 mutants showed reduced degradation of Cln2 in vivo. 68.

Yaalom J, Linskens HK, Sadis S, Rubin DM, Futcher 8, Finlev D:p34c~2s-mediated control of Cln3 cyclin degradation. Md/ Cell Biol 1995, 15:731-741. Derivatives of Cln3, a yeast C, cyclin, and Cln3-fi-galactosidase were shown to be degraded in viva by a pathway that requires both the Cdc34 E2 enzyme and the Cdc28 kinase. A carboxy-terminal domain containing multiple PEST sequences (single letter code for amino acids) plays a critical role in degradation.

.

M Hochstrasser, ology, University 60637, USA.

Department of Chicago,

of Biochemistry 920 East 58th

and Molecular Street, Chicago,

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