Voet D., Voet Ju.G. Biochemistry

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(b)

as adapters that position the reactive E2–ubiquitin

thioester bond for the direct transfer of the ubiquitin to

the substrate protein Lys side chain.

In order for a target protein to be efficiently degraded, it

must be linked to a chain of at least four tandemly linked

ubiquitin molecules in which Lys 48 of each ubiquitin forms

an isopeptide bond with the C-terminal carboxyl group of

the succeeding ubiquitin (Fig. 32-78). These polyubiquitin

(polyUb) chains, which can reach lengths of 50 or more

ubiquitin molecules, are generated by the E3s, although

how they switch from transferring a ubiquitin to the target

protein to processively synthesizing a polyubiquitin chain

is unknown.

c. Ubiquitinated Proteins Are Hydrolyzed

in the Proteasome

A ubiquitinated protein is proteolytically degraded to

short peptides in an ATP-dependent process mediated by a

large (2000 kD, 26S) multisubunit protein complex named

the 26S proteasome (sometimes spelled “proteosome”) that

electron micrographic studies reveal has the shape of a bi-

capped hollow barrel (Fig. 32-79). Proteolysis occurs inside

the barrel, which permits this process to be extensive and

processive, while preventing nonspecific proteolytic dam-

age to other cellular components. PolyUb chains are the

signals that target a protein to the proteasome; the identity

of the target protein has little effect on the efficiency with

which it is degraded by the proteasome. Nevertheless, the

proteasome does not degrade ubiquitin molecules; they are

returned to the cell. The size and functional complexity of

this entire proteolytic system, which occurs in the nucleus

as well as the cytosol, rivals that of the ribosome (Section

32-3) and the spliceosome (Section 31-4A) and hence is

Section 32-6. Protein Degradation 1411

Figure 32-78 X-ray structure of human tetraubiquitin. (a) A

ribbon drawing in which the isopeptide bonds connecting

successive ubiquitin molecules, together with the Lys side chains

making them, are drawn in stick form with C green, N blue, and

O red. However, since the C-terminal three residues of ubiquitin

2 are disordered, the isopeptide bond connecting it to ubiquitin 3

is represented by a thin gray bond (this isopeptide bond

nevertheless exists, as was demonstrated by SDS–PAGE of

dissolved crystals).The monomer units in a multiubiquitin chain

of any length are likely to be arranged with the repeating

symmetry of the tetraubiquitin structure, although the weak

interactions between adjacent ubiquitin units suggests that this

chain is conformationally flexible. (b) A surface diagram, viewed

as in Part a, in which basic residues (Arg, Lys, His) are blue,

acidic residues (Asp, Glu) are red, uncharged polar residues

(Gly, Ser, Thr, Asn, Gln) are purple, and hydrophobic residues

(Ile, Leu,Val,Ala, Met, Phe,Tyr, Pro) are tan (ubiquitin lacks Cys

and Trp residues). Note the unusually large solvent-exposed

surface occupied by the hydrophobic residues. [Based on an

X-ray structure by William Cook, University of Alabama at

Birmingham, and Cecile Pickart, Johns Hopkins University.

PDBid 1TBE.]

Figure 32-79 Electron microscopy–based image of the

Drosophila melanogaster 26S proteasome. The complex is

around 450  190 Å.The central portion of this 2-fold symmetric

multiprotein complex (yellow), the 20S proteasome, consists of

four stacked 7-membered rings of subunits that form a hollow

barrel in which the proteolysis of ubiquitin-linked proteins

occurs.The 19S caps (cyan), which may attach to one or both

ends of the 20S proteasome, control the access of condemned

proteins to the 20S proteasome (see below). [Courtesy of

Wolfgang Baumeister, Max Planck Institute of Biochemistry,

Martinsreid, Germany.]

(a)

JWCL281_c32_1338-1428.qxd 9/7/10 2:23 PM Page 1411

indicative of the importance of properly managing protein

degradation. Indeed, ⬃5% of the proteins expressed by

yeast participate in protein degradation. We discuss the

structure and function of the 26S proteasome below.

d. Many E3s Have Elaborate Modular Structures

The proto-oncogene product c-Cbl (906 residues) is a

single-subunit, RING domain–containing E3 that functions

to ubiquitinate certain activated receptor tyrosine kinases

(RTKs; Section 19-3A), thereby terminating their signaling.

Nikola Pavletich determined the X-ray structure of the N-

terminal half of c-Cbl (residues 47–447) in its ternary com-

plex with the E2 protein UbcH7 (which consists of little

more than the ⬃150-residue E2 catalytic core) and a 9-

residue peptide containing the ubiquitination signal from a

nonreceptor tyrosine kinase (NRTK) named ZAP-70

(Fig. 19-44). The structure (Fig. 32-80) reveals that UbcH7

and c-Cbl’s RING domain and SH2-containing tyrosine ki-

nase–binding (TKB) domains interact with one another

across multiple interfaces to form a compact and apparently

rigid structure.The RING domain consists of a 3-stranded 

sheet, an  helix, and two large loops that are held together

by two tetrahedrally coordinated Zn

2

ions. UbcH7 adopts

the characteristic / fold of other E2s of known structure

(e.g., Fig. 32-77).The ZAP-70 peptide is bound on the oppo-

site side of the TKB domain from the UbcH7 active site Cys

residue (Cys 86) and is ⬃60 Å distant from it.

SCF complexes are multisubunit RING E3s that consist

of Cul1 (a member of the cullin family; 776 residues), Rbx1

(which contains the complex’s RING domain; 108

residues), Skp1 (163 residues), and a member of the F-box

protein family (⬃430 to 1000 residues; SCF for

Skp1–cullin–F-box protein). Rbx1 and Cul1 form the com-

plex’s catalytic core that binds E2; F-box proteins consist

of an ⬃40-residue F-box that binds Skp1 followed by

protein–protein interaction modules such as leucine-rich

repeats (LRRs) or WD40 repeats (Section 19-2C) that bind

substrate protein; and Skp1 functions as an adapter that

links the F-box to Cul1. Cells contain numerous different

F-box proteins (at least 38 in humans) that presumably

permit the specific ubiquitination of a diverse variety of

protein substrates (see below).

Pavletich has also determined the X-ray structures of

two segments of the SCF

Skp2

complex (where the super-

script identifies the complex’s F-box protein, here Skp2,

436 residues). The structure of the Skp1–Skp2 complex

(Fig. 32-81) reveals that it has the shape of a sickle with the

Skp1 and the 3-helix F-box of Skp2 forming the handle and

its 10 LRRs (⬃26 residues each) forming the curved blade.

The structure of the Cul1–Rbx1–Skp1–F-box

Skp2

quater-

nary complex (Fig. 32-82) shows that Cul1 is an elongated

protein that consists of a long stalk formed by three repeats

of a novel five-helix motif known as a cullin repeat fol-

lowed by a globular domain that binds Rbx1. Apparently

Cul1 acts like a rigid scaffold that organizes the Skp1–F-

box

Skp2

complex and Rbx1 so as to hold them over 100 Å

1412 Chapter 32. Translation

Figure 32-80 X-ray structure of the human c-Cbl–UbcH7–ZAP-

70 peptide ternary complex drawn in tube-and-arrow form.

UbcH7, an E2 that consists almost entirely of the E2 catalytic

core, is colored orange with the side chain of its active site Cys 86

shown in stick form with C green and S yellow. c-Cbl (residues

47–447 of the 903-residue protein), a monomeric RING E3, is

colored according to domain with its TKB domain green, its

RING domain red, and the linker joining them yellow. The

RING domain’s two bound Zn

2

ions are represented by cyan

spheres.The 9-residue ubiquitination site of the RTK ZAP-70,

whose fourth residue is phospho-Tyr, is drawn in stick form with

C magenta, N blue, and O red, and P orange. [Based on an X-ray

structure by Nikola Pavletich, Memorial Sloan-Kettering Cancer

Center, New York, New York. PDBid 1FBV.]

Figure 32-81 X-ray structure of the human Skp1–Skp2

complex. Skp1 and Skp2 are drawn in tube-and-arrow form in

blue and magenta. Skp2 consists of an N-terminal F-box that

forms three helices, followed by 3 noncanonical so-called linker

leucine-rich repeats (LRRs) that are contiguous with 7 LRRs

that were predicted from their amino acid sequences for a total

of 10 LRRs.After the tenth LRR, Skp2’s ⬃30-residue C-terminal

tail extends back past the first LRR by packing under the con-

cave surface of the LRR domain. [Based on an X-ray structure

by Nikola Pavletich, Memorial Sloan-Kettering Cancer Center,

New York, New York. PDBid 1FQV.]

JWCL281_c32_1338-1428.qxd 9/7/10 2:23 PM Page 1412

apart.The Rbx1 RING domain contains a 20-residue insert

that forms the binding site for a third tetrahedrally lig-

anded Zn

2

ion.

The apparent rigidity of the foregoing three structures

has enabled Pavletich to construct a model of the intact

SCF

Skp2

–E2 complex by superimposing Skp1–Skp2 on

Cul1–Rbx1–Skp1–F-box

Skp2

and docking the E2 UbcH7

onto the Rbx1 RING domain based on the c-Cbl–UbcH7

structure (Fig. 32-83). The model indicates that E2 and the

LRR-containing domain of Skp2 are on the same side of

the SCF complex but separated by a distance of ⬃50 Å.

This suggests that Cul1’s long stalk functions to separate

the complex’s substrate-binding and catalytic sites so that

substrates with different sizes and various distances be-

tween their ubiquitinated Lys residues and their ubiquiti-

nation signals can be accommodated.

e. The Ubiquitin System Has Both Housekeeping

and Regulatory Functions

Until the mid-1990s, it appeared that the ubiquitin sys-

tem functioned mainly in a “housekeeping” capacity to

maintain the proper balance among metabolic proteins

and to eliminate damaged proteins. Indeed, as Alexander

Varshavsky discovered, the half-lives of many cytoplasmic

proteins vary with the identities of their N-terminal residues

(Table 32-12). Thus, in a selection of 208 cytoplasmic pro-

teins known to be long lived, all have a “stabilizing”

residue, Met, Ser, Ala, Thr, Val, or Gly, at their N-termini.

This so-called N-end rule is true for both eukaryotes and

prokaryotes, which suggests the system that selects pro-

teins for degradation is conserved in eukaryotes and

prokaryotes, even though prokaryotes lack ubiquitin. The

N-end rule results from the actions of the single-subunit,

RING E3 named E3 (⬃1950 residues; also known as

Ubr1) whose ubiquitination signals are the destabilizing

N-terminal residues in Table 32-12.

Similarly, it has long been known that proteins with seg-

ments rich in Pro (P), Glu (E), Ser (S), and Thr (T), the so-

called PEST proteins, are rapidly degraded.This is because

these PEST elements often contain phosphorylation sites

that target their proteins for ubiquitination.

Section 32-6. Protein Degradation 1413

Table 32-12 Half-Lives of Cytoplasmic Enzymes as a

Function of Their N-Terminal Residues

N-Terminal Residue Half-Life

Stabilizing

Met 20 h

Ser

Ala

Thr

Va l

Gly

Destabilizing

Ile ⬃30 min

Glu

Ty r ⬃10 min

Gln

Highly Destabilizing

Phe ⬃3 min

Leu

Asp

Lys

Arg ⬃2 min

Source: Bachmair,A., Finley, D., and Varshavsky,A., Science 234,

180 (1986).

Figure 32-82 X-ray structure of the human

Cul1–Rbx1–Skp1–F-box

Skp2

quaternary complex. Cul1, Rbx1,

Skp1, and the Skp2 F-box are drawn in tube-and-arrow form and

respectively colored green, red, blue, and magenta.The three

cullin repeats of Cul1 are indicated.The three Zn

2

ions bound

to Rbx1 are represented by cyan spheres. [Based on an X-ray

structure by Nikola Pavletich, Memorial Sloan-Kettering Cancer

Center, New York, New York. PDBid 1LDK.]

Figure 32-83 Model of the SCF

Skp2

–E2 complex. This model,

which is based on the X-ray structures in Figs. 32-80, 32-81, and

32-82, is colored and viewed as in Fig. 32-82. E2 is yellow with its

active site Cys residue, to which ubiquitin would be covalently

linked, drawn in space-filling form in cyan.The Zn

2

ions

associated with the Rbx1 RING domain are represented by

yellow spheres.The gray arrow indicates the 50-Å gap between

the tip of the Skp2 LRR domain and the E2 active site. [Courtesy

of Nikola Pavletich, Memorial Sloan-Kettering Cancer Center,

New York, New York.]

JWCL281_c32_1338-1428.qxd 9/7/10 2:23 PM Page 1413

It is now clear, however, that the ubiquitin system is far

more sophisticated than a simple garbage disposal system.

Thus, the known E3s each respond to certain ubiquitina-

tion signals that often occur on a quite limited range of

target proteins, many of which have regulatory functions.

For example, the ubiquitination system has an essential

function in cell cycle progression. The cell cycle, as we have

seen in Section 30-4Aa and will further discuss in Section

34-4D, is regulated by a series of proteins known as cyclins.

A given cyclin, which is expressed immediately preceding

and/or during a specific phase of the cell cycle, binds to a

corresponding cyclin-dependent protein kinase (Cdk),

which then phosphorylates its target proteins so as to acti-

vate them to carry out the processes of that phase of the

cell cycle. Moreover, many cyclins also inhibit the transi-

tion to the subsequent phase of the cell cycle (e.g., DNA

replication or mitosis). Consequently, for a cell to progress

from one phase of the cell cycle to the next, the cyclin(s)

governing that phase must be eliminated. This occurs via

the specific ubiquitination of the cyclin, thereby condemn-

ing it to be destroyed by the proteasome.The E3s respon-

sible for this process are the SCF complexes containing

F-box proteins targeted to a corresponding cyclin and a

multisubunit complex known as the anaphase-promoting

complex (APC; alternatively the cyclosome; Section 34-

4Da).APC, an ⬃1500-kD RING domain–containing par-

ticle that in yeast consists of 11 subunits, specifically

ubiquitinates proteins that contain the 9-residue consen-

sus sequence RTALGDIGN, the so-called destruction

box, near their N-termini.

The transcription factor NF-B, which plays a central

role in immune and inflammatory responses (Section 34-

3Bs), is maintained in an inactive state in the cytosol

through its binding to the inhibitor IB in a way that oc-

cludes the short internal basic sequence that directs NF-

B’s import into the nucleus (its nuclear localization signal;

NLS). However, the stimulation of cell-surface receptors

by proinflammatory cytokines such as tumor necrosis

factor- (TNF; Section 19-3Db) and interleukin-1 (IL-1;

Section 19-3Eb) initiate a signal transduction pathway

(Section 19-3D) that phosphorylates IB bound to NF-

B at both Ser residues in the sequence DSGLDS. This

phosphorylated sequence is the ubiquitination signal for

the SCF complex containing the F-box protein -TrCP

(605 residues), which mediates the ubiquitination of the

phosphorylated IB.The consequent destruction of IB

exposes the NLS of NF-B, which is then translocated to

the nucleus where it activates the transcription of its target

genes (Section 34-3Bs).

Some viruses usurp the ubiquitin system. Oncogenic

forms of human papillomavirus (HPV), the cause of

nearly all cervical cancers (a leading cause of death of

women in developing countries), encode the ⬃150-residue

E6 protein, which combines with the 875-residue cellular

protein named E6-associated protein (E6AP; the first E3

known to contain a HECT domain) to ubiquitinate p53,

thereby marking it for destruction. This latter protein is a

transcription factor that monitors genome integrity and

hence is important in preventing malignant transforma-

tion and the proliferation of cancer cells (Section 34-4Ca),

that is, it is a tumor suppressor (a protein whose loss of

function is a cause of cancer). Consequently, HPV pro-

vokes the uncontrolled growth of the cells it infects and

hence its own proliferation. E6AP normally functions to

ubiquitinate certain members of the Src family of protein

tyrosine kinases (Section 19-3Ba), including Src itself. The

deletion of the segment of chromosome 15 that contains

the E6AP gene causes Angelman syndrome, which as we

have seen (Section 30-7d) is characterized by severe men-

tal retardation and is exclusively maternally inherited due

to genomic imprinting.

The foregoing are only a few examples of the numerous

cellular processes that are regulated by the ubiquitin-medi-

ated proteolysis system. Not surprisingly, therefore, many

pathological conditions in humans, including inflammatory,

neurodegenerative, and muscle-wasting diseases, are at-

tributable to malfunctioning ubiquitination systems.

f. The 20S Proteasome Catalyzes Proteolysis Inside

a Hollow Barrel

The 26S proteasome (Fig. 32-79) is an ⬃2100-kD multi-

subunit protein that catalyzes the ATP-dependent hydroly-

sis of ubiquitin-linked proteins. This yields oligopeptides

with lengths of 4 to 25 residues and averaging 7 to 9

residues that are subsequently degraded to their compo-

nent amino acids by cytosolic exopeptidases. The 26S pro-

teasome consists of a 20S proteasome (⬃670 kD), the bar-

rel-shaped catalytic core of the 26S proteasome, and its 19S

caps (⬃700 kD; also known as PA700 and the 19S regula-

tor), which associate with the ends of the 20S proteasome

and stimulate its activity (PA for proteasome activator).

The 20S proteasome only hydrolyzes unfolded proteins in

an ATP-independent manner; the 19S caps function to

identify and unfold the ubiquitinated protein substrates.

The 20S proteasome occurs in the nuclei and cytosol of

all eukaryotic cells and in all archaebacteria yet examined.

However,the only eubacteria in which it occurs are those of

the class Actinobacteria, which suggests that they obtained

it via horizontal gene transfer from some other organism.

The 20S proteasome of Thermoplasma acidophilum (an

archaebacterium) consists of 14 copies each of  and  sub-

units (233 and 203 residues) that electron microscopy stud-

ies revealed form a 150-Å long and 110-Å-diameter barrel

in which the subunits are arranged in four stacked rings (as

is evident in the central portion of the 26S proteasome seen

in Fig. 32-79).The  and  subunits are 26% identical in se-

quence except for an ⬃35-residue N-terminal tail of the 

subunit, which the  subunit lacks. Eukaryotic 20S protea-

somes are more complex in that they consist of 7 different

-like and 7 different -like subunits versus only one of

each type for the T. acidophilum 20S proteasome.

The X-ray structure of the T. acidophilum 20S protea-

some, determined by Baumeister and Robert Huber, re-

veals that its two inner rings each consist of 7  subunits

and its two outer rings each consist of 7  subunits, all

arranged with D

symmetry (Fig. 32-84). Thus the overall

1414 Chapter 32. Translation

JWCL281_c32_1338-1428.qxd 8/4/10 4:45 PM Page 1414

structure of the 20S proteasome superficially resembles

that of the unrelated molecular chaperone GroEL (Section

9-2Ca). The structures of the  and  subunits are remark-

ably similar (Fig. 32-84a) except, of course, for the  sub-

unit’s N-terminal tail (blue in Fig. 32-84a), which extends

radially inward to contact the N-terminal tail of a neigh-

boring  subunit. This accounts for the observation that 

subunits alone spontaneously assemble into 7-membered

rings (a capacity that is abolished by the deletion of their

N-terminal 35 residues), whereas  subunits alone remain

monomeric.

The central cavity of the T. acidophilum 20S proteasome

consists of three large chambers (Fig.32-84c): Two are located

at the interfaces between adjoining rings of  and  subunits,

with the third, larger chamber centrally located between the

two rings of  subunits. Unfolded polypeptide substrates en-

ter the central chamber of the barrel (where the protea-

some’s active sites are located; see below) through ⬃13-Å-

diameter axially located apertures in the  rings that are

lined with hydrophobic residues. This allows only unfolded

proteins to enter the central chamber, thereby protecting

properly folded proteins from indiscriminant degradation

by this omnivorous protein-dismantling machine.

The X-ray structure of the yeast 20S proteasome, deter-

mined by Huber, demonstrates that its outer and inner

rings respectively consist of seven different -type subunits

and seven different -type subunits, all of which are

uniquely arranged (Fig. 32-85). The -like subunits have

folds that are similar to one another as well as to that of the

T. acidophilum 20S proteasome and likewise for the -like

subunits. Consequently, this 28-subunit, 6182-residue pro-

tein complex has exact 2-fold rotational symmetry relating

its two pairs of rings but only pseudo-7-fold rotational sym-

metry relating the subunits within each ring. The narrow

axial apertures in the  rings through which unfolded

polypeptides enter the hydrolytic chamber (Fig. 32-85d)

are occluded in the closed state (Fig. 32-85c) by a plug

formed by the interdigitation of its  subunits’ N-terminal

tails.This indicates that the 19S caps of the 26S proteasome,

which have been shown to activate the 20S proteasome,

Section 32-6. Protein Degradation 1415

Figure 32-84 X-ray structure of the T. acidophilum 20S

proteasome. (a) Ribbon diagram, viewed with its 7-fold axis

tipped toward the viewer by 20°, in which the  subunits are pink

and the  subunits are light blue except for one  subunit (left)

and a similarly oriented  subunit (middle), which are both

colored in rainbow order from their N-termini (blue) to their

C-termini (red). Note their close structural resemblance. (b)

Diagram, viewed as in Part a, in which the subunits are

represented by equal-sized spheres with  subunits pink and 

subunits light blue. (c) Surface diagram, viewed along a 2-fold

axis, with the subunits nearest the viewer removed to expose the

proteasome’s triple-chambered internal cavity, which is

maximally ⬃100 Å long and ⬃60 Å in diameter.The active sites

on the  subunits are marked by the bound inhibitor, LLnL,

which is drawn in stick form in yellow. [Part a based on an X-ray

structure by and Part c courtesy of Robert Huber, Max-Planck-

Institut für Biochemie, Martinsried, Germany. PDBid 1PMA.]

(b)

(c)

(a)

JWCL281_c32_1338-1428.qxd 9/7/10 2:23 PM Page 1415

1416 Chapter 32. Translation

(a)

(b)

JWCL281_c32_1338-1428.qxd 9/7/10 2:23 PM Page 1416

control the access to it by inducing conformational changes

in its  rings (see below).The X-ray structure of the bovine

20S proteasome, determined by Tomitake Tsukihara, re-

veals that its arrangement of seven -type and seven

-type subunits is similar to that in yeast.

g. The Proteasome Catalyzes Peptide Hydrolysis via

a Novel Mechanism

The X-ray structure of the T. acidophilum 20S protea-

some in complex with the aldehyde inhibitor acetyl-Leu-

Leu-norleucinal (LLnL)

reveals that its active sites are on the inner surfaces of its

rings of  subunits, with the aldehyde function of the LLnL

close to the side chain of the highly conserved Thr 1.

Deletion of this Thr or its mutation to Ala yields properly

assembled 20S proteasomes that are completely inactive.

Evidently, 20S proteasomes catalyze peptide hydrolysis by

a novel mechanism in which the hydroxyl group of its Thr

1 is the attacking nucleophile. This, as yet, poorly under-

stood mechanism, in which the amino group at the N-

terminus and possibly a bound water molecule act to nucle-

ophilically activate the hydroxyl side chain, is now known

to be employed by other hydrolases (e.g., glutamate syn-

thase; Section 26-5Aa), which are collectively known as the

N-terminal nucleophile (Ntn) family of hydrolases. The T.

acidophilum  subunits preferably cleave polypeptides af-

ter hydrophobic residues. However, in the yeast and the

bovine 20S proteasomes, only subunits 1, 2, and 5 are

catalytically active. Their respective preferences for cleav-

age after acidic (caspase-like), basic (trypsin-like), and hy-

drophobic (chymotrypsin-like) residues are explained by

Acetyl-Leu-Leu-norleucinal (LLnL)

C Leu Leu NH CH CH

(CH

)

the respective basic, acidic, and nonpolar characters of

their pockets that bind the side chain of the residue preced-

ing the scissile peptide bond, although this specificity is rel-

atively low. The functions of the four different catalytically

inactive  subunits are unknown, although mutagenically

modifying an inactive  subunit can abolish the catalytic

activity of an active  subunit.

h. The 19S Caps Control the Access of Ubiquitinated

Proteins to the 20S Proteasome

The 20S proteasome probably does not exist alone in

vivo; it is most often in complex with two 19S caps that

function to recognize ubiquitinated proteins, unfold them,

and feed them into the 20S proteasome in an ATP-depend-

ent manner (it may also associate with other regulatory

complexes; see below). The 19S cap, which consists of ⬃18

different subunits, is poorly characterized due in large part

to its low intrinsic stability. Its so-called base complex con-

sists of 9 different subunits, 6 of which are ATPases that

form a ring that abuts the  ring of the 20S proteasome

(Fig. 32-79). Each of these ATPases contains an ⬃230-

residue ATPase module that is a member of the AAA

family (Section 30-2Ca). Cecile Pickart demonstrated via

cross-linking experiments that one of these ATPases,

named S6 (alternatively Rpt5), contacts the polyUb signal

that targets a condemned protein to the 26S proteasome.

This suggests that the recognition of the polyUb chain as

well as substrate protein unfolding are ATP-driven

processes. Moreover, the ring of ATPases must function to

open (gate) the otherwise closed axial aperture of the 20S

proteasome so as to permit the entry of the unfolded sub-

strate protein.

Eight additional subunits form the so-called lid com-

plex, the portion of the 19S cap that is more distal to (dis-

tant from) the 20S proteasome. The functions of the lid

subunits are largely unknown, although a truncated 26S

proteasome that lacks the lid subunits is unable to degrade

polyubiquitinated substrates. Several other subunits may

be transiently associated with the 19S cap and/or with the

20S proteasome.

i. Deubiquitinating Enzymes Have Several Functions

The enzymes that hydrolytically cleave the isopeptide

bonds linking successive ubiquitin units in polyUb are

known as deubiquitinating enzymes (DUBs). Cells contain

a surprisingly large number of DUBs (at least 17 in yeast

and ⬃100 in humans). Nearly all known DUBs are cysteine

proteases, enzymes whose catalytic mechanism resembles

that of serine proteases (Section 15-3C) but whose attack-

ing nucleophile is Cys¬S



rather than Ser¬OH.

DUBs may release entire polyUb chains from a con-

demned protein or sequentially release ubiquitin units

from the chain terminus. It has been proposed that this lat-

ter process functions as a clock to time the protein degra-

dation process. If a polyUb chain is trimmed to less than

four ubiquitin units before degradation begins, then its at-

tached protein is likely to escape destruction. This would

spare proteins that had been inappropriately tagged with

only short polyUb chains.

Section 32-6. Protein Degradation 1417

Figure 32-85 (Opposite) X-ray structure of the yeast 20S

proteasome. (a) Surface diagram viewed along the 28-mer’s

2-fold axis. Each pair of identical subunits has the same color

except for 1 and the symmetry related 1¿, which are colored

differently. (b) Cutaway surface diagram sliced along the

complex’s pseudo-7-fold axis. The slice surface is green and the

active sites of the 1, 2, and 5¿ subunits are marked in red. The

brackets indicate the approximate positions of the channel entry

ports as seen in the open state. (c) End view of the 20S

proteasome showing its -ring in its closed state represented as

in Part a.The pockets where PA26 binds (see below) are

highlighted in blue. (d) The open state represented as in Part c

and with the C-termini of its bound PA26 subunits shown in

worm form in yellow. [Courtesy of Daniel Finley, Harvard

Medical School. Based on X-ray structures by Robert Huber,

Max-Planck-Institut für Biochemie, Martinsried, Germany

(closed state), and Christopher Hill, University of Utah (open

state). PDBids 1RYP and 1FNT.]

JWCL281_c32_1338-1428.qxd 8/4/10 4:45 PM Page 1417

(a)

The mammalian 19S lid subunit known as POH1

(Rpn11 for the 65% identical yeast subunit) appears to be

responsible for the deubiquitination of target proteins

prior to their degradation; its inactivation prevents target

protein degradation. Curiously, this DUB is a Zn

2

-de-

pendent protease (as is carboxypeptidase A; Fig. 15-42)

rather than a cysteine protease.

Certain DUBs function to dismember polyUb chains that

have been released from substrate proteins by sequentially

removing ubiquitin units from the end of the chain that is

nearest to the substrate protein (that with a free C-termi-

nus). Consequently, these DUBs cannot remove ubiquitin

units from polyUb chains that are still attached to substrate

proteins, thereby preventing their premature removal.

Cells express ubiquitin as polyproteins containing sev-

eral ubiquitin units (Section 32-5Ac) or with ubiquitin fused

to certain ribosomal subunits (there is no gene that encodes

a single ubiquitin unit). These polyproteins are rapidly

processed by certain DUBs to yield free ubiquitin.

j. The 11S Activator Forms a Heptameric Barrel That

Opens the 20S Proteasome

Higher eukaryotes contain an 11S activator (alterna-

tively, 11S regulator) that functions to open the channel

into the 20S proteasome in an ATP-independent manner

so as to permit the entrance of polypeptides (but not

folded proteins). The mammalian 11S activator, which

functions in the generation of peptides for presentation to

the immune system (Section 35-2E), is named REG (alter-

natively PA28). It is a heteroheptameric complex of two

⬃245-residue subunits, REG and REG, that exhibit

⬃50% sequence identity except for a highly variable inter-

nal 18-residue segment that is thought to confer subunit-

specific properties. Indeed REG alone forms a heptamer

whose biochemical properties are similar to that of REG

(although both subunits must be present in vivo).

The trypanosome Trypanosoma brucei, which lacks 19S

caps, expresses a homoheptameric 11S activator named

PA26 that is only 14% identical to human REG. Neverthe-

less, the various 11S activators activate 20S proteasomes

from widely divergent species.Thus,rat 20S proteasome is ac-

tivated by PA26 and the yeast 20S proteasome is activated by

human REG despite the fact that yeast lacks 11S activators.

1418 Chapter 32. Translation

Figure 32-86 X-ray structure of T. brucei PA26 in complex

with the yeast 20S proteasome. (a) The PA26 heptamer in ribbon

form viewed with its 7-fold axis vertical. Each of its subunits are

differently colored with that closest to the viewer colored in

rainbow order from its N-terminus (blue) to its C-terminus (red).

(b) Cutaway diagram of the entire complex drawn in worm form

and viewed with its 7-fold axis vertical.The PA26 is yellow, the 

and  subunits of the 20S proteasome are magenta and blue, its

-annulus is green, and its N-terminal segments that are ordered

and partially disordered are red and pink. [Part a based on an

X-ray structure by and Part b courtesy of Christopher Hill,

University of Utah. PDBid 1FNT.]

(b)

JWCL281_c32_1338-1428.qxd 8/19/10 10:06 PM Page 1418

The X-ray structure of PA26 in complex with the yeast

20S proteasome, determined by Christopher Hill, reveals

that each PA26 monomer consists of an up–down–up–down

4-helix bundle. These monomers form a 7-fold symmetric

heptameric barrel that is 90 Å in diameter, 70 Å long, and

has a 33-Å-diameter central pore (Fig. 32-86a) and which

closely resembles the previously determined X-ray struc-

ture of human REG.Two PA26 barrels associate coaxially

with the 20S proteasome, one at each end (Fig. 32-86b).The

conformation of the 20S proteasome in this complex, for

the most part, is closely similar to that of the 20S protea-

some alone (Fig. 32-85). However, the C-terminal tails of

the PA26 subunits insert into pockets on the 20S protea-

some’s  subunits in a way that induces conformational

changes in its N-terminal tails that clear the 20S protea-

some’s otherwise blocked central aperture (Fig. 32-85c,d),

thus permitting unfolded polypeptides to enter the protea-

some’s central chamber.

k. Bacteria Contain a Variety of Self-

Compartmentalized Proteases

Nearly all eubacteria lack 20S proteasomes. Neverthe-

less they have ATP-dependent proteolytic assemblies that

share the same barrel-shaped architecture and carry out

similar functions. For example, in E. coli, two proteins

known as Lon and Clp mediate up to 80% of the bac-

terium’s protein degradation,with additional contributions

from at least three other proteins including heat shock lo-

cus UV (HslUV). Thus, all cells appear to contain proteases

whose active sites are only available from the inner cavity of

a hollow particle to which access is controlled. These so-

called self-compartmentalized proteases appear to have

arisen early in the history of cellular life, before the advent

of eukaryotic membrane-bound organelles such as the

lysosome, which similarly carry out degradative processes

in a way that protects the cell contents from indiscriminant

destruction.

Clp protease consists of two components, the proteolyti-

cally active ClpP and one of several ATPases, which in E.

coli are ClpA and ClpX. The X-ray structure of ClpP, deter-

mined by John Flanagan, reveals that it oligomerizes to

form an ⬃90-Å-long and -wide hollow barrel that consists

of two back-to-back 7-fold symmetric rings of 193-residue

subunits (Fig.32-87) and thereby has the same D

symmetry

as does the 20S proteasome. Nevertheless, the ClpP subunit

has a novel fold that is different from that of the 20S protea-

some’s homologous  and  subunits. The ClpP active site,

which is only exposed on the inside of the barrel, contains a

catalytic triad composed of Ser 97, His 122, and Asp 171,

and hence is a serine protease (Section 15-3Ab).

HslUV protease appears to be a hybrid of Clp and the

26S proteasome. Its HslV subunits in Haemophilus influen-

zae (174 residues) are 18% identical to the  subunits of

the T. acidophilum 20S proteasome, whereas its regulatory

HslU caps (444 residues) have ATPase activity and are ho-

mologous to E. coli ClpX. The X-ray structure of H. in-

fluenzae HslUV, determined by David McKay, indi-

cates that HslV forms a dimer of hexameric rather than

Section 32-6. Protein Degradation 1419

Figure 32-87 X-ray structure of E. coli ClpP. (a) View of the

heptameric complex along its 7-fold axis, drawn in ribbon form in

which the lower ring is pale cyan and the upper ring is pink with

one subunit colored in rainbow order from its N-terminus (blue)

(a)

(b)

to its C-terminus (red). (b) View along the complex’s 2-fold axis

(rotated 90° about a horizontal axis with respect to Part a).

[Based on an X-ray structure by John Flanagan, Brookhaven

National Laboratory, Upton, New York. PDBid 1TYF.]

JWCL281_c32_1338-1428.qxd 8/19/10 10:06 PM Page 1419

heptameric rings (Fig. 32-88).A hexameric ring of HslU sub-

units binds to both ends of the HslV dodecamer to form a

24-subunit assembly with D

symmetry, rather than the D

symmetry of the 26S proteasome. Nevertheless, both the

fold and the intersubunit contacts of the HslV subunits are

closely similar to those of the 20S proteasome  subunits.

In addition, both have N-terminal Thr residues.Thus, HslV

can be regarded as the eubacterial homolog of archaebac-

terial and eukaryotic 20S proteasomes.

Thermoplasma acidophilum contains another large pro-

teolytic complex, which is unrelated to the proteasome.The

X-ray structure of this protease (Fig. 32-89), determined by

Huber, indicates that it forms a 730-kD toroidal hexameric

ring with D

symmetry that has a peculiar triangular shape

reminiscent of a tricorn (a hat whose brim is turned up on

three sides) and hence was named tricorn protease. Cryo-

EM studies indicate that 20 of these tricorn hexamers asso-

ciate to form a 14,600-kD hollow icosahedron (Fig. 32-89c;

an icosahedron is shown in Fig. 8-65c), making it by far the

largest homooligomeric enzyme complex known (it is even

larger than some virus particles, many of which also have

icosahedral symmetry; Section 33-2Aa).

l. Ubiquitination Has Multiple Proteasome-

Independent Functions

Proteins may be monoubiquitinated or polyubiquiti-

nated or even monoubiquitinated on more than one Lys

residue. Moreover, ubiquitin has seven Lys residues so that

seven types of polyubiquitin chains are possible.

Many types of ubiquitination mediate processes other

than directing their associated proteins to the proteasome.

This occurs through mechanisms reminiscent of protein

phosphorylation, but instead of being recognized by spe-

cialized phosphoprotein-binding motifs such as SH2 (Sec-

tion 19-3Cb),ubiquitinated proteins are recognized by con-

served ubiquitin-binding domains (UBDs). Moreover,

whereas protein phosphorylation is reversible through the

action of protein phosphatases (Section 19-3F), ubiquitina-

tion is reversible through the agency of DUBs.

Ubiquitination participates in regulating such diverse

cellular processes as endocytosis, protein trafficking,

DNA repair, intracellular signaling, and transcription.

For example, during S phase of the cell cycle, the mo-

noubiquitination of PCNA (the sliding clamp associated

with the eukaryotic DNA replication fork; Section 30-

4Ba) at its Lys 164 recruits an error-prone translesion

DNA polymerase to the replication fork at a DNA dam-

age site, whereas the polyubiquitination of the same site

with a Lys 63–linked chain recruits DNA polymerases

that mediate error-free lesion repair. In another example,

in the signal transduction pathway that activates NF-B

(Section 32-6Be), IB is phosphorylated by IB kinase

(IKK), which has a regulatory subunit named NEMO (for

NF-B essential modulator). The binding of cytokines

such as TNF and IL-1 to their transmembrane receptors

in this pathway activates the receptor to Lys 63–linked

polyubiquitinate NEMO, which in turn activates IKK to

phosphorylate IB. This, as we saw, induces the forma-

tion of the Lys 48–linked polyubiquitination signal that

leads to the destruction of IB and hence the transloca-

tion of NF-B to the nucleus. Yersinia pestis, the bac-

terium that causes bubonic plague (Section 19-3Fc), pro-

duces a virulence factor named YopJ, which functions as

a DUB that prevents the activation of NF-B (which is an

1420 Chapter 32. Translation

Figure 32-88 X-ray structure of H. influenzae HslVU in

complex with ATP. The 821-kD complex is drawn in ribbon form

viewed along a 2-fold axis with its 6-fold axis vertical.The D

symmetric dodecamer of HslV subunits is coaxially bound at

both ends by C

symmetric HslU hexamers to yield a complex

with overall D

symmetry. The subunits of the lower HslU

hexamer are alternately orange and cyan, whereas those of the

upper HslU hexamer are pale orange except for one subunit,

which is colored in rainbow order from its N-terminus (blue) to

its C-terminus (red).The subunits of the lower HslV hexamer are

alternately green and magenta, whereas those of the upper

hexamer are pale blue except for one subunit, which is colored in

rainbow order.The ATPs, which are bound at the interfaces

between neighboring HslU subunits, are drawn in space-filling

form with C green, N blue, O red, and P orange. [Based on an

X-ray structure by David McKay, Stanford University School of

Medicine. PDBid 1G3I.]

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