Biochemistry, 4th Edition P104 docx

31.3 How Do Proteins Find Their Proper Place in the Cell?Proteins are targeted to their proper cellular locations by signal sequences: Proteins destined for service in membranous organel

sine kinases, and steroid hormone receptors are some of the signal transduction

mol-ecules (see Chapter 32) that must associate with Hsp90 in order to become fully

com-petent; proteins fitting this description are called Hsp90 “client proteins.” The

matu-ration of Hsp70 client proteins requires other proteins as well, and together with

Hsp90, these proteins come together to form an assembly that has been called a

foldosome.CFTR (cystic fibrosis transmembrane regulator), telomerase, and nitric

oxide synthase are also Hsp90-dependent

Association of nascent polypeptide chains with proteins of the various chaperone

systems commits them to a folding pathway, redirecting them away from

degrada-tion pathways that would otherwise eliminate them from the cell However, if these

protein chains fail to fold, they are recognized as non-native and targeted for

destruction

Aside from these folding events, release of the completed polypeptide from the

ri-bosome is not necessarily the final step in the covalent construction of a protein

Many proteins must undergo covalent alterations before they become functional

In the course of these post-translational modifications, the primary structure of a

protein may be altered, and/or novel derivations may be introduced into its

amino acid side chains Hundreds of different amino acid variations have been

described in proteins, virtually all arising post-translationally The list of such

modifications is very large; some are rather commonplace, whereas others are

pe-culiar to a single protein The diphthamide moiety in elongation factor eEF-2 is

one example of an amino acid modification (see the Human Biochemistry box on

page 980 in Chapter 30); the fluorescent group of green fluorescent protein

(GFP; see Chapter 4) is another In addition, common chemical groups such as

carbohydrates and lipids may be covalently attached to a protein during its

matu-ration Phosphorylation, acetylation, and methylation of proteins are common

mechanisms for regulating protein function Interestingly, many proteins are

modified in multiple ways, and many post-translational modifications act in

com-binations—a phenomenon termed cross-talk (The majority of proteins in cells

can be phosphorylated on one or more residues A survey of some of the more

prominent chemical groups conjugated to proteins is given in Chapter 5.) To put

a number on the significance of post-translational modifications, we have seen

that the number of human proteins is estimated to exceed the number of human

genes (20,000 or so) by more than an order of magnitude

Proteolytic Cleavage Is the Most Common Form

of Post-Translational Processing

Proteolytic cleavage, as the most prevalent form of protein post-translational

modification, merits special attention The very occurrence of proteolysis as a

processing mechanism seems strange: Why join a number of amino acids in

se-quence and then eliminate some of them? Three reasons can be cited First,

di-versity can be introduced where none exists For example, a simple form of

pro-teolysis, enzymatic removal of N-terminal Met residues, occurs in many proteins

Met-aminopeptidase, by removing the invariant Met initiating all polypeptide

chains, introduces diversity at N-termini Second, proteolysis serves as an

activa-tion mechanism so that expression of the biological activity of a protein can be

delayed until appropriate A number of metabolically active proteins, including

digestive enzymes and hormones, are synthesized as larger inactive precursors

termed pro-proteins that are activated through proteolysis (see zymogens,

Chap-ter 15) The N-Chap-terminal pro-sequence on such proteins may act as an

intramole-cular chaperone to ensure correct folding of the active site Third, proteolysis is

involved in the targeting of proteins to their proper destinations in the cell, a

process known as protein translocation.

31.3 How Do Proteins Find Their Proper Place in the Cell?

Proteins are targeted to their proper cellular locations by signal sequences: Proteins

destined for service in membranous organelles or for export from the cell are syn-thesized in precursor form carrying an N-terminal stretch of amino acid residues,

or leader peptide, that serves as a signal sequence In effect, signal sequences serve as

“zip codes” for sorting and dispatching proteins to their proper compartments Thus, the information specifying the correct cellular localization of a protein is found within its structural gene Once the protein is routed to its destination, the signal sequence is often, but not always, proteolytically clipped from the protein by

a signal sequence-specific endopeptidase called a signal peptidase.

Proteins Are Delivered to the Proper Cellular Compartment

by Translocation

Protein translocationis the name given to the process whereby proteins are inserted into membranes or delivered across membranes Protein translocation occurs in all cells Newly synthesized chains of membrane proteins or secretory proteins are tar-geted to the plasma membrane (in prokaryotes) or the endoplasmic reticulum (in eukaryotes) by their signal sequences In addition to the ER, a number of eukary-otic membrane systems are competent in protein translocation, including the mem-branes of the nucleus, mitochondria, chloroplasts, and peroxisomes Several com-mon features characterize protein translocation systems:

1 Proteins to be translocated are made as preproteins containing contiguous blocks of amino acid sequence that act as organelle-specific sorting signals

2 Signal recognition particles (SRPs) recognize the presence of a nascent protein chain in the ribosomal exit tunnel and, together with signal receptors (SRs),

de-liver the nascent chain to the membrane If the nascent sequence emerging from the ribosome is a signal sequence, it is delivered to a specific membrane protein

complex, the translocon, that mediates protein integration into the membrane or

protein translocation across the membrane

3 Translocons are selectively permeable protein-conducting channels that catalyze

movement of the proteins across the membrane, and metabolic energy in the form of ATP, GTP, or a membrane potential is essential In eukaryotes, ATP-dependent chaperone proteins within the membrane compartment usually as-sociate with the entering polypeptide and provide the energy for translocation Proteins destined for membrane integration contain amino acid sequences that

act as stop-transfer signals, allowing diffusion of transmembrane segments into

the bilayer

4 Preproteins are maintained in a loosely folded, translocation-competent confor-mation through interaction with molecular chaperones

Prokaryotic Proteins Destined for Translocation Are Synthesized

as Preproteins

Gram-negative bacteria typically have four compartments: cytoplasm, plasma (or in-ner) membrane, periplasmic space (or periplasm), and outer membrane Most pro-teins destined for any location other than the cytoplasm are synthesized with amino-terminal leader sequences 16 to 26 amino acid residues long These leader sequences,

or signal sequences, consist of a basic N-terminal region, a central domain of 7 to 13

hydrophobic residues, and a nonhelical C-terminal region (Figure 31.4) The

con-Leader sequence 16–26 residues N

Basic region 7–13

hydrophobic residues

0 Nonhelical C-terminal region

Gly or Pro

Cleavage site

FIGURE 31.4 General features of the N-terminal signal

sequences on E coli proteins destined for translocation:

a basic N-terminal region, a central apolar domain, and a

nonhelical C-terminal region.

served features of the last part of the leader, the C-terminal region, include a

helix-breaking Gly or Pro residue and amino acids with small side chains located one and

three residues before the proteolytic cleavage site Unlike the basic N-terminal and

nonpolar central regions, the C-terminal features are not essential for translocation

but instead serve as recognition signals for the leader peptidase, which removes the

leader sequence The exact amino acid sequence of the leader peptide is

unimpor-tant Nonpolar residues in the center and a few Lys residues at the amino terminus

are sufficient for successful translocation The functions of leader peptides are to

re-tard the folding of the preprotein so that molecular chaperones have a chance to

in-teract with it and to provide recognition signals for the translocation machinery and

leader peptidase

Eukaryotic Proteins Are Routed to Their Proper Destinations

by Protein Sorting and Translocation

Eukaryotic cells are characterized by many membrane-bounded compartments In

general, signal sequences targeting proteins to their appropriate compartments are

located at the N-terminus as cleavable presequences, although many proteins have

N-terminal localization signals that are not cleaved and others have internal targeting

sequences that may or may not be cleaved Proteolytic removal of the leader sequences

is also catalyzed by specialized proteases, but removal is not essential to translocation

No sequence similarity is found among the targeting signals for each compartment

Thus, the targeting information resides in more generalized features of the leader

se-quences such as charge distribution, relative polarity, and secondary structure For

ex-ample, proteins destined for secretion enter the lumen of the endoplasmic reticulum

(ER) and reach the plasma membrane via a series of vesicles that traverse the

endo-membrane system Recognition by the ER depends on an N-terminal amino acid

se-quence that contains one or more basic amino acids followed by a run of 6 to 12

hydro-phobic amino acids An example is serum albumin, which is synthesized in precursor

form (preproalbumin) having a MK W VTFLLLLFISGSAFSR N-terminal signal

se-quence The italicized K highlights the basic residue in the sequence, and the bold

residues denote a continuous stretch of (mostly) hydrophobic residues A signal

pep-tidase in the ER removes the signal sequence by cleaving the preproprotein between

the S and R

The Synthesis of Secretory Proteins and Many Membrane Proteins Is Coupled to

Translocation Across the ER Membrane The signals recognized by the ER

translo-cation system are virtually indistinguishable from bacterial signal sequences; indeed,

the two are interchangeable in vitro In addition, the translocon systems in

prokary-otes and eukaryprokary-otes are highly analogous In higher eukaryprokary-otes, translation and

translocation of many proteins destined for processing via the ER are tightly coupled

Translocation across the ER occurs co-translationally (that is, as the protein is being

translated on the ribosome) As the N-terminal sequence of a protein undergoing

syn-thesis enters the exit tunnel of the ribosome, it is detected by a signal recognition

particle (SRP; Figure 31.5) SRP is a 325-kD nucleoprotein assembly that contains six

polypeptides and a 300-nucleotide 7S RNA SRP54, a 54-kD subunit of SRP and a

G-protein family member, recognizes the nascent protein’s signal sequence, and SRP

binding of the signal sequence causes the ribosome to cease translation This arrest

prevents release of the growing protein into the cytosol before it reaches the ER

and its intended translocation The SRP–ribosome complex is referred to as the

RNC–SRP (ribosome nascent chain⬊SRP complex).

Interaction Between the RNC–SRP and the SR Delivers the RNC to the Membrane

The RNC–SRP is then directed to the cytosolic face of the ER, where it binds to the

signal receptor (SR), an  heterodimeric protein The 70-kD -subunit is anchored

to the membrane by the transmembrane -subunit; both subunits are G-protein

family members, and both have bound GTP When SRP54 docks with SR, the

RNC–SRP becomes membrane associated (Figure 31.5) If the nascent chain

emerg-ing from the ribosome is not a signal sequence, the RNC is released from the SRP

and the membrane If the nascent chain emerging from the ribosome is a signal se-quence, the complex remains intact, and SRP54 and SR function together as reci-procal GTPase-activating proteins GTP hydrolysis causes the dissociation of SRP from SR and transfer of the RNC to the translocon

The Ribosome and the Translocon Form a Common Conduit for Transfer of the Nascent Protein Through the ER Membrane and into the Lumen Through inter-actions with the translocon, the ribosome resumes protein synthesis, delivering its growing polypeptide through the ER membrane The peptide exit tunnel of the large ribosomal subunit and the protein-conducting channel of the translocon are aligned with one another, forming a continuous conduit from the peptidyl trans-ferase center of the ribosome to the ER lumen

The mammalian translocon is a complex, multifunctional entity that has as its

core the Sec61 complex, a heterotrimeric complex of membrane proteins, and a unique fourth subunit, TRAM, that is required for insertion of nascent integral

membrane proteins into the membrane The 53-kD -subunit of Sec61p has ten membrane-spanning segments, whereas the - and -subunits are single TMS pro-teins Sec61 forms the transmembrane protein-conducting channel through which the nascent polypeptide is transported into the ER lumen (Figure 31.5) The pore size of Sec61p is very dynamic, ranging from about 0.6 to 6 nm in diameter Thus, a great variety of protein structures could be accommodated easily within the translocon This flexibility allows the Sec61p translocon complex to function in post-translational translocation (translocation of completely formed proteins) as well as co-translational translocation

As the protein is threaded through the Sec61p channel into the lumen, an

Hsp70 chaperone family member called BiP binds to it and mediates proper

fold-ing BiP function, like that of other Hsp70 proteins, is dependent, and ATP-dependent protein folding provides the driving force for translocation of the polypeptide into the lumen When the ribosome dissociates from the translocon, BiP serves as a plug to block the protein-conducting channel, preventing ions and other substances from moving between the ER lumen and the cytosol

A Signal Peptidase Within the ER Lumen Clips Off the Signal Peptide Soon after

it enters the ER lumen, the signal peptide is clipped off by membrane-bound signal

peptidase(also called leader peptidase), which is a complex of five proteins Other

modifying enzymes within the lumen introduce additional post-translational alter-ations into the polypeptide, such as glycosylation with specific carbohydrate residues ER-processed proteins destined for secretion from the cell or inclusion in

Translating ribosome

Signal recognition particle (SRP)

Signal sequence

Signal receptor (SR)

Signal peptidase

Clipped signal sequence

Translocon

BiP

Cytosol

1 2

FIGURE 31.5 Synthesis of a eukaryotic secretory protein

and its translocation into the endoplasmic reticulum.

(1) The signal recognition particle (SRP, red) recognizes

the signal sequence emerging from a translating

ribo-some (riboribo-some nascent complex [RNC], gray) (2) The

RNC-SRP interacts with the signal receptor (SR, purple)

and is transferred to the translocon (pink) (3) Release of

the SRP and alignment of the peptide exit tunnel of the

RNC with the protein-conducting channel of the

trans-locon stimulates the ribosome to resume translation.

(4) The membrane-associated signal peptidase (purple

circle) clips off the N-terminal signal sequence, and BiP

(the ER lumen Hsp70 chaperone, blue) binds the nascent

chain mediating its folding into its native conformation.

(5) Following dissociation of the ribosome, BiP plugs the

translocon channel Not shown are subsequent secretory

protein maturation events, such as glycosylation (Adapted

from Figures 1a and 2a in Frydman, J., 2001 Folding of newly

translated proteins in vivo: The role of molecular chaperones.

Annual Review of Biochemistry 70:603–647.)

vesicles such as lysosomes end up contained within the soluble phase of the ER

lu-men On the other hand, polypeptides destined to become membrane proteins

carry stop-transfer sequences within their mature domains The stop-transfer

se-quence is typically a 20-residue stretch of hydrophobic amino acids that arrests the

passage across the ER membrane Proteins with stop-transfer sequences remain

em-bedded in the ER membrane with their C-termini on the cytosolic face of the ER

Such membrane proteins arrive at their intended destinations via subsequent

pro-cessing of the ER

Retrograde Translocation Prevents Secretion of Damaged Proteins and Recycles

Old ER Proteins To prevent secretion of inappropriate proteins, fragmented or

misfolded secretory proteins are passed from the ER back into the cytosol via

Sec61p Thus, Sec61p also serves as a channel for aberrant secretory proteins to be

returned to the cytosol so that they can be destroyed by the proteasome

degrada-tion apparatus (see Secdegrada-tion 31.4) Among these proteins are ER membrane proteins

that are damaged or no longer needed

Mitochondrial Protein Import Most mitochondrial proteins are encoded by the

nuclear genome and synthesized on cytosolic ribosomes Mitochondria consist of

four principal subcompartments: the outer membrane, the intermembrane space,

the inner membrane, and the matrix Thus, not only must mitochondrial proteins

find mitochondria, they must gain access to the proper subcompartment; and once

there, they must attain a functionally active conformation As a consequence,

mi-tochondria possess multiple preprotein translocons and chaperones Similar

con-siderations apply to protein import to chloroplasts, organelles with five principal

subcompartments (outer membrane, intermembrane space, inner/thylakoid

membrane, stroma, and thylakoid lumen; see Chapter 21)

Signal sequences on nuclear-encoded proteins destined for the mitochondria

are N-terminal cleavable presequences 10 to 70 residues long These mitochondrial

presequences lack contiguous hydrophobic regions Instead, they have positively

charged and hydroxy amino acid residues spread along their entire length These

sequences form amphipathic␣-helices (Figure 31.6) with basic residues on one side

of the helix and uncharged and hydrophobic residues on the other; that is,

mito-chondrial presequences are positively charged amphiphatic sequences In general,

mitochondrial targeting sequences share no sequence homology Once synthesized,

mitochondrial preproteins are retained in an unfolded state with their target

se-quences exposed, through association with Hsp70 molecular chaperones Import

involves binding of a preprotein to the mitochondrial outer membrane translocon

(TOM)(Figure 31.7) If the protein is destined to be an outer mitochondrial

mem-brane protein, it is transferred from the TOM to the sorting and assembly complex

TOM complex

TIM23

complex

Matrix protein

TIM22 complex

SAM complex

Mitochondrial precursor protein

Inter-membrane space

Matrix

Cytosol

TOM 40 Mas37 20

Outer-membrane protein

Outer membrane

Inner membrane

Inner-membrane protein

+

+

+

R R R R

FIGURE 31.6 Structure of an amphipathic -helix having

basic ( ) residues on one side and uncharged and hydrophobic (R) residues on the other.

FIGURE 31.7 Translocation of mitochondrial preproteins involves distinct translocons All mitochondrial proteins must interact with the outer mitochondrial membrane (TOM) From there, depending on their destiny, they are (1) passed to the SAM complex if they are in-tegral proteins of the outer mitochondrial membrane or (2) traverse the TOM and enter the intermembrane space, where they are taken up by either TIM22 or TIM23, depending

on whether they are integral membrane pro-teins of the inner mitochondrial membrane (TIM22) or mitochondrial matrix proteins (TIM23) (Adapted from Figure 1 in Mihara, K., 2003.

E

3

(SAM)and inserted in the outer membrane If it is an integral protein of the inner mitochondrial membrane, it traverses the TOM complex, enters the

intermem-brane space, and is taken up by the inner mitochondrial memintermem-brane translocon

(TIM22)and inserted into the inner membrane On the other hand, if it is destined

to be a mitochondrial matrix protein, a different TIM complex, TIM23, binds the

preprotein and threads it across the inner mitochondrial membrane into the

ma-trix Chloroplasts have TOCs (translocon outer chloroplast membrane) and TICs

(translocon inner chloroplast membrane) for these purposes

Levels of Specific Proteins?

Cellular proteins are in a dynamic state of turnover, with the relative rates of protein synthesis and protein degradation ultimately determining the amount of protein present at any point in time In many instances, transcriptional regulation deter-mines the concentrations of specific proteins expressed within cells, with protein degradation playing a minor role In other instances, the amounts of key enzymes and regulatory proteins, such as cyclins and transcription factors, are controlled via selective protein degradation In addition, abnormal proteins arising from biosyn-thetic errors or postsynbiosyn-thetic damage must be destroyed to prevent the deleterious consequences of their buildup The elimination of proteins typically follows first-order kinetics, with half-lives (t1/2) of different proteins ranging from several min-utes to many days A single, random proteolytic break introduced into the polypep-tide backbone of a protein is believed sufficient to trigger its rapid disappearance be-cause no partially degraded proteins are normally observed in cells

Protein degradation poses a real hazard to cellular processes To control this haz-ard, protein degradation is compartmentalized, either in macromolecular structures

known as proteasomes or in degradative organelles such as lysosomes Protein

degra-dation within lysosomes is largely nonselective; selection occurs during lysosomal up-take Proteasomes are found in eukaryotic as well as prokaryotic cells The protea-some is a functionally and structurally sophisticated counterpart to the riboprotea-some Regulation of protein levels via degradation is an essential cellular mechanism Reg-ulation by degradation is both rapid and irreversible

Eukaryotic Proteins Are Targeted for Proteasome Destruction

by the Ubiquitin Pathway

Ubiquitinationis the most common mechanism to label a protein for proteasome

degradation in eukaryotes Ubiquitin is a highly conserved, 76-residue (8.5-kD)

polypeptide widespread in eukaryotes Proteins are condemned to degradation through ligation to ubiquitin Three proteins in addition to ubiquitin are involved

in the ligation process: E 1 , E 2 , and E 3(Figure 31.8) E1is the ubiquitin-activating

enzyme(105-kD dimer) It becomes attached via a thioester bond to the C-terminal Gly residue of ubiquitin through ATP-driven formation of an activated ubiquitin-adenylate intermediate Ubiquitin is then transferred from E1to an SH group on

E2, the ubiquitin-carrier protein (E2is actually a family of at least seven different small proteins, several of which are heat shock proteins; there is also a variety of E3

proteins.) In protein degradation, E2-S⬃ ubiquitin transfers ubiquitin to free amino groups on proteins selected by E3 (180 kD), the ubiquitin-protein ligase.

Upon binding a protein substrate, E3catalyzes the transfer of ubiquitin from E2-S⬃ ubiquitin to free amino groups (usually Lys 2) on the protein More than one ubiquitin may be attached to a protein substrate, and tandemly linked chains

of ubiquitin also occur via isopeptide bonds between the C-terminal glycine residue of

one ubiquitin and the residues, at positions 6, 11, 27, 29, 33, 48, and 63 Only isopeptide linkages to K11,

K29, K48, and K63have been found, with the K48-type being most common as a degra-dation signal

E1

(Top) A ubiquitin⬊E1 heterodimer complex (pdb id 

1R4N; ubiquitin is shown in blue) (Middle) A ubiquitin⬊

E2 complex (pdb id  1FXT; E2 is shown in orange,

ubiquitin in blue) (Bottom) The clamp-shaped E3

heteromultimer (pdb id  1LDK and 1FQV).The target

protein is bound between the jaws of the clamp.

E3

E3plays a central role in recognizing and selecting proteins for degradation E3

selects proteins by the nature of the N-terminal amino acid Proteins must have a

free-amino terminus to be susceptible Proteins having either Met, Ser, Ala, Thr,

Val, Gly, or Cys at the amino terminus are resistant to the ubiquitin-mediated

degradation pathway However, proteins having Arg, Lys, His, Phe, Tyr, Trp, Leu,

Asn, Gln, Asp, or Glu N-termini have half-lives of only 2 to 30 minutes

Interestingly, proteins with acidic N-termini (Asp or Glu) show a tRNA

require-ment for degradation (Figure 31.9) Transfer of Arg from Arg-tRNA to the

N-terminus of these proteins alters their N-terminus from acidic to basic, rendering

the protein susceptible to E3 It is also interesting that Met is less likely to be cleaved

from the N-terminus if the next amino acid in the chain is one particularly

suscep-tible to ubiquitin-mediated degradation

Most proteins with susceptible N-terminal residues are not normal intracellular

proteins but tend to be secreted proteins in which the susceptible residue has

been exposed by action of a signal peptidase Perhaps part of the function of the

E 2

E 2

: Ubiquitin-activating enzyme

E 1

P P

C Ubiquitin

O–

O

O

Ubiquitinyl-acyladenylate (C-term Gly)

C Ubiquitin

O

E 1

S C

O Ubiquitin

S C

O Ubiquitin

(Thioester)

: Ubiquitin-carrier protein

E 2

E 1 S C

O

: Ligase

E 3

E 3

E 3

+ Protein

(substrate)

E 3 : Protein

: Protein + E 2 S C E 2

O Ubiquitin SH + E 3 + Protein

Ubiquitin

ATP

1

2

ACTIVE FIGURE 31.8 Enzymatic reac-tions in the ligation of ubiquitin to proteins Ubiquitin is attached to selected proteins via isopeptide bonds formed between the ubiquitin carboxy-terminus and free amino groups (-NH2 terminus, Lys 2 side

chains) on the protein Test yourself on the concepts

in this figure at www.cengage.com/login.

Arg Arg-tRNAArg

Arg

tRNAArg

(Asp, Glu)

Protein with acidic

N-terminal residue

(Asp or Glu)

(Asp, Glu)

Protein with N-terminal Arg

Protein transferase

FIGURE 31.9 Proteins with acidic N-termini show a tRNA requirement for degradation Arginyl-tRNA Arg ⬊protein transferase catalyzes the transfer of Arg to the free

-NH2 of proteins with Asp or Glu N-terminal residues Arg-tRNA Arg ⬊protein transferase serves as part of the protein degradation recognition system.

N-terminal recognition system is to recognize and remove from the cytosol any in-vading “foreign” or secreted proteins

Other proteins targeted for ubiquitin ligation and proteasome degradation

con-tain PEST sequences—short, highly conserved sequence elements rich in proline

(P), glutamate (E), serine (S), and threonine (T) residues

Proteins Targeted for Destruction Are Degraded by Proteasomes

Proteasomes are large oligomeric structures enclosing a central cavity where

prote-olysis takes place The 20S proteasome from the archaeon Thermoplasma acidophilum

is a 700-kD barrel-shaped structure composed of two different kinds of polypeptide chains, and , arranged to form four stacked rings of 7777-subunit organiza-tion The barrel is about 15 nm in height and 11 nm in diameter, and it contains a three-part central cavity (Figure 31.10a) The proteolytic sites of the 20S proteasome are found within this cavity Access to the cavity is controlled through a 1.3-nm open-ing formed by the outer 7rings These rings are believed to unfold proteins des-tined for degradation and transport them into the central cavity The -subunits pos-sess the proteolytic activity Proteolysis occurs when the -subunit N-terminal threonine side-chain O atom makes nucleophilic attack on the carbonyl-C of a pep-tide bond in the target protein The products of proteasome degradation are oligo-peptides seven to nine residues long

Eukaryotic cells contain two forms of proteasomes: the 20S proteasome, and its larger counterpart, the 26S proteasome The eukaryotic 26S proteasome is a

45-nm-long structure composed of a 20S proteasome plus two additional substructures

known as 19S regulators (also called 19S caps or PA700 [for proteasome

activator-700kD]) (Figure 31.10b) Overall, the 26S proteasome (approximately 2.5 megadal-tons) has 2 copies each of 32 to 34 distinct subunits, 14 in the 20S core and 18 to

20 in the cap structures Unlike the archaeal 20S proteasome, the eukaryotic 20S core structure contains seven different kinds of -subunits and seven different kinds

( b )

19S

890 kD

20S

720 kD

19S

890 kD

lid

base

base

lid

15 nm

( a )

FIGURE 31.10 The structure of the 26S proteasome (a) The yeast (Saccharomyces cerevisiae) 20S proteasome

core with bortezomib bound (red) (pdb id  2F16) Bortezomib is the first therapeutic proteasome inhibitor used in humans It is approved in the United States for treatment of relapsed multiple myeloma and mantle

cell lymphoma (b) Composite model of the 26S proteasome The 20S proteasome core is shown in yellow; the

19S regulator (19S cap) structures are in blue (Adapted from Figure 5 in Voges, D., Zwickl, P., and Baumeister, W., 1999 The

26S proteasome: A molecular machine designed for controlled proteolysis Annual Review of Biochemistry 68:1015–1068.)

of-subunits Interestingly, only three of the seven different -subunits have

pro-tease active sites The 26S proteasome forms when the 19S regulators dock to the two

outer7rings of the 20S proteasome cylinder Many of the 19S regulator subunits

have ATPase activity Replacement of certain 19S regulator subunits with others

changes the specificity of the proteasome The 19S regulators cause the proteolytic

function of the 20S proteasome to become ATP-dependent and specific for

ubiqui-tinylated proteins as substrates That is, these 19S caps act as regulatory complexes

for the recognition and selection of ubiquitinylated proteins for degradation by the

20S proteasome core (Figure 31.11) The 26S proteasome shows a preference for

proteins having four or more ubiquitin molecules attached to them The 19S

regu-lators also carry out the unfolding and transport of ubiquitinylated protein substrates

into the proteolytic central cavity

The 19S regulators consist of two parts: the base and the lid The base subcomplex

connects to the 20S proteasome and contains the six ATPase subunits that unfold

pro-teasome substrates These subunits are members of the AAA family of ATPases

(ATPases associated with various cellular activities); AAA-ATPases are an

evolutionar-ily ancient famevolutionar-ily of proteins involved in a variety of cellular functions requiring

energy-dependent unfolding, disassembly, and remodeling of proteins The lid

sub-complex acts as a cap on the base subsub-complex and one of its subunits functions in

recognition and ubiquitin-chain processing of proteasome protein substrates

ATPase Modules Mediate the Unfolding of Proteins in the Proteasome

The base of the 19S regulators that cap the 26S proteasome consists of a hexameric

ring of AAA–ATPases that mediate the ATP-dependent unfolding of ubiquitinated

proteins targeted for destruction in the proteasome Structural studies have

re-vealed the presence of loops extending from these AAA–ATPase subunits; these

loops face the central channel of the hexameric ring The loops are in an “up”

po-sition when an AAA–ATPase subunit has ATP bound in its active site, but they shift

to a “down” position when ADP occupies the active site Apparently, these loops

bind protein substrates and act like the levers of a machine, using the energy of ATP

hydrolysis to tug the protein into an unfolded state and thread it down through the

narrow central channel This channel leads into the cavity of the 20S proteasome,

where proteolytic degradation takes place The AAA–ATPase cycle of ATP binding,

protein substrate binding, ATP hydrolysis, and protein unfolding is reminiscent of

the ATP-dependent action of Hsp70 chaperones

Ubiquitination Is a General Regulatory Protein Modification

Protein ubiquitination is a signal for protein degradation, as described on page 998

Ubiquitin conjugation to proteins is also used for other purposes in cells

Non-degradative functions for ubiquitination include roles in chromatin remodeling,

DNA repair, transcription, signal transduction, endocytosis, spliceosome assembly,

and sorting of proteins to specific organelles and cell structures In addition, cells

possess a variety of protein modifiers attached to target proteins by processes

simi-lar to the ubiquitin pathway, as described in the following section

Small Ubiquitin-Like Protein Modifiers Are Post-transcriptional

Regulators

Small ubiquitin-like protein modifiers (SUMOs) are a highly conserved family of

proteins found in all eukaryotic cells Like ubiquitin, SUMO family members are

covalently ligated to lysine residues in target proteins by a three-enzyme

conjugat-ing system (Figure 31.12) SUMO proteins share only limited homology to

ubiqui-tin, and sumoylated proteins are not targeted for destruction Instead, sumoylation

alters the ability of the modified protein to interact with other proteins This ability

to change protein–protein interactions is believed to be the biological purpose of

SUMO proteins

26S proteasome

Substrate

Substrate

E2

E2 E1

E3 Ubiquitin

ACTIVE FIGURE 31.11 Diagram of the ubiquitin-proteasome degradation pathway Pink “lolli-pop” structures symbolize ubiquitin molecules (Adapted from Figure 1 in Hilt, W., and Wolf, D H., 1996 Proteasomes:

Destruction as a program Trends in Biochemical Sciences

21:96–102.) Test yourself on the concepts in this fig-ure at www.cengage.com/login.

Sumoylation can have three general consequences for modified proteins (Figure 31.13): (1) sumoylation can interfere with the interactions between the target and its partner so that the interaction can only occur in the absence of sumoylation; (2) sumoylation can create a binding site for an interacting partner protein; and (3) sumoylation can induce a conformational change in the modified target pro-tein, altering its interactions with partner proteins The regulatory opportunities as-sociated with sumoylation are significant for many cellular functions, including transcriptional regulation, chromosome organization, nuclear transport, and signal transduction (see Chapter 32)

Many E2 enzymes participate in ubiquitination processes, but the only known SUMO E2 enzyme is Ubc9 (Figure 31.14a) Ubc9 recognizes a KXD/E consensus sequence in proteins destined for sumoylation In this sequence,  is an aliphatic branched amino acid (such as Leu), K is the lysine to which SUMO is conjugated, and X is any amino acid, with an acidic D or E completing the sequence Recogni-tion by Ubc9 is only possible if the consensus sequence is in a relatively unstruc-tured part of a target protein or if it is part of an extended loop, as in RanGAP1 (Figure 31.14a) In the Ubc9–RanGAP1 complex, Leu525of RanGAP1 is in van der Waals contact with several nonpolar Ubc9 residues, whereas RanGAP1 Lys526lies in

a hydrophobic groove of Ubc9, juxtaposed with the catalytic Cys93(Figure 31.14b)

GG

GG

GG

GG

GG XXXX

SUMO

C 173

UBA2 SUMO

SENP

SENP

ATP

AMP+

PPi

E3 AOS1

K Target

C UBC9

E1

E2 SUMO

SUMO SUMO

FIGURE 31.12 The mechanism of reversible

sumoyla-tion Before conjugation, small ubiquitin-like protein

modifiers (SUMOs) need to be proteolytically processed,

removing anywhere from 2 to 11 amino acids to reveal

the C-terminal Gly–Gly motif SUMOs are then activated

by the E1 enzyme in an ATP-dependent reaction to

form a thioester bond between SUMO and E1 SUMO is

then transferred to the catalytic Cys residue of the E2

enzyme, Ubc9 Finally, an “isopeptide bond” is formed,

between the C-terminal Gly of SUMO and a Lys residue

on the substrate protein, through the action of an E3

enzyme The SUMO-specific protease SENP can

decon-jugate SUMO from target proteins.

SUMO

E1, E2, E3 enzymes, ATP

Isopeptidases

(c)

Partner A

Partner B

SUMO

SUMO SUMO

Target Target

Target

FIGURE 31.13 The molecular consequences of sumoylation The process can affect a modified protein in three

ways: (a) Sumoylation can interfere with the interaction between a target protein and its binding partner.

(b) Sumoylation can provide a new binding site for an interacting partner (c) Sumoylation can induce a

con-formational change in the modified target protein.