31.2 How Are Proteins Processed Following Translation? 993 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. 31.2 How Are Proteins Processed Following Translation? 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-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. 994 Chapter 31 Completing the Protein Life Cycle: Folding, Processing, and Degradation 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 translocation is 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. 31.3 How Do Proteins Find Their Proper Place in the Cell? 995 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 WVTFLLLLFISGSAFSR 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 eukaryotes are highly analogous. In higher eukaryotes, 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 chainϺ 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 996 Chapter 31 Completing the Protein Life Cycle: Folding, Processing, and Degradation 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 ATP-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 ER lumen Secretory protein Translocon BiP Cytosol 1 2 3 45 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 (ribosome 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.) 31.3 How Do Proteins Find Their Proper Place in the Cell? 997 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 Section 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 Mas 37 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 (ϩ) 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. Moving inside membranes. Nature 424:505–506.) E 2 E 3 998 Chapter 31 Completing the Protein Life Cycle: Folding, Processing, and Degradation (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 membrane 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. 31.4 How Does Protein Degradation Regulate Cellular 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 postsynthetic damage must be destroyed to prevent the deleterious consequences of their buildup. The elimination of proteins typically follows first- order kinetics, with half-lives (t 1/2 ) 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 ribosome. 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 Ubiquitination is 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). E 1 is 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 E 1 to an SH group on E 2 , the ubiquitin-carrier protein. (E 2 is actually a family of at least seven different small proteins, several of which are heat shock proteins; there is also a variety of E 3 proteins.) In protein degradation, E 2 -S ϳ ubiquitin transfers ubiquitin to free amino groups on proteins selected by E 3 (180 kD), the ubiquitin-protein ligase. Upon binding a protein substrate, E 3 catalyzes the transfer of ubiquitin from E 2 -S ϳ ubiquitin to free amino groups (usually Lys ⑀-NH 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 ⑀-amino of Lys residues in another. Ubiquitin has seven lysine residues, at positions 6, 11, 27, 29, 33, 48, and 63. Only isopeptide linkages to K 11 , K 29 , K 48 , and K 63 have been found, with the K 48 -type being most common as a degra- dation signal. E 1 (Top) A ubiquitinϺE1 heterodimer complex (pdb id ϭ 1R4N; ubiquitin is shown in blue). (Middle) A ubiquitinϺ E2 complex (pdb id ϭ 1FXT; E2 is shown in orange, ubiquitin in blue). (Bottom) The clamp-shaped E3 heteromultimer (pdb id ϭ 1LDK and 1FQV).The target protein is bound between the jaws of the clamp. E 3 31.4 How Does Protein Degradation Regulate Cellular Levels of Specific Proteins? 999 E 3 plays a central role in recognizing and selecting proteins for degradation. E 3 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 E 3 . 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 1 E 1 E 2 E 2 : Ubiquitin-activating enzyme E 1 P P C Ubiquitin O – O ++ Ubiquitin C AMP O Ubiquitinyl-acyladenylate(C-term. Gly) C Ubiquitin O + SH + E 1 S C O Ubiquitin S C O Ubiquitin (Thioester) : Ubiquitin-carrier protein E 2 + SH E 1 SC O Ubiquitin E 1 SH + : Ligase E 3 E 3 E 3 + Protein (substrate) E 3 : Protein : Protein + E 2 E 2 C S O Ubiquitin SH + E 3 + Protein Ubiquitin AMP AMP 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 (␣-NH 2 terminus, Lys ⑀-NH 2 side chains) on the protein. Test yourself on the concepts in this figure at www.cengage.com/login. Arg Arg-tRNA Arg Arg tRNA Arg (Asp, Glu) Protein with acidic N-terminal residue (Asp or Glu) (Asp, Glu) Protein with N-terminal Arg Arg-tRNA Arg Protein transferase FIGURE 31.9 Proteins with acidic N-termini show a tRNA requirement for degradation. Arginyl-tRNA Arg Ϻprotein transferase catalyzes the transfer of Arg to the free ␣-NH 2 of proteins with Asp or Glu N-terminal residues. Arg-tRNA Arg Ϻprotein transferase serves as part of the protein degradation recognition system. 1000 Chapter 31 Completing the Protein Life Cycle: Folding, Processing, and Degradation 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 ␣ 7  7  7 ␣ 7 -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 ␣ 7 rings. 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- 700 kD]) (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 ϭ 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.) 31.4 How Does Protein Degradation Regulate Cellular Levels of Specific Proteins? 1001 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 outer ␣ 7 rings 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 family 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 subcomplex 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. 1002 Chapter 31 Completing the Protein Life Cycle: Folding, Processing, and Degradation 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, Leu 525 of RanGAP1 is in van der Waals contact with several nonpolar Ubc9 residues, whereas RanGAP1 Lys 526 lies in a hydrophobic groove of Ubc9, juxtaposed with the catalytic Cys 93 (Figure 31.14b). GG GG GG GG GGXXXX SUMO C 173 UBA2 SUMO SENP SENP ATP AMP + PP i E3 AOS1 K Target C UBC9 E 1 E 2 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 (a) (b) (c) Partner A Partner B SUMO SUMO SUMO Target Target 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.