27.3 Protein Targeting and Degradation in Chapter 27 Protein Metabolism

27.3 Protein Targeting and Degradation

The eukaryotic cell is made up of many structures, compartments, and organelles, each with specific functions that require distinct sets of proteins and enzymes. These proteins (with the exception of those produced in mitochondria and plastids) are synthesized on ribosomes in the cytosol, so how are they directed to their final cellular destinations?

We are now beginning to understand this complex and fascinating process. Proteins destined for secretion, integration in the plasma membrane, or inclusion in lysosomes generally share the first few steps of a pathway that begins in the endoplasmic reticulum. Proteins destined for mitochondria, chloroplasts, or the nucleus use three separate mechanisms. And proteins destined for the cytosol simply remain where they are synthesized. The thermodynamic cost of protein synthesis is magnified by the processes used by cells to transport proteins to their correct cellular locations.

The most important element in many of these targeting pathways is a short sequence of amino acids called a signal sequence, whose function was first postulated by Günter Blobel and colleagues in 1970. The signal sequence directs a protein to its appropriate location in the cell and, for many proteins, is removed during transport or after the protein has reached its final destination. In proteins slated for transport into mitochondria, chloroplasts, or the ER, the signal sequence is at the amino terminus of a newly synthesized polypeptide. In many cases, the targeting capacity of particular signal sequences has been confirmed by fusing the signal sequence from one protein to a second protein and showing that the signal directs the second protein to the location where the first protein is normally found. The selective degradation of proteins no longer needed by the cell also relies largely on a set of molecular signals embedded in each protein’s structure.

In this concluding section we examine protein targeting and degradation, emphasizing the underlying signals and molecular regulation that are so crucial to cellular metabolism. Except where noted, the focus is now on eukaryotic cells.

Posttranslational Modification of Many Eukaryotic Proteins Begins in the Endoplasmic Reticulum

Perhaps the best-characterized targeting system begins in the ER. Most lysosomal, membrane, or secreted proteins have an amino-terminal signal sequence (Fig. 27-37) that marks them for translocation into the lumen of the ER; hundreds of such signal sequences have been determined. The carboxyl terminus of the signal sequence is defined by a cleavage site, where protease action removes the sequence after the protein is imported into the ER. Signal sequences vary in length from 13 to 36 amino acid residues, but all have the following features: (1) about 10 to 15 hydrophobic amino acid residues; (2) one or more positively charged residues, usually near the amino terminus, preceding the hydrophobic sequence; and (3) a short sequence at the carboxyl terminus (near the cleavage site) that is relatively polar, typically having amino acid residues with short side chains (especially Ala) at the positions closest to the cleavage site.

A figure shows amino-terminal signal sequence of five eukaryotic proteins. — FIGURE 27-37 Amino-terminal signal sequences of some eukaryotic proteins that direct their translocation into the ER. The hydrophobic core (yellow) is preceded by one or more basic residues (blue). Polar and short-side-chain residues immediately precede (to the left, as shown here) the cleavage sites (indicated by red arrows).

The sequences are shown in five rows. From top to bottom, the rows are labeled human influenza virus A, human preproinsulin, bovine growth hormone, bee promellitin, and italicized Drosophila end italics glue protein. The sequences are aligned with a total of 28 spaces possible. A red arrow indicating a cleavage site points between the amino acids in the twenty sixth and twenty seventh positions of each sequence. Human influenza virus A: The sequence begins after 11 spaces and consists of 17 amino acids. The sequence is yellow M e t blue L y s yellow A l a blue L y s, then a yellow region of L e u L e u V a l L e u L e u T y r A l a P h e V al A la, then the yellow region ends and the sequence continues as G l y cleavage site A s p G ln three dashes. Human preproinsulin: The sequence begins after two spaces and consists of 26 amino acids. The sequence is M e t A l a L e u T r p M e t blue A r g L e u L e u P r o, then a yellow region of L e u L e u A l a L e u L e u A l a L e u T r p, then the yellow region ends and the sequence continues as G l y P r o A s p P r o A l a A l a A l a cleavage site P h e V a l three dashes. Bovine growth hormone: The sequence begins without any spaces and consists of 28 amino acids. The sequence is M e t M e t A l a A l a G l y P r o blue A r g T h r S e r, then a yellow region begins of L e u L e u L e u A l a P h e A l a L e u L e u C y s L e u P r o T r p, then the yellow region ends and the sequence continues as T h r G l n V al V al G l y cleavage site A l a P h e three dashes. Bee promellitin: The sequence begins after five spaces and consists of 23 amino acids. The sequence is M e t blue L y s P h e L e u V a l, then a yellow region of A s n V a l A l a L e u V a l P h e M e t V a l V a l T y r I l e, then the yellow region ends and the sequence continues as S e r T y r I l e T y r A l cleavage site A l a P r o three dashes. Italicized Drosophila end italics glue protein: The sequence begins after four spaces and consists of 24 amino acids. The sequence is M e t blue L y s, then a yellow region of L e u L e u V a l V a l A l a V a l I l e A l a C y s M e t L e u I l e G l y P h e A l a, then the yellow region ends and the sequence continues as A s p P r o A l a S e r G l y cleavage site C y s L y s three dashes.

As originally demonstrated by the cell biologist George Palade, proteins with these signal sequences are synthesized on ribosomes attached to the ER. The signal sequence itself helps to direct the ribosome to the ER, as illustrated in Figure 27-38. The targeting pathway begins in step , with initiation of protein synthesis on free ribosomes. The signal sequence appears early in the synthetic process (step ), because it is at the amino terminus, which, as we have seen, is synthesized first. As it emerges from the ribosome (step ), the signal sequence — and the ribosome itself — is bound by the large signal recognition particle (SRP). The SRP is a rod-shaped complex containing a 300 nucleotide RNA (7SL-RNA) and six different proteins (combined $M_{r} 325,000$ $upper M Subscript r Baseline 325,000$ ). The SRP then binds GTP and halts elongation of the polypeptide when it is about 70 amino acids long and the signal sequence has completely emerged from the ribosome. In step , the GTP-bound SRP directs the ribosome (still bound to the mRNA) and the incomplete polypeptide to GTP-bound SRP receptors in the cytosolic face of the ER; the nascent polypeptide is delivered to a peptide translocation complex in the ER, which interacts directly with the ribosome. In step , the SRP dissociates from the ribosome, accompanied by hydrolysis of GTP in both the SRP and the SRP receptor. The SRP receptor is a heterodimer of $α (M_{r} 69,000)$ $alpha left-parenthesis upper M Subscript r Baseline 69,000 right-parenthesis$ and $β (M_{r} 30,000)$ $beta left-parenthesis upper M Subscript r Baseline 30,000 right-parenthesis$ subunits, both of which bind and hydrolyze multiple GTP molecules during this process.

Elongation of the polypeptide now resumes (step ), with the ATP-driven translocation complex feeding the growing polypeptide into the ER lumen until the complete protein has been synthesized. In step , the signal sequence is removed by a signal peptidase within the ER lumen. The ribosome dissociates (step ) and is recycled (step ).

A figure shows how eukaryote proteins with the appropriate signals are directed to the endoplasmic reticulum. — FIGURE 27-38 Directing eukaryotic proteins with the appropriate signals to the endoplasmic reticulum. This process involves the SRP cycle and the translocation and cleavage of the nascent polypeptide. One protein subunit of SRP binds directly to the signal sequence, obstructing elongation by sterically blocking the entry of aminoacyl-tRNAs and inhibiting peptidyl transferase. Another protein subunit binds and hydrolyzes GTP.

An oval bar with a yellow membrane is labeled endoplasmic reticulum and the purple interior is labeled E R lumen. A piece of m R N A is shown with a short red 5 prime cap at the one o’clock position that becomes green and loops all the way around in an oval to the five o’clock position to end at A A A (A) italicized subscript n end italics end subscript 3 prime. Arrow 1 points to a ribosome near the start of this m R N A that has a pink half-circle on top with its flat base against a purple roughly rectangular piece below. m R N A runs through this purple part, where it is dark green and reads G U A. An arrow points left to a similar ribosome with a growing blue polypeptide extending into the pink region that ends with an orange piece labeled signal sequence. Arrow 2 points down to another ribosome at the nine o’clock position that has a longer blue polypeptide with the orange piece extending beyond the pink piece. A green rectangle labeled S R P is nearby and has a half-circle cutout at its lower end. Arrow 3 points down to a similar ribosome at the seven o’clock position that has a longer polypeptide and green S R P in the lower right pink part with its cutout facing outward. This is shown above structures in the yellow E R membrane. There are two adjacent vertical brown bars labeled ribosome receptor that each have curved cutouts on top, with the left-hand one curving down to the right and the right-hand one curving up to the left. A brown triangle is at the lower left of the left-hand brown bar. Each of these brown bars is connected to a brown vertical oval that crosses the membrane and that is labeled peptide translocation complex. There is a white oval vertical bar extending up from the membrane to the right of these brown bars. This white bar is shorter and more rounded and is labeled S R P receptor. An arrow labeled 4 points right and is accompanied by a curved line showing the addition of G T P. This yields a ribosome that is pushed against the ribosome receptors, which fit against its rounded lower pink side, and which has the S R P receptor fitted into he cutout in S R P. A bright blue diamond labeled signal peptidase is shown to the lower right of a brown oval peptide translocation complex. The blue polypeptide extends through the space between eh two ribosome receptors and coils within the lumen, ending at the orange signal sequence. Two arrows point from this ribosome. Arrow 5 curves upward accompanied by an arrow that branches off to show the loss of G D P plus P subscript i end subscript. It then continues up to show S R P and then loops back to the ribosome at the nine o’clock position that had not yet bound S R P. This is labeled the S R P cycle. Arrow 6 points to the right from the ribosome bound to the ribosome receptors. This yields a similar ribosome with a longer polypeptide inside the lumen that ends near the bright blue triangle with the orange signal sequence now separate beneath number 7. To the lower right, the full polypeptide is shown beneath empty ribosome receptors and an empty S R P receptor. Arrow 8 points up to show the pink and purple subunits of the ribosome separated at the three o’clock position. Arrow 9 points back up through m R N A to the start of arrow 1 to restart the cycle. All data are approximate.

Glycosylation Plays a Key Role in Protein Targeting

In the ER lumen, newly synthesized proteins are further modified in several ways. Following the removal of signal sequences, polypeptides are folded, disulfide bonds are formed, and many proteins are glycosylated to form glycoproteins. In many glycoproteins, the linkage to their oligosaccharides is through Asn residues. These N-linked oligosaccharides are diverse (Chapter 7), but the pathways by which they form have a common first step. A 14 residue core oligosaccharide is built up stepwise, first on the cytosolic face of the membrane and then on the lumenal face. Once completed, it is transferred from a dolichol phosphate donor molecule to certain Asn residues in the protein (Fig. 27-39). The transferase is on the lumenal face of the ER and thus cannot catalyze glycosylation of cytosolic proteins. After transfer, the core oligosaccharide is trimmed and elaborated in different ways on different proteins, but all N-linked oligosaccharides retain a pentasaccharide core derived from the original 14 residue oligosaccharide.

A figure shows the synthesis of the core oligosaccharide of glycoproteins in 9 steps. — FIGURE 27-39 Synthesis of the core oligosaccharide of glycoproteins. The core oligosaccharide is built up by the successive addition of monosaccharide units. , The first steps occur on the cytosolic face of the ER. Translocation moves the incomplete oligosaccharide across the membrane (mechanism not shown), and completion of the core oligosaccharide occurs within the lumen of the ER. The precursors that contribute additional mannose and glucose residues to the growing oligosaccharide in the lumen are dolichol phosphate derivatives. In the first step in construction of the N-linked oligosaccharide moiety of a glycoprotein, the core oligosaccharide is transferred from dolichol phosphate to an Asn residue of the protein, and protein synthesis continues. The core oligosaccharide is then further modified in the ER and the Golgi complex in pathways that differ for different proteins. The five sugar residues shown surrounded by a beige screen, after step , are retained in the final structure of all N-linked oligosaccharides. The released dolichol pyrophosphate is again translocated so that the pyrophosphate is on the cytosolic face of the ER, then a phosphate is hydrolytically removed to regenerate dolichol phosphate.

FIGURE 27-39 Synthesis of the core oligosaccharide of glycoproteins. The core oligosaccharide is built up by the successive addition of monosaccharide units. , The first steps occur on the cytosolic face of the ER. Translocation moves the incomplete oligosaccharide across the membrane (mechanism not shown), and completion of the core oligosaccharide occurs within the lumen of the ER. The precursors that contribute additional mannose and glucose residues to the growing oligosaccharide in the lumen are dolichol phosphate derivatives. In the first step in construction of the N-linked oligosaccharide moiety of a glycoprotein, the core oligosaccharide is transferred from dolichol phosphate to an Asn residue of the protein, and protein synthesis continues. The core oligosaccharide is then further modified in the ER and the Golgi complex in pathways that differ for different proteins. The five sugar residues shown surrounded by a beige screen, after step , are retained in the final structure of all N-linked oligosaccharides. The released dolichol pyrophosphate is again translocated so that the pyrophosphate is on the cytosolic face of the ER, then a phosphate is hydrolytically removed to regenerate dolichol phosphate.

An oval with a yellow membrane is labeled endoplasmic reticulum and the surrounding region is labeled cytosol. The interior of the E R is purple. A yet at the upper left shows that a blue square represents italicized N end italics-acetylglucosamine (G l c N A c), a green circle represents mannose (M a n), and a blue circle represents glucose (G l c). The structure of dolichol phosphate (italicized n end italics equals 9 to 22) is shown as a circle labeled P bonded to a zigzag chain of C H 2 C H 2 C H bonded to C H 3 above bonded to C H 2 bonded through an open parenthesis to C H 2 bonded to C H double bonded to C bonded to C H 3 below and bonded to C H 2 above bonded across the close parenthesis with subscript italicized n end subscript end italics to C H 2 bonded to C H double bonded to C (C H 3) 2. Dolichol bonded to a circle labeled P is shown as an orange diamond with its top vertex at the base of the top E R membrane and a gray strand extending across the membrane to P on the other side. Arrow 1 points left to similar structure with P bonded to another P above further bonded to a chain of two blue squares. A curved arrow above arrow 1 shows the addition of 2 U D P-G l c N A c and loss of U M P plus U D P. A red “X” in a red circle above the inflection point of this arrow is indicated by a dashed arrow from tunicamycin. Arrow 2 is accompanied by a curved arrow showing the addition of 5 G D P-M a n and loss of 5 G D P. This yields a similar structure in which the top blue square is bonded to a green circle bonded to one green circle on the right and to a chain of three green circles to the left. An arrow labeled 3 accompanied by the word translocation points down to the upper right side of the membrane to a similar structure that has changed position. The orange diamond is outside of the membrane and connected by a gray strand to P bonded to the rest of the same molecule inside of the E R. Arrow 4 points down and curves to the right accompanied by two curved arrows. The first curved arrow shows the addition of 4 dolichol bonded to a circle labeled P bonded to M a n and loss of 4 dolichol bonded to a circle labeled P. The second curved arrow shows the addition of 3 dolichol bonded to a circle labeled P bonded to G l y and loss of 3 dolichol bonded to a circle labeled P. This yields a similar molecule in which the bottom green circle is now bonded to a green circle on the right further bonded to two chains of two green circles each and is bonded to a green circle on the left further bonded to a chain of two green circles further bonded to a chain of three blue circles. Arrow 5 points right to show a blue polypeptide extending through the membrane to N H 3 with a positive charge on N at the top within the E R and with the bottom extending down to a ribosome on a green strand of m R N A running from its 5 prime end on the left to its 3 prime end on the right. Just above the place that the blue polypeptide crosses the membrane, it has a bond to A s n with a red pair of electrons. A red arrow points from this red pair of electrons back to the top P of the structure before arrow 5. Arrow 6 points to a similar polypeptide extending up from a ribosome but the polypeptide is longer and there is a bond from the piece of the strand where A s n had been shown previously to a blue square bonded to a blue square bonded to a green circle bonded to a green circle on the right further bonded to two chains of two green circles each and bonded to a green circle on the left further bonded to a chain of two green circles further bonded to a chain of three blue circles. Arrow 7 points right to show the polypeptide loose inside the E R. The two blue squares bonded to a green circle bonded to green circles to the upper left and right is highlighted in yellow. An orange diamond in the cytosol has its top vertex against the membrane and is connected by a gray strand to a chain of to circles labeled P within the E R. An arrow numbered 1 points right to show the same structure inverted at the three o’clock position so that the diamond is inside the membrane and the phosphates are outside of the membrane. An arrow points up with a curved arrow branching off labeled 9 dolichol phosphate recycled that shows the loss of P subscript i end subscript. The arrow points back to the original orange diamond within the E R with its top vertex against the lumen connected by a gray strand to a single P outside of the membrane. All data are approximate.

Several antibiotics act by interfering with one or more steps in this process and have aided in elucidating the steps of protein glycosylation. The best characterized is tunicamycin, which mimics the structure of UDP-N-acetylglucosamine and blocks the first step of the process (Fig. 27-39, step ). A few proteins are O-glycosylated in the ER, but most O-glycosylation occurs in the Golgi complex or in the cytosol (for proteins that do not enter the ER).

Suitably modified proteins can now be moved to a variety of intracellular destinations. Proteins travel from the ER to the Golgi complex in transport vesicles (Fig. 27-40). In the Golgi complex, oligosaccharides are O-linked to some proteins, and N-linked oligosaccharides are further modified. By mechanisms not yet fully understood, the Golgi complex also sorts proteins and sends them to their final destinations. The processes that segregate proteins targeted for secretion from those targeted for the plasma membrane or lysosomes must distinguish among these proteins on the basis of structural features other than signal sequences, which were removed in the ER lumen.

A figure shows the structure of tunicamycin. A figure shows the pathways taken by proteins destined for lysosomes, the plasma membrane, or secretion. — FIGURE 27-40 Pathway taken by proteins destined for lysosomes, the plasma membrane, or secretion. Proteins are moved from the ER to the cis side of the Golgi complex in transport vesicles. Sorting occurs primarily in the trans side of the Golgi complex.

The molecule is shown with four highlighted regions. A light red region at the upper left is labeled italicized N end italics-acetylglucosamine, a central green region is labeled tunicamine, a blue region to the upper right of the green region is labeled uracil, and a yellow vertical region to the lower left is labeled fatty acyl side chain. The light red region, italicized N end italics-acetylglucosamine, contains a six-membered sugar with O at the upper right vertex; the upper left vertex bonded to C H 2 O H above and H below; the left side vertex bonded to H above and O H below; the lower left vertex bonded to O H above and H below; and lower right vertex bonded to H above and to N H below further bonded to C double bonded to O to the left and bonded to C H 3 below; and the right side vertex bonded to H above and to O in the green region below (tunicamine) that is bonded to the upper left vertex of a six-membered ring. This six-membered ring has O at the upper right vertex; an upper left vertex bonded to O above further bonded to the part in the light red region and bonded to H below; a left side vertex bonded to H above and to N H below further bonded to C at the top of the fatty acyl side chain the yellow region; a lower left vertex bonded to O H above and H below; a lower right vertex bonded to H above and O H below; and a right side vertex bonded to H above and to C H 2 below further bonded to C H O H bonded to the left side vertex of a five-membered ring with O at its top vertex. This five-membered ring has a left side vertex that is also bonded to H below; a lower left vertex boned to H above and O H below; a lower right vertex bonded to H above and O H below, and a right side vertex bonded to H below and bonded to N at the bottom vertex of a six-membered ring in the blue region above, uracil. This six-membered ring has N substituted for C at position 1 at the bottom vertex bonded to the right side vertex of the sugar ring below, C 2 at the lower left vertex double bonded to O, N substituted for C at position 3 at the upper left vertex and bonded to H, C 4 at the top vertex double bonded to O, and a double bond between C 5 and C 6 at the right side. The fatty acyl side chain, highlighted in yellow, begins with the C that is bonded above to N H in the green region. This C is double bonded to O and boned to C subscript alpha end subscript below that is bonded to H to the left and double bonded to C subscript beta end subscript below that I bonded to H to the left and bonded to (C H 2) subscript italicized n end subscript end italics below that is further bonded to C H bonded to C H 3 to the lower left and right. (italicized n end italics equals 8 to 11).

Within a background of mostly yellow cytosol, an irregularly-shaped blue nucleus is shown near the top of the figure with a dashed line representing the nuclear envelope and many blue lines inside. It is surrounded by bars representing the rough E R. These bars curve around the nucleus and are connected by small vertical pieces. There are three to four layers of bars. The bars have dots on their external surfaces. The interior of the bars is pinkish purple. A circular protrusion from one of these bars contains a blue thread and is near irregular pieces with rounded and curved shapes. This is labeled smooth E R. An arrow points from this protrusion to a circle containing a blue thread, from which an arrow points to a crescent-shaped piece that contains the same blue thread, from which an arrow points to another circle containing the same blue thread and labeled transport vesicle. The transport vesicle has a more purple color that resembles the color of most of the Golgi complex. An arrow points from this transport vesicle to a rounded protrusion from the cis side of the Golgi complex. This protrusion contains the blue thread. The Golgi complex consists of four layers of bars that all curve down on their left and right sides. They become progressively shorter from top to bottom. The bottom piece is more pink than the others, which are more purple in color. An empty protrusion is on the upper right side of the top bar of the Golgi complex. The third bar from the top has a similar blue thread in its right side and a sphere to the right. The bottom piece of the Golgi has text below reading sorting with arrows pointing to the left and right. This is labeled the trans side of the Golgi complex. The left side contains many green dots and an arrow pointing from it splits to point to a green sphere labeled lysosome and to a pinkish sphere containing green dots labeled secretory granule. An arrow points from the secretory granule to a protrusion up from the plasma membrane that contains the green dots, which are leaving the protrusion and spreading out. The right side of the bottom piece of the Golgi is rounded and tan. AN arrow points from it to a tan irregular oval shape from which an arrow points to a larger tan oval. These are labeled transport vesicles. A small tan oval is nearby.

This sorting process is best understood in the case of hydrolases destined for transport to lysosomes. On arrival of a hydrolase (a glycoprotein) in the Golgi complex, an as yet undetermined feature (sometimes called a signal patch) of the three-dimensional structure of the hydrolase is recognized by a phosphotransferase, which phosphorylates terminal mannose residues in the oligosaccharides (Fig. 27-41). The presence of one or more mannose 6-phosphate residues in its N-linked oligosaccharide is the structural signal that targets a protein to lysosomes. A receptor protein in the membrane of the Golgi complex recognizes the mannose 6-phosphate signal and binds the hydrolase so marked. Vesicles containing these receptor-hydrolase complexes bud from the trans side of the Golgi complex and make their way to sorting vesicles. Here, the receptor-hydrolase complex dissociates in a process facilitated by the lower pH in the vesicle and by phosphatase-catalyzed removal of phosphate groups from the mannose 6-phosphate residues. The receptor is then recycled to the Golgi complex, and vesicles containing the hydrolases bud from the sorting vesicles and move to the lysosomes. In cells treated with tunicamycin (Fig. 27-39, step ), hydrolases that should be targeted to lysosomes are instead secreted, confirming that the N-linked oligosaccharide plays a key role in targeting these enzymes to lysosomes.

A figure shows how mannose residues are phosphorylated on lysosome-targeted enzymes. — FIGURE 27-41 Phosphorylation of mannose residues on lysosome-targeted enzymes. N-Acetylglucosamine phosphotransferase recognizes some as yet unidentified structural feature of hydrolases destined for lysosomes.

U D P-italicized N end italics-acetylglucosamine (U D P-G l c N A c) is a six-membered ring with O at the upper right vertex, the upper left vertex bonded to C H 2 O H above and H below, the left side vertex bonded to H above and O H below, the lower left vertex bonded to O H above and H below, the lower right vertex bonded to H above and to N H below further bonded to C double boned to O to the right and to C H 3 below, and the right side vertex bonded to H above and to O to the right in a light red box further bonded to P double bonded to O above, bonded to O minus below, and bonded to O to the right that is partially in the light red box and partially in the green box to the right, where it is bonded to P double bonded to O above, bonded to O minus below, and bonded to O to the right further bonded to a box labeled uridine all within the green box. This is added to a mannose residue. It is a six-membered ring with O at the upper right vertex, the upper left vertex bonded to C H 2 O H above and H below, the left side vertex bonded to H above and O H below, the lower left vertex bonded to O H above and H below, the lower right vertex bonded to O H above and H below, and the right side vertex bonded to H above and to O to the lower right bonded to a box labeled oligosaccharide further bonded to N H bonded to a blue oval labeled enzyme above the word hydrolase. An arrow points downward accompanied by blue text reading italicized N end italics-acetylglucosamine phosphotransferase and by an arrow branching off to show the loss of a green box labeled U M P. This yields a structure similar to U D P-G l c N A c except that there is only one phosphate with its right-hand O in the light red box and further bonded to C H 2 bonded to the upper left vertex of the six-membered ring of a mannose residue similar to the original residue except that C H 2 O H has been replaced by C H 2 bonded to phosphate. An arrow pointing downward is accompanied by blue text reading phosphodiesterase and an arrow that branches off to show the loss of G l c N A c. This yields mannose 6-phosphate residue, which is similar to the mannose residue except that the upper left vertex of the ring is bonded to C H 2 above further bonded to O in a light red box bonded to P double bonded to O above and bonded to O minus to the left and below, all within the light red box.

The pathways that target proteins to mitochondria and chloroplasts also rely on amino-terminal signal sequences. Although mitochondria and chloroplasts contain DNA, most of their proteins are encoded by nuclear DNA and must be targeted to the appropriate organelle. Unlike other targeting pathways, however, the mitochondrial and chloroplast pathways begin only after a precursor protein has been completely synthesized and released from the ribosome. Precursor proteins destined for mitochondria or chloroplasts are bound by cytosolic chaperone proteins and delivered to receptors on the exterior surface of the target organelle. Specialized translocation mechanisms then transport the protein to its final destination in the organelle, after which the signal sequence is removed.

Signal Sequences for Nuclear Transport Are Not Cleaved

Molecular communication between the nucleus and the cytosol requires the movement of macromolecules through nuclear pores. RNA molecules synthesized in the nucleus are exported to the cytosol. Ribosomal proteins synthesized on cytosolic ribosomes are imported into the nucleus and assembled into 60S and 40S ribosomal subunits in the nucleolus; completed subunits are then exported back to the cytosol (Fig. 27-17). A variety of nuclear proteins (RNA and DNA polymerases, histones, topoisomerases, proteins that regulate gene expression, and so forth) are synthesized in the cytosol and imported into the nucleus. This traffic is modulated by a complex system of molecular signals and transport proteins that is gradually being elucidated.

In most multicellular eukaryotes, the nuclear envelope breaks down at each cell division, and once division is completed and the nuclear envelope reestablished, the dispersed nuclear proteins must be reimported. To allow this repeated nuclear importation, the signal sequence that targets a protein to the nucleus — the nuclear localization sequence (NLS) — is not removed after the protein arrives at its destination. An NLS, unlike other signal sequences, may be located almost anywhere along the primary sequence of the protein. NLSs can vary considerably in structure, but many consist of four to eight amino acid residues and include several consecutive basic (Arg or Lys) residues.

Nuclear importation is mediated by several proteins that cycle between the cytosol and the nucleus (Fig. 27-42), including importin α and β and a small GTPase known as Ran (Ras-related nuclear protein). A heterodimer of importin α and β functions as a soluble receptor for proteins targeted to the nucleus, with the α subunit binding NLS-bearing proteins in the cytosol. The complex of the NLS-bearing protein and the importin docks at a nuclear pore and is translocated through the pore by an energy-dependent mechanism. In the nucleus, the importin β is bound by Ran GTPase, releasing importin β from the imported protein. Importin β is bound by Ran and by CAS (cellular apoptosis susceptibility protein) and separated from the NLS-bearing protein. Importin α and β, in their complexes with Ran and CAS, are then exported from the nucleus. Ran hydrolyzes GTP in the cytosol to release the importins, which are then free to begin another importation cycle. Ran itself is also cycled back into the nucleus by the binding of Ran-GDP to nuclear transport factor 2 (NTF2). Inside the nucleus, the GDP bound to Ran is replaced with GTP through the action of Ran guanosine nucleotide–exchange factor (Ran-GEF).

A two-part figure shows process involved in the targeting of nuclear proteins in part a and a micrograph showing nuclear pore complexes in a nuclear envelope in part b. — FIGURE 27-42 Targeting of nuclear proteins. (a) A protein with an appropriate nuclear localization signal (NLS) is bound by a complex of importins α and β. The resulting complex binds to a nuclear pore and translocates. Inside the nucleus, dissociation of importin β is promoted by the binding of Ran-GTP. Importin α binds to Ran-GTP and CAS (cellular apoptosis susceptibility protein), releasing the nuclear protein. Importins α and β and CAS are transported out of the nucleus and recycled. They are released in the cytosol when Ran hydrolyzes its bound GTP. Ran-GDP is bound by NTF2, and transported back into the nucleus. Ran-GEF promotes the exchange of GDP for GTP in the nucleus, and Ran-GTP is ready to process another NLS-bearing protein-importin complex. (b) Transmission electron micrograph of a freeze-fractured nucleus, showing numerous nuclear pores. The nuclear pore complex is one of the largest molecular aggregates in the cell $(M_{r} ~ 5 \times 10^{7})$ $left-parenthesis upper M Subscript r Baseline tilde zero width space 5 times 10 Superscript 7 Baseline right-parenthesis$ . It is made up of multiple copies of more than 30 different proteins. [(a) Information from C. Strambio-De-Castillia et al., *Nat. Rev. Mol. Cell Biol.* 11:490, 2010, Fig. 1.]

FIGURE 27-42 Targeting of nuclear proteins. (a) A protein with an appropriate nuclear localization signal (NLS) is bound by a complex of importins α and β. The resulting complex binds to a nuclear pore and translocates. Inside the nucleus, dissociation of importin β is promoted by the binding of Ran-GTP. Importin α binds to Ran-GTP and CAS (cellular apoptosis susceptibility protein), releasing the nuclear protein. Importins α and β and CAS are transported out of the nucleus and recycled. They are released in the cytosol when Ran hydrolyzes its bound GTP. Ran-GDP is bound by NTF2, and transported back into the nucleus. Ran-GEF promotes the exchange of GDP for GTP in the nucleus, and Ran-GTP is ready to process another NLS-bearing protein-importin complex. (b) Transmission electron micrograph of a freeze-fractured nucleus, showing numerous nuclear pores. The nuclear pore complex is one of the largest molecular aggregates in the cell $(M_{r} ~ 5 \times 10^{7})$ $left-parenthesis upper M Subscript r Baseline tilde zero width space 5 times 10 Superscript 7 Baseline right-parenthesis$ . It is made up of multiple copies of more than 30 different proteins. [(a) Information from C. Strambio-De-Castillia et al., *Nat. Rev. Mol. Cell Biol.* 11:490, 2010, Fig. 1.]

Part a has a top half labeled cytosol and a bottom half labeled nucleoplasm. These are separated by a horizontal boundary with ovals with yellow boundaries in between cylindrical structures. The ovals are labeled nuclear envelope. The cylindrical structures are labeled nuclear pore complex. Each is wider at the top and bottom, has sides made of ovals, has four vertical wavy threads extending upward, and has four vertical lines extending down to meet a curved horizontal line across the bottom boundary. Step 1: A nuclear protein is shown as a coiled blue strand with a red region labeled N L S. An arrow from this nuclear protein joins an arrow from two structures labeled importin. One is a dark green structure labeled beta that resembles an oval with a cutout in the top. It has a rectangular cutout in the center with angled sides to the left and right. The second is a light green structure labeled alpha with a rectangular base and protrusions to the upper left and right. These come together so that alpha fits into the cutout in beta and the red part of the nuclear protein fits against the curved top of alpha. An arrow points down to the left to show this structure above a nuclear pore complex next to the arrow 2 with an arrow pointing down through the nuclear pore complex to show the structure in the nucleoplasm. Two arrows point fro this structure. Arrow 3 points to a light green circle labeled R a n – G T P attached to the top of beta, from which another arrow labeled 5 points up through a second nuclear pore to show beta next to alpha, together labeled importin, ready to repeat the cycle. The other arrow points down to show alpha with the protein still attached. Arrow 4 points right and splits to show that the protein separates and alpha binds to a light green circle labeled R a n – G T P above and to an orange rectangle labeled C A S below. An arrow points from this structure back through a nuclear pore complex to show alpha ready to repeat the process. Two arrows point away from alpha and beta above the nuclear pore complex to the left. Both arrows point to a light green circle labeled R a n – G D P, but the bottom arrow has an arrow that branches off to show the loss of an orange rectangle labeled C A S. An arrow points to the right from R a n – G D P and is joined by a curved line from blue N T F 2, which resembles a rectangular with a half-circle cutout. These join together so that the curved bottom of N T F 2 fits against the top of R a n – G T P. This is labeled 6 and an arrow points through a nuclear pore complex to show the same structure in the nucleoplasm. Arrow 7 points down and is accompanied by a curved line showing the addition of G T P, next to a purple oval labeled R a n – G E F, and then two arrows that branch away to show the loss of N T F 2 and G D P. This yields R a n - G T P. Two arrows point away from R an – G T P. One points to the structure produced by arrow 4 in which R a n – G T P binds to alpha below, which is bound to C A S below, before this structure moves back through a nuclear pore complex to produce alpha ready to repeat the process. The second arrow points up to the product of arrow 3, R a n G T P bonded to beta below ready to follow arrow 5 back across the nuclear pore complex to produce beta ready to repeat the process. In part b, a micrograph shows a rough background containing many circular structures that are each about 0.2 micrometers in diameter. All data are approximate.

During mitosis, when the nuclear envelope transiently breaks down, the Ran GTPase and the importins play additional roles. The Ran GTPase–importin β complex helps to position the spindle microtubules on the cell perimeter to facilitate chromosome segregation as the cell divides, and this complex also regulates microtubule interaction with other cellular structures.

Bacteria Also Use Signal Sequences for Protein Targeting

Bacteria can target proteins to their inner or outer membranes, to the periplasmic space between these membranes, or to the extracellular medium. They use signal sequences at the amino terminus of the proteins (Fig. 27-43), much like those on eukaryotic proteins targeted to the ER, mitochondria, and chloroplasts.

A figure shows eight signal sequences that target proteins to different locations in bacteria. — FIGURE 27-43 Signal sequences that target proteins to different locations in bacteria. Basic amino acids near the amino terminus are highlighted in blue, hydrophobic core amino acids in yellow. Cleavage sites marking the ends of the signal sequences are indicated by red arrows. Note that the inner bacterial cell membrane is where phage fd coat proteins and DNA are assembled into phage particles. OmpA is outer membrane protein A; LamB is a cell surface receptor protein for λ phage.

The sequences are shown in eight rows separated into three categories. The top category is inner membrane proteins and includes phage fd, major coat protein and phage fd, minor coat protein. The middle category is periplasmic proteins and includes alkaline phosphatase, leucine-specific binding protein, and beta-lactamase of p B R 322. The bottom category is outer membrane proteins and includes lipoprotein, L a m B, and O m p A. The sequences are aligned with a total of 25 spaces possible. A red arrow indicating a cleavage site points between the twenty third and twenty fourth amino acids of each sequence. The two inner membrane proteins are listed first. Phage fd, minor coat protein: M e t, blue L y s, blue L y s, S e r, L e u, V a l, L e u, blue L y s, then a yellow region of A l a S e r V a l A l a V a l A l a T h r L e u V a l P r o, then the yellow region ends and the sequence continues as M e t L e u S e r P h e A l a cleavage site A la a G l u three dashes. Phage fd, minor coat protein: The sequence begins after five spaces and consists of 20 amino acids. The sequence is M e t blue L y s blue L y s, then a yellow region of L e u L e u P h e A l A I l e P r o L e u V a l V a l P r o P h e, then the yellow region ends and the sequence continues as T y r S e r H i s S e r cleavage site A l a G l u three dashes. The three periplasmic proteins are listed next. Alkaline phosphatase: The sequence begins after two spaces and consists of 23 proteins. The sequence is M e t blue L y s G l n S e r T h r, then a yellow region of I l e A l A L e u A l a L e u L e u P r o L e u L e u P h e, then the yellow region ends and the sequence continues as T h r P r o V a l T h r L y s A l a cleavage site A r g T h r three dashes. Leucine-specific binding protein: The sequence begins with no spaces as M e t blue L y s A l a A s n A l a blue L y s T h r I l e, then a yellow region of I l e A l a G l y M e t I l e A l a L e u A l a I l e, then the yellow region ends and the sequence continues as S e r H i s T h r A l a M e t A l a cleavage site A s p A s p three dashes. Beta-lactamase of p BR322: The sequence begins with no spaces as M e t S e r I l e G l n blue H i s P h e blue A r g, then a yellow region of V a l A l a L e u I l e P r o P h e P h e A l a A l a P h e, then the yellow region ends and the sequence continues as C y s L e u P r o V a l P h e A l a cleavage site H i s P r o three dashes. The three outer membrane proteins are listed next. Lipoprotein: The sequence begins after three spaces and consists of 22 amino acids. The sequence is M e t blue L y s A l a T h r blue L y s, then a yellow region of L e u V a l L e u G l y A l a V a l I l e L e u G l y, then the yellow region ends and the sequence continues as S e r T h r L e u L e u A l a G l y cleavage site C y s S e r three dashes. L a m B begins after two spaces and consists of 23 amino acids. The sequence is L e u blue A r g blue L y s, then a yellow region of L e u P r o L e u A l a V a l A l a V a l A l a A l a G l y V a l, then the yellow region ends and the sequence continues as M e t S e r A l a G l n A l a M e t A l a cleavage site V al A s p three dashes. O m p A: The sequence begins with no spaces as M e t M et I l e T h r M e t blue L y s blue L y s, then a yellow region of T h r A l a I l e A l a I l e A l a V a l A l a L e u A l a G l y P h e A l a, then the yellow region ends and the sequence continues as T h r V a l A l a G l n A l a cleavage site A l a P r o three dashes.

Most proteins exported from E. coli make use of the pathway shown in Figure 27-44. Following translation, a protein to be exported may fold only slowly, the amino-terminal signal sequence impeding the folding. The soluble chaperone protein SecB binds to the protein’s signal sequence or other features of its incompletely folded structure. The bound protein is then delivered to SecA, a protein associated with the inner surface of the plasma membrane. SecA acts as both a receptor and a translocating ATPase. Released from SecB and bound to SecA, the protein is delivered to a translocation complex in the membrane, made up of SecY, E, and G, and is translocated stepwise through the membrane at the SecYEG complex in lengths of about 20 amino acid residues. Each step requires the hydrolysis of ATP, catalyzed by SecA.

A figure shows a model for protein export in bacteria with 5 steps. — FIGURE 27-44 Model for protein export in bacteria. A newly translated polypeptide binds to the cytosolic chaperone protein SecB, which delivers it to SecA, a protein associated with the translocation complex (SecYEG) in the bacterial cell membrane. SecB is released, and SecA inserts itself into the membrane, forcing about 20 amino acid residues of the protein to be exported through the translocation complex. Hydrolysis of an ATP by SecA provides the energy for a conformational change that causes SecA to withdraw from the membrane, releasing the polypeptide. SecA binds another ATP, and the next stretch of 20 amino acid residues is pushed across the membrane through the translocation complex. Steps and are repeated until the entire protein has passed through and is released to the periplasm. The electrochemical potential across the membrane (denoted by + and −) also provides some of the driving force required for protein translocation.

FIGURE 27-44 Model for protein export in bacteria. A newly translated polypeptide binds to the cytosolic chaperone protein SecB, which delivers it to SecA, a protein associated with the translocation complex (SecYEG) in the bacterial cell membrane. SecB is released, and SecA inserts itself into the membrane, forcing about 20 amino acid residues of the protein to be exported through the translocation complex. Hydrolysis of an ATP by SecA provides the energy for a conformational change that causes SecA to withdraw from the membrane, releasing the polypeptide. SecA binds another ATP, and the next stretch of 20 amino acid residues is pushed across the membrane through the translocation complex. Steps and are repeated until the entire protein has passed through and is released to the periplasm. The electrochemical potential across the membrane (denoted by + and −) also provides some of the driving force required for protein translocation.

The figure shows a horizontal membrane with cytosol above and the periplasmic space below. A blue folded polypeptide is shown next to a blue sphere labeled S e c B at the upper left. Arrow 1 points down to show the polypeptide bound to S e c B. Arrow 2 points down to show S e c B with bound polypeptide attached to a vertical rectangular orange bar in the membrane immediately adjacent to a similar bar labeled S e c Y E C that is bound to S e c A above. S e c A overlaps the blue sphere of S e c B and has a horizontal rectangle at the bottom with a vertical protrusion at the right side. To the left, three plus symbols are shown above the membrane and three minus symbols are shown beneath the membrane. In step 3, an arrow points right accompanied by a curved arrow showing the addition of A T P and loss of S e c B. This yields two vertical orange bars with S e c A inverted so that its right side extends down over the bar instead of upward. The polypeptide is shown passing through the space between the two orange vertical bars. Arrow 4 points right accompanied by an arrow that branches to show the loss of A D P plus P subscript i end subscript. This yields a similar structure in which S e c A now has its right side protruding upward again. Arrow 5 points right accompanied by a curved lien showing the addition of A T P . S e c A is shown with its right side protruding downward and the polypeptide has moved much farther through the opening so that only a small amount is still in the cytosol.

Although most exported bacterial proteins use this pathway, some follow an alternative pathway that uses signal recognition and receptor proteins homologous to components of the eukaryotic SRP and the SRP receptor (see Fig. 27-38).

Cells Import Proteins by Receptor-Mediated Endocytosis

Some proteins are imported into eukaryotic cells from the surrounding medium; examples include low-density lipoprotein (LDL), the iron-carrying protein transferrin, peptide hormones, and circulating proteins destined for degradation. There are several importation pathways (Fig. 27-45). In one path, proteins bind to receptors in invaginations of the membrane called coated pits, which concentrate endocytic receptors in preference to other cell-surface proteins. The pits are coated on their cytosolic side with a lattice of the protein clathrin, which forms closed polyhedral structures (Fig. 27-46). The clathrin lattice grows as more receptors are occupied by target proteins. Eventually, a complete membrane-bounded endocytic vesicle is pinched off the plasma membrane with the aid of the large GTPase dynamin, and it enters the cytoplasm. The clathrin is quickly removed by uncoating enzymes, and the vesicle fuses with an endosome. ATPase activity in the endosomal membranes reduces the pH therein, facilitating dissociation of receptors from their target proteins. In a related pathway, caveolin causes invagination of patches of membrane containing lipid rafts associated with certain types of receptors (see Fig. 11-23). These endocytic vesicles then fuse with caveolin-containing internal structures called caveosomes, where the internalized molecules are sorted and redirected to other parts of the cell and the caveolins are prepared for recycling to the membrane surface. There are also clathrin- and caveolin-independent pathways; some make use of dynamin and others do not.

A figure summarizes endocytosis pathways in eukaryotic cells. — FIGURE 27-45 Summary of endocytosis pathways in eukaryotic cells. Pathways dependent on clathrin or caveolin make use of the GTPase dynamin to pinch vesicles from the plasma membrane. Some pathways do not use clathrin or caveolin; some of these make use of dynamin and some do not.

A figure shows a membrane across the top. Across the bottom, a long bar is labeled early endosome. At the left, clathirin-dependent endocytosis is shown. A small opening in the membrane extends down through a ring of yellow spheres labeled dynamin to a sphere surrounded by hexagonal structures labeled clathrin that form an outer coat around an inner spherical membrane. An arrow points down to show the same structure with pieces of the hexagonal structures coming off. This is labeled uncoating. An arrow points down to the early endosome. To the right, caveolin-dependent endocytosis is shown. A smaller membranous sphere is attached to the membrane by a small opening surrounded by a ring of yellow spheres. The membrane is surrounded by blue paired structures. Each structure, labeled caveolin, has a thicker outer part that curves down and out to form a piece that crosses the membrane and a thinner piece that runs up across the membrane. These pairs have long thin pieces that project in opposite directions. Two arrows point away from this structure. One points to a similar structure below that is fully enclosed, from which an arrow points to the early endosome. The second arrow points to a very large sphere labeled a caveosome that has a small protrusion to the upper left with caveolin along its borders. An arrow points down from the caveosome to the early endosome. To the right, clathrin- and caveolin-independent pathways are shown. The left-hand pathway shows a sphere with a small opening to the outside connecting it with the membrane and surrounded by a ring of yellow spheres. An arrow points down to a circular structure below, from which an arrow points to the early endosome. To its right, a similar process is shown except that the structure in the first step does not have a ring of yellow spheres around its opening at its place of attachment to the membrane.

A two-part figure shows the structure of clathrin as a drawing and in an electron micrograph. — FIGURE 27-46 Clathrin. (a) The protein has three light (L) chains $(M_{r} 35,000)$ $left-parenthesis upper M Subscript r Baseline 35,000 right-parenthesis$ and three heavy (H) chains $(M_{r} 180,000)$ $left-parenthesis upper M Subscript r Baseline 180,000 right-parenthesis$ of the ${(HL)}_{3}$ $left-parenthesis HL right-parenthesis Subscript 3$ clathrin unit, organized as a three-legged structure called a triskelion. Triskelions tend to assemble into polyhedral lattices. (b) Electron micrograph of a coated pit on the cytosolic face of the plasma membrane of a fibroblast. [(a) Information from S. Mayor and R. E. Pagano, *Nat. Rev. Mol. Cell Biol.* 8:603, 2007. (b) ©1980 Heuser. The Rockefeller University Press. J. Heuser, *J. Cell Biol.* 84:560, 1980.]

FIGURE 27-46 Clathrin. (a) The protein has three light (L) chains $(M_{r} 35,000)$ $left-parenthesis upper M Subscript r Baseline 35,000 right-parenthesis$ and three heavy (H) chains $(M_{r} 180,000)$ $left-parenthesis upper M Subscript r Baseline 180,000 right-parenthesis$ of the ${(HL)}_{3}$ $left-parenthesis HL right-parenthesis Subscript 3$ clathrin unit, organized as a three-legged structure called a triskelion. Triskelions tend to assemble into polyhedral lattices. (b) Electron micrograph of a coated pit on the cytosolic face of the plasma membrane of a fibroblast. [(a) Information from S. Mayor and R. E. Pagano, *Nat. Rev. Mol. Cell Biol.* 8:603, 2007. (b) ©1980 Heuser. The Rockefeller University Press. J. Heuser, *J. Cell Biol.* 84:560, 1980.]

Part a shows a structure with many hexagonal structures joined together to make up its side. It is approximately 80 n m in diameter. Red highlighting shows that a heavy chain runs from a central point to the upper left and then vertically up before bending to a rounded point, to the upper right before bending to the lower left to curve to end at a rounded point, and vertically down before bending to the lower left before curving to form a rounded point. These structures join together to make the overall highly structured sphere. From the central point, blue lines labeled light chain run to the upper left, upper right, and vertically down. Part b shows a circular structure in a micrograph with many evenly spaced rounded structures. The diameter of the entire sphere is 0.3 micrometers. All data are approximate.

The imported proteins and receptors then go their separate ways, their fates varying with the cell and protein type. Transferrin and its receptor are eventually recycled. Some hormones, growth factors, and immune complexes, after eliciting the appropriate cellular response, are degraded along with their receptors. LDL is degraded after the associated cholesterol has been delivered to its destination, but the LDL receptor is recycled (see Fig. 21-42).

Receptor-mediated endocytosis is exploited by some toxins and viruses to gain entry to cells. Influenza virus, diphtheria toxin, SARS-CoV-2 (the virus that causes COVID-19), and cholera toxin all enter cells in this way.

Protein Degradation Is Mediated by Specialized Systems in All Cells

Protein degradation is critical to overall cellular proteostasis, preventing the buildup of abnormal or unwanted proteins and permitting the recycling of amino acids. The half-lives of eukaryotic proteins vary from 30 seconds to many days. Most proteins turn over rapidly relative to the lifetime of a cell, although a few (such as hemoglobin) can last for the life of the cell (about 110 days for an erythrocyte). Rapidly degraded proteins include those that are defective because of incorrectly inserted amino acids or because of damage accumulated during normal functioning. And enzymes that act at key regulatory points in metabolic pathways often turn over rapidly.

Defective proteins and those with characteristically short half-lives are generally degraded in both bacterial and eukaryotic cells by selective ATP-dependent cytosolic systems. A second system in vertebrates, operating in lysosomes, recycles the amino acids of membrane proteins, extracellular proteins, and proteins with characteristically long half-lives.

In E. coli, many proteins are degraded by one of several proteolytic systems that contain $AAA + ATPases$ $AAA plus ATPases$ (see Chapter 25), including Lon (the name refers to the “long form” of proteins, observed only when this protease is absent), ClpXP, ClpAP, ClpCP, ClpYQ, and FtsH. Each system targets particular proteins distinguished by their structure or subcellular location or both. Typically, ATP hydrolysis is used to maneuver a target protein through a pore into a proteolytic chamber, unfolding the protein in the process. Proteins are cleaved within the chamber. Once a protein has been reduced to small, inactive peptides, other ATP-independent proteases complete their degradation.

The ATP-dependent pathway in eukaryotic cells is quite different, involving the protein ubiquitin, which, as its name suggests, occurs throughout the eukaryotic kingdoms. One of the most highly conserved proteins known, ubiquitin (76 amino acid residues) is essentially identical in organisms as different as yeasts and humans and is key to proteostasis (see Figs. 4-23 and 13-29) and cell cycle regulation (see Fig. 12-38). Ubiquitin is covalently linked to proteins slated for destruction via an ATP-dependent pathway that includes three separate types of enzymes: E1 activating enzymes, E2 conjugating enzymes, and E3 ligases (Fig. 27-47).

A figure shows how ubiquitin is attached to a protein in three steps. — FIGURE 27-47 Three-step pathway by which ubiquitin is attached to a protein. The pathway includes two different enzyme-ubiquitin intermediates. First, the free carboxyl group of ubiquitin’s carboxyl-terminal Gly residue becomes linked to an E1-class activating enzyme through a thioester. The ubiquitin is then transferred to an E2 conjugating enzyme. An E3 ligase ultimately catalyzes transfer of the ubiquitin from E2 to the target, linking ubiquitin through an amide (isopeptide) bond to the ε-amino group of a Lys residue in the target protein. Additional cycles produce polyubiquitin, a covalent polymer of ubiquitin subunits that targets the attached protein for destruction in eukaryotes. Multiple pathways of this sort, with different protein targets, are present in most eukaryotic cells.

A box labeled ubiquitin is bonded to C to the right double bonded to O to the upper right and bonded to O minus to the lower right. An arrow points downward accompanied by a curved arrow showing the addition of H red highlighted S bonded to a purple oval labeled E 1 plus an orange oval labeled A T P and the loss of A M P plus P P subscript i end subscript. This yields ubiquitin bonded to C double bonded to O above and bonded to red highlighted S to the right further bonded to the purple oval labeled E 1. An arrow points down accompanied by a curved arrow showing the addition of H red highlighted S bonded to a small vertical purple oval labeled E 2 and shows the loss of H red highlighted S bonded to E 1. This yields ubiquitin bonded to C double bonded to O above and bonded to red highlighted S to the right further bonded to E 2. An arrow points down ward accompanied by purple oval E 3 and a curved arrow showing the addition of H 2 N bonded to L y s bonded to a long light purple oval labeled target protein and loss of H red highlighted S bonded to E 2. This yields ubiquitin bonded to double bonded to O above and bonded to N H bonded to L y s bonded to the target protein. An arrow points down to text that reads, Repeated cycles lead to attachment of additional ubiquitin.

Ubiquitinated proteins are degraded by a large complex known as the 26S proteasome $(M_{r} 2.5 \times 10^{6})$ $left-parenthesis upper M Subscript r Baseline 2.5 times 10 Superscript 6 Baseline right-parenthesis$ (Fig. 27-48). The eukaryotic proteasome consists of two copies each of at least 32 different subunits, most of which are highly conserved from yeasts to humans. The proteasome contains two main types of subcomplexes: a barrel-like core particle and regulatory particles at each end of the barrel. The 19S regulatory particle on each end of the core particle contains approximately 18 subunits, including some that recognize and bind to ubiquitinated proteins. Six of the subunits are $AAA + ATPases$ $AAA plus ATPases$ that probably function in unfolding the ubiquitinated proteins and translocating the unfolded polypeptide into the core particle for degradation. The 19S particle also deubiquitinates the proteins as they are degraded in the proteasome. Most cells have additional regulatory complexes that can replace the 19S particle. These alternative regulators do not hydrolyze ATP and do not bind to ubiquitin, but they are important for the degradation of particular cellular proteins. The 26S proteasome can be effectively “accessorized” with regulatory complexes that change with changing cellular conditions.

A two-part figure shows the three-dimensional structure of the eukaryotic proteosome as a molecular structure in part a and in a schematic form in part b. — FIGURE 27-48 Three-dimensional structure of the eukaryotic proteasome. The 20S core particle and the 19S regulatory particle, or cap, are shown (a) as a molecular structure and (b) in schematic form. The core particle consists of four rings arranged to form a barrel-like structure. The outer rings are formed from seven α subunits, and the inner rings from seven β subunits. Three of the β subunits have protease activities, each with different substrate specificity. A regulatory particle forms a cap on each end of the core particle. The regulatory particle binds ubiquitinated proteins, unfolds them, and translocates them into the core particle, where they are degraded to peptides of 3 to 25 amino acid residues. [(a) Data from PDB ID 3L5Q, K. Sadre-Bazzaz et al., *Mol. Cell* 37:728, 2010.]

Part a shows a surface contour view of a vertical oval structure that has a white rounded region at the top, a narrow light brown region, a rounded dark red central region, another narrow light brown region, and then another white rounded region. Part b shows a central region labeled beta that consists of brown ovals arranged in two horizontal layers. Above and below there are single layers of light brown ovals. These layers are labeled alpha. The layers of alpha and beta subunits combined are labeled 20 S core. A gray oval that is pointed toward the lower right is at the upper right corner overlapping the top light brown layer and is labeled 19 S regulatory particle. A similar piece is shown at the bottom with its rounded part facing downward and its top embedded in the lower layer of alpha subunits. To the left of the upper 19 S regulatory particle, there is a rounded blue structure containing a blue strand. This is labeled substrate protein interacts with proteasome. Between this blue region and the gray 19 S regulatory particle, there are four orange spheres labeled polyubiquitin that form a chain that runs along the top of the blue protein as it enters the structure. Once it passes into the layer of alpha subunits, the blue strand becomes dashed to show it is inside. The blue strand ends in the top layer of beta subunits.

Although we do not yet understand all the signals that trigger ubiquitination, one simple signal has been found. For many proteins, the identity of the first residue that remains after removal of the amino-terminal Met residue, and any other posttranslational proteolytic processing of the amino-terminal end, has a profound influence on half-life (Table 27-9). These amino-terminal signals have been conserved over billions of years of evolution and are the same in bacterial protein degradation systems and in the human ubiquitination pathway. More complex signals, such as the destruction box discussed in Chapter 12 (see Fig. 12-38), are also being identified.

TABLE 27-9 Relationship between Protein Half-Life and Amino-Terminal Amino Acid Residue
Amino-terminal residue	Half-life^a
Stabilizing
Ala, Gly, Met, Ser, Thr, Val	>20 h
Destabilizing
Gln, Ile	∼30 min
Glu, Tyr	∼10 min
Pro	∼7 min
Asp, Leu, Lys, Phe	∼3 min
Arg	∼2 min
Information from A. Bachmair et al., Science 234:179, 1986. ^aHalf-lives were measured in yeast for the β-galactosidase protein modified so that in each experiment it had a different amino-terminal residue. Half-lives may vary for different proteins and in different organisms, but this general pattern appears to hold for all organisms.

Ubiquitin-dependent proteolysis is as important for the regulation of cellular processes as it is for the elimination of defective proteins. Many proteins required at only one stage of the eukaryotic cell cycle are rapidly degraded by the ubiquitin-dependent pathway after fulfilling their function. Ubiquitin-dependent destruction of cyclin is critical to cell-cycle regulation. The E1, E2, and E3 components of the ubiquitination pathway (Fig. 27-47) are large families of proteins. Different E1, E2, and E3 enzymes exhibit different specificities for target proteins and thus regulate different cellular processes. Some of these enzymes are highly localized in certain cellular compartments, reflecting a specialized function.

Not surprisingly, defects in the ubiquitination pathway have been implicated in a wide range of disease states. An inability to degrade certain proteins that activate cell division (the products of oncogenes) can lead to tumor formation, and a too-rapid degradation of proteins that act as tumor suppressors can have the same effect. The ineffective or overly rapid degradation of cellular proteins also seems to play a role in a range of other conditions, including renal diseases, asthma, and neurodegenerative disorders such as Alzheimer and Parkinson diseases that are associated with the formation of characteristic proteinaceous structures in neurons. Cystic fibrosis is caused in some cases by a too-rapid degradation of a chloride ion channel, with resultant loss of function (see Box 11-2). Liddle syndrome, in which a sodium channel in the kidney is not degraded, leads to excessive ${Na}^{+}$ $Na Superscript plus$ absorption and early-onset hypertension. Drugs designed to inhibit proteasome function are being developed as potential treatments for some of these conditions. Many bacterial pathogens have found ways to hijack the eukaryotic ubiquitination system, evolving enzymes that ubiquitinate and thus eliminate host proteins as required to facilitate infection. In a changing metabolic environment, protein degradation is as important to a cell’s survival as is protein synthesis, and much remains to be learned about these interesting pathways.

SUMMARY 27.3 Protein Targeting and Degradation

After synthesis, many proteins are directed to particular locations in the cell, a process mediated by signal sequences embedded in the polypeptide chain. In eukaryotic cells, one class of signal sequences is recognized by the signal recognition particle (SRP), which binds the signal sequence as soon as it appears on the ribosome and transfers the entire ribosome and incomplete polypeptide to the endoplasmic reticulum. Polypeptides with these signal sequences are moved into the endoplasmic reticulum lumen as they are synthesized.
Once in the lumen of the ER, many proteins are glycosylated. So modified, they are moved to the Golgi complex, then sorted and sent to lysosomes, the plasma membrane, or transport vesicles.
Proteins targeted to the nucleus have an internal signal sequence that, unlike other signal sequences, is not cleaved once the protein is successfully targeted.
Proteins targeted to mitochondria and chloroplasts in eukaryotic cells, and those destined for export in bacteria, also make use of an amino-terminal signal sequence.
Some eukaryotic cells import proteins by receptor-mediated endocytosis.
All cells eventually degrade proteins, using specialized proteolytic systems. Defective proteins and those slated for rapid turnover are generally degraded by an ATP-dependent system. In eukaryotic cells, the proteins are first tagged by linkage to ubiquitin, a highly conserved protein. Ubiquitin-dependent proteolysis, critical to the regulation of many cellular processes, is carried out by proteasomes, which also are highly conserved.