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Cell_Biology_Alberts. The polypeptide Chain passes Through an aqueous Channel in the Translocator It had long been debated whether polypeptide chains are transferred across the ER membrane in direct contact with the lipid bilayer or through a channel in a protein translocator. The debate ended with the identification of the translocator, which was shown to form a water-filled channel in the membrane through common pool of ribosomal subunits in cytosol mRNA encoding a protein targeted to ER remains membrane-bound


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Cell_Biology_Alberts. Figure 12–38 Free and membrane-bound polyribosomes. (a) a common pool of ribosomes synthesizes the proteins that stay in the cytosol and those that are transported into the er. The er signal sequence on a newly formed polypeptide chain binds to srp, which directs the translating ribosome to the er membrane. The mrna molecule remains permanently bound to the er as part of a polyribosome, while the ribosomes that move along it are recycled; at the end of each round of protein synthesis, the ribosomal subunits are released and rejoin the common pool in the cytosol. (B) a thin section electron micrograph of polyribosomes attached to the er membrane. The plane of section in some places cuts through the er roughly parallel to the membrane, giving a face-on view of the rosettelike pattern of the polyribosomes. (B, courtesy of George palade.)


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Cell_Biology_Alberts. Figure 12–39 Structure of the Sec61 complex. (a) a side view (left) and a top view (right, seen from the cytosol) of the structure of the sec61 complex of the archaeon Methanococcus jannaschii. The sec61α subunit has an inverted repeat structure (see figure 11–10) and is shown in blue and beige to indicate this pseudo-symmetry; the two smaller β and γ subunits are shown in gray. in the side view, some helices in front have been omitted to make the inside of the pore visible. The yellow short helix is thought to form a plug that seals the pore when the translocator is closed. To open, the complex rearranges itself to move the plug helix out of the way, as indicated by the red arrow. a ring of hydrophobic amino acid side chains is thought to form a tight-fitting diaphragm around translocating polypeptide chain to prevent leaks of other molecules across the membrane. The pore of the sec61 complex can also open sideways at a lateral seam. (B) models of the closed and open states of the


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Cell_Biology_Alberts. polypeptide chain to prevent leaks of other molecules across the membrane. The pore of the sec61 complex can also open sideways at a lateral seam. (B) models of the closed and open states of the translocator are shown in top view, illustrating how a signal sequence (or a transmembrane segment) could be released into the lipid bilayer after opening of the seam. (pDB codes: 1rh5 and 1rhz.) which the polypeptide chain passes. The core of the translocator, called the Sec61 complex, is built from three subunits that are highly conserved from bacteria to eukaryotic cells. The structure of the Sec61 complex suggests that α helices contributed by the largest subunit surround a central channel through which the polypeptide chain traverses the membrane (Figure 12–39). The channel is gated by a short α helix that is thought to keep the translocator closed when it is idle and to move aside when it is engaged in passing a polypeptide chain. According to this view, the pore is a dynamic gated


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Cell_Biology_Alberts. short α helix that is thought to keep the translocator closed when it is idle and to move aside when it is engaged in passing a polypeptide chain. According to this view, the pore is a dynamic gated channel that opens only transiently when a polypeptide chain traverses the membrane. In an idle translocator, it is important to keep the channel closed, so that the membrane remains impermeable to ions, such as Ca2+, which otherwise would leak out of the ER. As a polypeptide chain is translocating, a ring of hydrophobic amino acid side chains is thought to provide a flexible seal to prevent ion leaks.


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Cell_Biology_Alberts. The structure of the Sec61 complex suggests that the pore can also open along a seam on its side. Indeed, some structures of the translocator show it locked in an open-seam conformation. This opening allows a translocating peptide chain lateral access into the hydrophobic core of the membrane, a process that is important both for the release of a cleaved signal peptide into the membrane (see Figure 12–35) and for the integration of transmembrane proteins into the bilayer, as we discuss later.


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Cell_Biology_Alberts. Figure 12–40 A ribosome (green) bound to the ER protein translocator (blue). (a) a side-view reconstruction of the complex from electron microscopic images. (B) a view of the translocator seen from the er lumen. The translocator contains sec61, accessory proteins, and detergent used in the preparation. Domains of accessory proteins extend across the membrane and form the lumenal bulge. (C) a schematic drawing of a membrane-bound ribosome attached to the translocator, indicating the location of the tunnel in the large ribosomal subunit through which the growing polypeptide chain exits from the ribosome. The mrna (not shown) would be located between the small and large ribosomal subunits. (adapted from J.f. ménétret et al., J. Mol. Biol. 348:445–457, 2005. With permission from academic press.)


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Cell_Biology_Alberts. In eukaryotic cells, four Sec61 complexes form a large translocator assembly that can be visualized on ER-bound ribosomes after detergent solubilization of the ER membrane (Figure 12–40). It is likely that this assembly includes other membrane complexes that associate with the translocator, such as enzymes that modify the growing polypeptide chain, including oligosaccharide transferase and the signal peptidase. The assembly of a translocator with these accessory components is called the translocon. Translocation across the er membrane Does not always require ongoing polypeptide Chain elongation As we have seen, translocation of proteins into mitochondria, chloroplasts, and peroxisomes occurs post-translationally, after the protein has been made and released into the cytosol, whereas translocation across the ER membrane usually occurs during translation (co-translationally). This explains why ribosomes are bound to the ER but not to other organelles.


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Cell_Biology_Alberts. Some completely synthesized proteins, however, are imported into the ER, demonstrating that translocation does not always require ongoing translation. Post-translational protein translocation is especially common across the yeast ER membrane and the bacterial plasma membrane (which is thought to be evolutionarily related to the ER). To function in post-translational translocation, the ER translocator needs accessory proteins that feed the polypeptide chain into the pore and drive translocation (Figure 12–41). In bacteria, a translocation motor protein, the SecA ATPase, attaches to the cytosolic side of the translocator, where it undergoes cyclic conformational changes driven by ATP hydrolysis. Each time an ATP is hydrolyzed, a portion of the SecA protein inserts into the pore of the translocator, pushing a short segment of the passenger protein with it. As a result of this ratchet mechanism, the SecA ATPase progressively pushes the polypeptide chain of the transported protein across


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Cell_Biology_Alberts. pushing a short segment of the passenger protein with it. As a result of this ratchet mechanism, the SecA ATPase progressively pushes the polypeptide chain of the transported protein across the membrane.


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Cell_Biology_Alberts. Eukaryotic cells use a different set of accessory proteins that associate with the Sec61 complex. These proteins span the ER membrane and use a small domain on the lumenal side of the ER membrane to deposit an hsp70-like chaperone protein (called BiP, for binding protein) onto the polypeptide chain as it emerges from the pore into the ER lumen. ATP-dependent cycles of BiP binding and release drive unidirectional translocation, as described earlier for the mitochondrial hsp70 proteins that pull proteins across mitochondrial membranes. Proteins that are transported into the ER by a post-translational mechanism are first released into the cytosol, where they bind to chaperone proteins to prevent folding, as discussed earlier for proteins destined for mitochondria and chloroplasts. in single-pass Transmembrane proteins, a single internal er signal sequence remains in the lipid Bilayer as a membrane-spanning α helix


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Cell_Biology_Alberts. in single-pass Transmembrane proteins, a single internal er signal sequence remains in the lipid Bilayer as a membrane-spanning α helix The ER signal sequence in the growing polypeptide chain is thought to trigger the opening of the pore in the Sec61 protein translocator: after the signal sequence is released from the SRP and the growing chain has reached a sufficient length, the CYTOSOL ER LUMEN CYTOSOL EXTRACELLULAR SPACE SecA ATPase SecY complex Sec62,63,71,72 complex BiP SRP SRP receptor Sec61 complex ATPADPER signal sequence Pi+ bacterial inner (plasma) membrane Figure 12–41 Three ways in which protein translocation can be driven through structurally similar translocators.


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Cell_Biology_Alberts. (a) Co-translational translocation. The ribosome is brought to the membrane by the srp and srp receptor and then engages with the sec61 protein translocator. The growing polypeptide chain is threaded across the membrane as it is made. no additional energy is needed, as the only path available to the growing chain is to cross the membrane. (B) post-translational translocation in eukaryotic cells requires an additional complex composed of sec62, sec63, sec71, and sec72 proteins, which is attached to the sec61 translocator and deposits Bip molecules onto the translocating chain as it emerges from the translocator in the lumen of the er. aTp-driven cycles of Bip binding and release pull the protein into the lumen, a mechanism that closely resembles the mechanism of mitochondrial import in figure 12–23. (C) post-translational translocation in bacteria. The completed polypeptide chain is fed from the cytosolic side into the bacterial homolog of the sec61 complex (called the secY complex in


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Cell_Biology_Alberts. 12–23. (C) post-translational translocation in bacteria. The completed polypeptide chain is fed from the cytosolic side into the bacterial homolog of the sec61 complex (called the secY complex in bacteria) in the plasma membrane by the seca aTpase. aTp hydrolysis-driven conformational changes drive a pistonlike motion in seca, each cycle pushing about 20 amino acids of the protein chain through the pore of the translocator. The sec pathway used for protein translocation across the thylakoid membrane in chloroplasts uses a similar mechanism (see figure 12–26B).


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Cell_Biology_Alberts. Whereas the sec61 translocator, srp, and srp receptor are found in all organisms, seca is found exclusively in bacteria, and the sec62, 63, 71, 72 complex is found exclusively in eukaryotic cells. (adapted from p. Walter and a.e. Johnson, Annu. Rev. Cell Biol. 10:87–119, 1994. With permission from annual reviews.) signal sequence binds to a specific site inside the pore itself, thereby opening the pore. An ER signal sequence is therefore recognized twice: first by an SRP in the cytosol and then by a binding site in the pore of the protein translocator, where it serves as a start-transfer signal (or start-transfer peptide) that opens the pore (for example, see Figure 12–35 for how this works for a soluble protein). Dual recognition may help ensure that only appropriate proteins enter the lumen of the ER.


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Cell_Biology_Alberts. While bound in the translocation pore, a signal sequence is in contact not only with the Sec61 complex, which forms the walls of the pore, but also, along the lateral seam, with the hydrophobic core of the lipid bilayer. This was shown in chemical cross-linking experiments in which the signal sequence and the hydrocarbon chains of lipids were covalently linked together. When the nascent polypeptide chain grew long enough, the ER signal peptidase cleaved off the signal sequence and released it from the pore into the membrane, where it was rapidly degraded to amino acids by other proteases in the ER membrane. To release the signal sequence into the membrane, the translocator opens laterally along the seam (see Figures 12–35 and 12–39). The translocator is therefore gated in two directions: it opens to form a pore across the membrane to let the hydrophilic portions of proteins cross the lipid bilayer, and it opens laterally within the membrane to let hydrophobic portions of proteins


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Cell_Biology_Alberts. it opens to form a pore across the membrane to let the hydrophilic portions of proteins cross the lipid bilayer, and it opens laterally within the membrane to let hydrophobic portions of proteins partition into the lipid bilayer. Lateral gating of the pore is an essential step during the integration of transmembrane proteins.


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Cell_Biology_Alberts. The integration of membrane proteins requires that some parts of the polypeptide chain be translocated across the lipid bilayer whereas others are not. Despite this additional complexity, all modes of insertion of membrane proteins are simply variants of the sequence of events just described for transferring a soluble protein into the lumen of the ER. We begin by describing the three ways in which single-pass transmembrane proteins (see Figure 10–17) become inserted into the ER membrane.


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Cell_Biology_Alberts. In the simplest case, an N-terminal signal sequence initiates translocation, just as for a soluble protein, but an additional hydrophobic segment in the polypeptide chain stops the transfer process before the entire polypeptide chain is trans-located. This stop-transfer signal anchors the protein in the membrane after the ER signal sequence (the start-transfer signal) has been cleaved off and released from the translocator (Figure 12–42). The lateral gating mechanism transfers the stop-transfer sequence into the bilayer, where it remains as a single α-helical membrane-spanning segment, with the N-terminus of the protein on the lumenal side of the membrane and the C-terminus on the cytosolic side.


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Cell_Biology_Alberts. In the other two cases, the signal sequence is internal, rather than at the N-terminal end of the protein. As for an N-terminal ER signal sequence, the SRP binds to an internal signal sequence by recognizing its hydrophobic α-helical features. The SRP brings the ribosome making the protein to the ER membrane, and the ER signal sequence then serves as a start-transfer signal that initiates the protein’s translocation. After release from the translocator, the internal start-transfer sequence remains in the lipid bilayer as a single membrane-spanning α helix.


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Cell_Biology_Alberts. Internal start-transfer sequences can bind to the translocation apparatus in either of two orientations; this in turn determines which protein segment (the one preceding or the one following the start-transfer sequence) is moved across the membrane into the ER lumen. In one case, the resulting membrane protein has its C-terminus on the lumenal side (pathway A in Figure 12–43), while in the other, it has its N-terminus on the lumenal side (pathway B in Figure 12–43). The orientation of the start-transfer sequence depends on the distribution of nearby charged amino acids, as described in the figure legend. Combinations of start-Transfer and stop-Transfer signals Determine the Topology of multipass Transmembrane proteins


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Cell_Biology_Alberts. Combinations of start-Transfer and stop-Transfer signals Determine the Topology of multipass Transmembrane proteins In multipass transmembrane proteins, the polypeptide chain passes back and forth repeatedly across the lipid bilayer as hydrophobic α helices (see Figure 10–17). It is thought that an internal signal sequence serves as a start-transfer signal in these proteins to initiate translocation, which continues until the translocator encounters a stop-transfer sequence; in double-pass transmembrane proteins, for example, the polypeptide can then be released into the bilayer


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Cell_Biology_Alberts. Figure 12–42 How a single-pass transmembrane protein with a cleaved ER signal sequence is integrated into the ER membrane. in this protein, the co-translational translocation process is initiated by an n-terminal er signal sequence (red) that functions as a start-transfer signal, opening the translocator as in figure 12–35. in addition to this start-transfer sequence, however, the protein also contains a stop-transfer sequence (orange); when this sequence enters the translocator and interacts with a binding site within the pore, the translocator opens at the seam and discharges the protein laterally into the lipid bilayer, where the stop-transfer sequence remains to anchor the protein in the membrane. (in this figure and the two figures that follow, the ribosomes have been omitted for clarity.) (Figure 12–44). In more complex multipass proteins, in which many hydrophobic α helices span the bilayer, a second start-transfer sequence reinitiates translocation further down the polypeptide


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Cell_Biology_Alberts. (Figure 12–44). In more complex multipass proteins, in which many hydrophobic α helices span the bilayer, a second start-transfer sequence reinitiates translocation further down the polypeptide chain until the next stop-transfer sequence causes polypeptide release, and so on for subsequent start-transfer and stop-transfer sequences (Figure 12–45 and Movie 12.5).


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Cell_Biology_Alberts. Hydrophobic start-transfer and stop-transfer signal sequences both act to fix the topology of the protein in the membrane by locking themselves into the membrane as membrane-spanning α helices; and they can do this in either orientation. Whether a given hydrophobic signal sequence functions as a start-transfer or stop-transfer sequence must depend on its location in a polypeptide chain, since its function can be switched by changing its location in the protein by using recombinant DNA techniques. Thus, the distinction between start-transfer and stop-transfer sequences results mostly from their relative order in the growing polypeptide chain. It seems that the SRP begins scanning an unfolded polypeptide chain for hydrophobic segments at its N-terminus and proceeds toward the C-terminus, in the direction that the protein is synthesized. By recognizing the first appropriate hydrophobic segment to emerge from the ribosome, the SRP sets the “reading frame” for membrane integration: after


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Cell_Biology_Alberts. the direction that the protein is synthesized. By recognizing the first appropriate hydrophobic segment to emerge from the ribosome, the SRP sets the “reading frame” for membrane integration: after the SRP initiates trans-location, the translocator recognizes the next appropriate hydrophobic segment in the direction of transfer as a stop-transfer sequence, causing the region of the polypeptide chain in between to be threaded across the membrane. A similar


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Cell_Biology_Alberts. Figure 12–43 Integration of a single-pass transmembrane protein with an internal signal sequence into the ER membrane. an internal er signal sequence that functions as a start-transfer signal can bind to the translocator in one of two ways, leading to a membrane protein that has either its C-terminus (pathway a) or its n-terminus (pathway


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Cell_Biology_Alberts. B) in the er lumen. proteins are directed into either pathway by features in the polypeptide chain flanking the internal start-transfer sequence: if there are more positively charged amino acids immediately preceding the hydrophobic core of the start-transfer sequence than there are following it, the membrane protein is inserted into the translocator in the orientation shown in pathway a, whereas if there are more positively charged amino acids immediately following the hydrophobic core of the start-transfer sequence than there are preceding it, the membrane protein is inserted into the translocator in the orientation shown in pathway B. Because translocation cannot start before a start-transfer sequence appears outside the ribosome, translocation of the n-terminal portion of the protein shown in (B) can occur only after this portion has been fully synthesized.


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Cell_Biology_Alberts. note that there are two ways to insert a single-pass membrane-spanning protein whose n-terminus is located in the er lumen: that shown in figure 12–42 and that shown here in (B). scanning process continues until all of the hydrophobic regions in the protein have been inserted into the membrane as transmembrane α helices.


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Cell_Biology_Alberts. scanning process continues until all of the hydrophobic regions in the protein have been inserted into the membrane as transmembrane α helices. Because membrane proteins are always inserted from the cytosolic side of the ER in this programmed manner, all copies of the same polypeptide chain will have the same orientation in the lipid bilayer. This generates an asymmetrical ER membrane in which the protein domains exposed on one side are different from those exposed on the other side. This asymmetry is maintained during the many membrane budding and fusion events that transport the proteins made in the ER to other cell membranes (discussed in Chapter 13). Thus, the way in which a newly synthesized protein is inserted into the ER membrane determines the orientation of the protein in all of the other membranes as well.


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Cell_Biology_Alberts. When proteins are extracted with detergent from a membrane and then reconstituted into artificial lipid vesicles, a random mixture of right-side-out and inside-out protein orientations usually results. Thus, the protein asymmetry observed in cell membranes seems not to be an inherent property of the proteins, but instead results solely from the process by which proteins are inserted into the ER membrane from the cytosol. Figure 12–44 Integration of a double-pass transmembrane protein with an internal signal sequence into the ER membrane. in this protein, an internal er signal sequence acts as a start-transfer signal (as in figure 12–43) and initiates the transfer of the C-terminal part of the protein. at some point after a stop-transfer sequence has entered the translocator, the translocator discharges the sequence laterally into the membrane.


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Cell_Biology_Alberts. Figure 12–45 The insertion of the multipass membrane protein rhodopsin into the ER membrane. rhodopsin is the light-sensitive protein in rod photoreceptor cells in the mammalian retina (discussed in Chapter 15). (a) a hydropathy plot (see figure 10–20) identifies seven short hydrophobic regions in rhodopsin. (B) The hydrophobic region nearest the n-terminus serves as a start-transfer sequence that causes the preceding n-terminal portion of the protein to pass across the er membrane. subsequent hydrophobic sequences function in alternation as start-transfer and stop-transfer sequences. The green arrows indicate the paired start and stop signals inserted into the translocator. (C) The final integrated rhodopsin has its n-terminus located in the er lumen and its C-terminus located in the cytosol. The blue hexagons represent covalently attached oligosaccharides. er Tail-anchored proteins are integrated into the er membrane by a special mechanism


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Cell_Biology_Alberts. Many important membrane proteins are anchored in the membrane by a C-terminal transmembrane, hydrophobic α helix. These ER tail-anchored proteins include a large number of SNARE protein subunits that guide vesicular traffic (discussed in Chapter 13). When such a tail-anchored protein inserts into the ER membrane from the cytosol, only a few amino acids that follow the transmembrane αhelix on its C-terminal side are translocated into the ER lumen, while most of the protein remains in the cytosol. Because of the unique position of the transmembrane α helix in the protein sequence, translation terminates while the C-terminal amino acids that will form the transmembrane α helix have not yet emerged from the ribosome exit tunnel. Recognition by SRP is therefore not possible. It was long thought that these proteins are released from the ribosome and the hydrophobic C-terminal tail spontaneously partitions into the ER membrane. Such a mechanism could not explain, however, why ER


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Cell_Biology_Alberts. long thought that these proteins are released from the ribosome and the hydrophobic C-terminal tail spontaneously partitions into the ER membrane. Such a mechanism could not explain, however, why ER tail-anchored proteins insert into the ER membrane selectively and not also into all other membranes in the cell. It is now clear that a specialized targeting machinery is involved that is fueled by ATP hydrolysis (Figure 12–46). Although the components and details differ, this post-translational targeting mechanism is conceptually similar to SRP-dependent protein targeting (see Figure 12–37).


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Cell_Biology_Alberts. Not all tail-anchored proteins are inserted into the ER, however. Some proteins contain a C-terminal membrane anchor that contains additional sorting information that directs the protein to mitochondria or peroxisomes. How these proteins are sorted there remains unknown. Translocated polypeptide Chains fold and assemble in the lumen of the rough er Many of the proteins in the lumen of the ER are in transit, en route to other destinations; others, however, normally reside there and are present at high concentrations. These ER resident proteins contain an ER retention signal of four amino acids at their C-terminus that is responsible for retaining the protein in the ER (see Table 12–3. p. 648; discussed in Chapter 13). Some of these proteins function as catalysts that help the many proteins that are translocated into the ER lumen to fold and assemble correctly.


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Cell_Biology_Alberts. One important ER resident protein is protein disulfide isomerase (PDI), which catalyzes the oxidation of free sulfhydryl (SH) groups on cysteines to form disulfide (S–S) bonds. Almost all cysteines in protein domains exposed to either the extracellular space or the lumen of organelles in the secretory and endocytic pathways are disulfide-bonded. By contrast, disulfide bonds form only very rarely in domains exposed to the cytosol, because of the reducing environment there.


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Cell_Biology_Alberts. Figure 12–46 The insertion mechanism for tail-anchored proteins. in this post-translational pathway for the insertion of tail-anchored er membrane proteins, a soluble pre-targeting complex captures the hydrophobic C-terminal α helix after it emerges from the ribosomal exit tunnel and loads it onto the Get3 aTpase. The resulting complex is targeted to the er membrane by interaction with the Get1– Get2 receptor complex that functions as a membrane protein insertion machine. after Get3 hydrolyzes its bound aTp, the tail-anchored protein is released from the receptor and inserted into the er membrane. aDp release and renewed aTp binding recycles Get3 back to the cytosol.


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Cell_Biology_Alberts. Another ER resident protein is the chaperone protein BiP. We have already discussed how BiP pulls proteins post-translationally into the ER through the Sec61 ER translocator. Like other chaperones (discussed in Chapter 13), BiP recognizes incorrectly folded proteins, as well as protein subunits that have not yet assembled into their final oligomeric complexes. It does so by binding to exposed amino acid sequences that would normally be buried in the interior of correctly folded or assembled polypeptide chains. An example of a BiP-binding site is a stretch of alternating hydrophobic and hydrophilic amino acids that would normally be buried in a β sheet with its hydrophobic side oriented towards the hydrophobic core of the folded protein. The bound BiP both prevents the protein from aggregating and helps keep it in the ER (and thus out of the Golgi apparatus and later parts of the secretory pathway). Like some other members of the hsp70 family of chaperone proteins, which bind unfolded


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Cell_Biology_Alberts. and helps keep it in the ER (and thus out of the Golgi apparatus and later parts of the secretory pathway). Like some other members of the hsp70 family of chaperone proteins, which bind unfolded proteins and facilitate their import into mitochondria and chloroplasts, BiP hydrolyzes ATP to shuttle between highand low-affinity binding states, which allow it to hold on to and let go of its substrate proteins in a dynamic cycle.


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Cell_Biology_Alberts. most proteins synthesized in the rough er are Glycosylated by the addition of a Common N-linked oligosaccharide The covalent addition of oligosaccharides to proteins is one of the major biosynthetic functions of the ER. About half of the soluble and membrane-bound proteins that are processed in the ER—including those destined for transport to the Golgi apparatus, lysosomes, plasma membrane, or extracellular space—are glycoproteins that are modified in this way. Many proteins in the cytosol and nucleus are also glycosylated, but not with oligosaccharides: they carry a much simpler sugar modification, in which a single N-acetylglucosamine group is added to a serine or threonine of the protein.


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Cell_Biology_Alberts. During the most common form of protein glycosylation in the ER, a preformed precursor oligosaccharide (composed of N-acetylglucosamine, mannose, and glucose, and containing a total of 14 sugars) is transferred en bloc to proteins. Because this oligosaccharide is transferred to the side-chain NH2 group of an asparagine in the protein, it is said to be N-linked or asparagine-linked (Figure 12–47A). The transfer is catalyzed by a membrane-bound enzyme complex, an protein with N-glycosylation site


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Cell_Biology_Alberts. Figure 12–47 N-linked protein glycosylation in the rough ER. (a) almost as soon as a polypeptide chain enters the er lumen, it is glycosylated on target asparagine amino acids. The precursor oligosaccharide (shown in color) is attached only to asparagines in the sequences asnX-ser and asn-X-Thr (where X is any amino acid except proline). These sequences occur much less frequently in glycoproteins than in nonglycosylated cytosolic proteins. evidently there has been selective pressure against these sequences during protein evolution, presumably because glycosylation at too many sites would interfere with protein folding. The five sugars in the gray box form the core region of this oligosaccharide. for many glycoproteins, only the core sugars survive the extensive oligosaccharide trimming that takes place in the Golgi apparatus. (B) The precursor oligosaccharide is transferred from a dolichol lipid anchor to the asparagine as an intact unit in a reaction catalyzed by a transmembrane


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Cell_Biology_Alberts. that takes place in the Golgi apparatus. (B) The precursor oligosaccharide is transferred from a dolichol lipid anchor to the asparagine as an intact unit in a reaction catalyzed by a transmembrane oligosaccharyl transferase enzyme. one copy of this enzyme is associated with each protein translocator in the er membrane. (The translocator is not shown.) oligosaccharyl transferase contains 13 transmembrane α helices and a large er lumenal domain that contains its substrate-binding sites. The asparagine binds a tunnel that penetrates the enzyme interior. There, the amino group of the asparagine is twisted out of the plane that stabilizes the otherwise poorly reactive amide bond, activating it for reaction with the dolichol–oligosaccharide. The structure shown is of a prokaryotic homolog that closely resembles the catalytic subunit of the eukaryotic oligosaccharyl transferase. (pDB code: 3rCe.) oligosaccharyl transferase, which has its active site exposed on the lumenal side of the ER


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Cell_Biology_Alberts. closely resembles the catalytic subunit of the eukaryotic oligosaccharyl transferase. (pDB code: 3rCe.) oligosaccharyl transferase, which has its active site exposed on the lumenal side of the ER membrane; this explains why cytosolic proteins are not glycosylated in this way. A special lipid molecule called dolichol anchors the precursor oligosaccharide in the ER membrane. The precursor oligosaccharide is transferred to the target asparagine in a single enzymatic step immediately after that amino acid has reached the ER lumen during protein translocation. The precursor oligosaccharide is linked to the dolichol lipid by a high-energy pyrophosphate bond, which provides the activation energy that drives the glycosylation reaction (Figure 12–47B). One copy of oligosaccharyl transferase is associated with each protein translocator, allowing it to scan and glycosylate the incoming polypeptide chains efficiently.


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Cell_Biology_Alberts. The precursor oligosaccharide is built up sugar by sugar on the membrane-bound dolichol lipid and is then transferred to a protein. The sugars are first activated in the cytosol by the formation of nucleotide (UDP or GDP)-sugar intermediates, which then donate their sugar (directly or indirectly) to the lipid in an orderly sequence. Part way through this process, the lipid-linked oligosaccharide is flipped, with the help of a transporter, from the cytosolic to the lumenal side of the ER membrane (Figure 12–48).


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Cell_Biology_Alberts. All of the diversity of the N-linked oligosaccharide structures on mature glycoproteins results from the later modification of the original precursor oligosaccharide. While still in the ER, three glucoses (see Figure 12–47) and one mannose are quickly removed from the oligosaccharides of most glycoproteins. We shall return to the importance of glucose trimming shortly. This oligosaccharide “trimming,” or “processing,” continues in the Golgi apparatus, as we discuss in Chapter 13. The N-linked oligosaccharides are by far the most common oligosaccharides, being found on 90% of all glycoproteins. Less frequently, oligosaccharides are linked to the hydroxyl group on the side chain of a serine, threonine, or hydroxy-lysine amino acid. A first sugar of these O-linked oligosaccharides is added in the ER and the oligosaccharide is then further extended in the Golgi apparatus (see Figure 13–32). lipid bilayer of ER membrane


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Cell_Biology_Alberts. Figure 12–48 Synthesis of the lipid-linked precursor oligosaccharide in the rough ER membrane. The oligosaccharide is assembled sugar by sugar onto the carrier lipid dolichol (a polyisoprenoid; see panel 2–5, pp. 98–99). Dolichol is long and very hydrophobic: its 22 five-carbon units can span the thickness of a lipid bilayer more than three times, so that the attached oligosaccharide is firmly anchored in the membrane. The first sugar is linked to dolichol by a pyrophosphate bridge. This high-energy bond activates the oligosaccharide for its eventual transfer from the lipid to an asparagine side chain of a growing polypeptide on the lumenal side of the rough er. as indicated, the synthesis of the oligosaccharide starts on the cytosolic side of the er membrane and continues on the lumenal face after the (man)5(Glcnac)2 lipid intermediate is flipped across the bilayer by a transporter (which is not shown). all the subsequent glycosyl transfer reactions on the lumenal side of the er


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Cell_Biology_Alberts. face after the (man)5(Glcnac)2 lipid intermediate is flipped across the bilayer by a transporter (which is not shown). all the subsequent glycosyl transfer reactions on the lumenal side of the er involve transfers from dolichol-p-glucose and dolichol-pmannose; these activated, lipid-linked monosaccharides are synthesized from dolichol phosphate and UDp-glucose or GDp-mannose (as appropriate) on the cytosolic side of the er and are then flipped across the er membrane. Glcnac = N-acetylglucosamine; man = mannose; Glc = glucose.


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Cell_Biology_Alberts. oligosaccharides are Used as Tags to mark the state of protein folding


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Cell_Biology_Alberts. It has long been debated why glycosylation is such a common modification of proteins that enter the ER. One particularly puzzling observation has been that some proteins require N-linked glycosylation for proper folding in the ER, yet the precise location of the oligosaccharides attached to the protein’s surface does not seem to matter. A clue to the role of glycosylation in protein folding came from studies of two ER chaperone proteins, which are called calnexin and calreticulin because they require Ca2+ for their activities. These chaperones are carbohydrate-binding proteins, or lectins, which bind to oligosaccharides on incompletely folded proteins and retain them in the ER. Like other chaperones, they prevent incompletely folded proteins from irreversibly aggregating. Both calnexin and calreticulin also promote the association of incompletely folded proteins with another ER chaperone, which binds to cysteines that have not yet formed disulfide bonds.


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Cell_Biology_Alberts. Calnexin and calreticulin recognize N-linked oligosaccharides that contain a single terminal glucose, and they therefore bind proteins only after two of the three glucoses on the precursor oligosaccharide have been removed during glucose trimming by ER glucosidases. When the third glucose has been removed, the glycoprotein dissociates from its chaperone and can leave the ER. How, then, do calnexin and calreticulin distinguish properly folded from incompletely folded proteins? The answer lies in yet another ER enzyme, a glucosyl transferase that keeps adding a glucose to those oligosaccharides that have lost their last glucose. It adds the glucose, however, only to oligosaccharides that are attached to unfolded proteins. Thus, an unfolded protein undergoes continuous cycles of glucose trimming (by glucosidase) and glucose addition (by glucosyl transferase), maintaining an affinity for calnexin and calreticulin until it has achieved its fully folded state (Figure 12–49).


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Cell_Biology_Alberts. improperly folded proteins are exported from the er and Degraded in the Cytosol Despite all the help from chaperones, many protein molecules (more than 80% for some proteins) translocated into the ER fail to achieve their properly folded or oligomeric state. Such proteins are exported from the ER back into the cytosol, where they are degraded in proteasomes (discussed in Chapter 6). In many ways, the mechanism of retrotranslocation is similar to other post-translational Figure 12–49 The role of N-linked glycosylation in ER protein folding.


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Cell_Biology_Alberts. The er-membrane-bound chaperone protein calnexin binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the er. removal of the terminal glucose by a glucosidase releases the protein from calnexin. a glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from UDp-glucose to the N-linked oligosaccharide, renewing the protein’s affinity for calnexin and retaining it in the er. The cycle repeats until the protein has folded completely. Calreticulin functions similarly, except that it is a soluble er resident protein. another er chaperone, erp57 (not shown), collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the er. erp57 recognizes free sulfhydryl groups, which are a sign of incomplete disulfide bond formation.


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Cell_Biology_Alberts. modes of translocation. For example, like translocation into mitochondria or chloroplasts, chaperone proteins are necessary to keep the polypeptide chain in an unfolded state prior to and during translocation. Similarly, a source of energy is required to provide directionality to the transport and to pull the protein into the cytosol. Finally, a translocator is necessary.


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Cell_Biology_Alberts. Selecting proteins from the ER for degradation is a challenging process: mis-folded proteins or unassembled protein subunits should be degraded, but folding intermediates of newly made proteins should not. Help in making this distinction comes from the N-linked oligosaccharides, which serve as timers that measure how long a protein has spent in the ER. The slow trimming of a particular man-nose on the core oligosaccharide tree by an enzyme (a mannosidase) in the ER creates a new oligosaccharide structure that ER-lumenal lectins of the retrotranslocation apparatus recognize. Proteins that fold and exit from the ER faster than the mannosidase can remove its target mannose therefore escape degradation.


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Cell_Biology_Alberts. In addition to the lectins in the ER that recognize the oligosaccharides, chaperones and protein disulfide isomerases (enzymes mentioned earlier that catalyze the formation and breakage of S–S bonds) associate with the proteins that must be degraded. The chaperones prevent the unfolded proteins from aggregating, and the disulfide isomerases break disulfide bonds that may have formed incorrectly, so that a linear polypeptide chain can be translocated back into the cytosol.


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Cell_Biology_Alberts. Multiple translocator complexes move different proteins from the ER membrane or lumen into the cytosol. A common feature is that they each contain an E3 ubiquitin ligase enzyme, which attaches polyubiquitin tags to the unfolded proteins as they emerge into the cytosol, marking them for destruction. Fueled by the energy derived from ATP hydrolysis, a hexomeric ATPase of the family of AAA-ATPases (see Figure 6–85) pulls the unfolded protein through the translocator into the cytosol. An N-glycanase removes its oligosaccharide chains en bloc. Guided by its ubiquitin tag, the deglycosylated polypeptide is rapidly fed into proteasomes, where it is degraded (Figure 12–50). misfolded proteins in the er activate an Unfolded protein response


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Cell_Biology_Alberts. misfolded proteins in the er activate an Unfolded protein response Cells carefully monitor the amount of misfolded protein in various compartments. An accumulation of misfolded proteins in the cytosol, for example, triggers a heat-shock response (discussed in Chapter 6), which stimulates the transcription of genes encoding cytosolic chaperones that help to refold the proteins. Similarly, an accumulation of misfolded proteins in the ER triggers an unfolded protein response, which includes an increased transcription of genes encoding proteins involved in retrotranslocation and protein degradation in the cytosol, ER chaperones, and many other proteins that help to increase the protein-folding capacity of the ER. Figure 12–50 The export and degradation of misfolded ER proteins.


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Cell_Biology_Alberts. Figure 12–50 The export and degradation of misfolded ER proteins. misfolded soluble proteins in the er lumen are recognized and targeted to a translocator complex in the er membrane. They first interact in the er lumen with chaperones, disulfide isomerases, and lectins. They are then exported into the cytosol through the translocator. in the cytosol, they are ubiquitylated, deglycosylated, and degraded in proteasomes. misfolded membrane proteins follow a similar pathway but use a different translocator. How do misfolded proteins in the ER signal to the nucleus? There are three parallel pathways that execute the unfolded protein response (Figure 12–51A). The first pathway, which was initially discovered in yeast cells, is particularly remarkable. Misfolded proteins in the ER activate a transmembrane protein kinase in the ER, called IRE1, which causes the kinase to oligomerize and phosphorylate itself. (Some cell-surface receptor kinases in the plasma membrane are activated in a


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Cell_Biology_Alberts. Figure 12–51 The unfolded protein response. (a) By three parallel intracellular signaling pathways, the accumulation of misfolded proteins in the er lumen signals to the nucleus to activate the transcription of genes that encode proteins that help the cell cope with misfolded proteins in the er. (B) regulated rna splicing is a key regulatory switch in pathway 1 of the unfolded protein response (Movie 12.6).


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Cell_Biology_Alberts. 1 MISFOLDED PROTEINS IN ER SIGNAL THE NEED FOR MORE ER CHAPERONES. THEY BIND TO AND ACTIVATE A TRANSMEMBRANE KINASE 2 ACTIVATED KINASE UNMASKS AN ENDORIBONUCLEASE ACTIVITY 3 ENDORIBONUCLEASE CUTS SPECIFIC RNA MOLECULES AT TWO POSITIONS, REMOVING AN INTRON 6 TRANSCRIPTION REGULATOR ENTERS NUCLEUS AND ACTIVATES GENES ENCODING ER CHAPERONES 7 CHAPERONES ARE MADE IN ER, WHERE THEY HELP FOLD PROTEINS 4 TWO EXONS ARE LIGATED TO FORM AN ACTIVE mRNA 5 mRNA IS TRANSLATED TO MAKE A TRANSCRIPTION REGULATOR similar way, as discussed in Chapter 15.) The oligomerization and autophosphorylation of IRE1 activates an endoribonuclease domain in the cytosolic portion of the same molecule, which cleaves a specific cytosolic mRNA molecule at two positions, excising an intron. (This is a unique exception to the rule that introns are spliced out while the RNA is still in the nucleus.) The separated exons are then joined by an RNA ligase, generating a spliced mRNA, which is translated to produce an active


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Cell_Biology_Alberts. the rule that introns are spliced out while the RNA is still in the nucleus.) The separated exons are then joined by an RNA ligase, generating a spliced mRNA, which is translated to produce an active transcription regulatory protein. This protein activates the transcription of genes encoding the proteins that help mediate the unfolded protein response (Figure 12–51B).


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Cell_Biology_Alberts. Misfolded proteins also activate a second transmembrane kinase in the ER, PERK, which inhibits a translation initiation factor by phosphorylating it, thereby reducing the production of new proteins throughout the cell. One consequence of the reduction in protein synthesis is to reduce the flux of proteins into the ER, thereby reducing the load of proteins that need to be folded there. Some proteins, however, are preferentially translated when translation initiation factors are scarce (discussed in Chapter 7, p. 424), and one of these is a transcription regulator that helps activate the transcription of the genes encoding proteins active in the unfolded protein response.


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Cell_Biology_Alberts. Finally, a third transcription regulator, ATF6, is initially synthesized as a trans-membrane ER protein. Because it is embedded in the ER membrane, it cannot activate the transcription of genes in the nucleus. When misfolded proteins accumulate in the ER, however, the ATF6 protein is transported to the Golgi apparatus, where it encounters proteases that cleave off its cytosolic domain, which can now migrate to the nucleus and help activate the transcription of genes encoding proteins involved in the unfolded protein response. (This mechanism is similar to that described in Figure 12–16 for activation of the transcription regulator that controls cholesterol biosynthesis.) The relative importance of each of these three pathways in the unfolded protein response differs in different cell types, enabling each cell type to tailor the unfolded protein response to its particular needs.


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Cell_Biology_Alberts. As discussed in Chapter 10, several cytosolic enzymes catalyze the covalent addition of a single fatty acid chain or prenyl group to selected proteins. The attached lipids help direct and attach these proteins to cell membranes. A related process is catalyzed by ER enzymes that covalently attach a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of some membrane proteins destined for the plasma membrane. This linkage forms in the lumen of the ER, where, at the same time, the transmembrane segment of the protein is cleaved off (Figure 12–52). A large number of plasma membrane proteins are modified in this way. Since they are attached to the exterior of the plasma membrane only by their GPI anchors,


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Cell_Biology_Alberts. Figure 12–52 The attachment of a GPI anchor to a protein in the ER. Gpianchored proteins are targeted to the er membrane by an n-terminal signal sequence (not shown), which is removed (see figure 12–42). immediately after the completion of protein synthesis, the precursor protein remains anchored in the er membrane by a hydrophobic C-terminal sequence of 15–20 amino acids; the rest of the protein is in the er lumen. Within less than a minute, an enzyme in the er cuts the protein free from its membrane-bound C-terminus and simultaneously attaches the new C-terminus to an amino group on a preassembled Gpi intermediate. The sugar chain contains an inositol attached to the lipid from which the Gpi anchor derives its name. it is followed by a glucosamine and three mannoses. The terminal mannose links to a phosphoethanolamine that provides the amino group to attach the protein. The signal that specifies this modification is contained within the hydrophobic C-terminal sequence and a few


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Cell_Biology_Alberts. links to a phosphoethanolamine that provides the amino group to attach the protein. The signal that specifies this modification is contained within the hydrophobic C-terminal sequence and a few amino acids adjacent to it on the lumenal side of the er membrane; if this signal is added to other proteins, they too become modified in this way. Because of the covalently linked lipid anchor, the protein remains membrane-bound, with all of its amino acids exposed initially on the lumenal side of the er and eventually on the exterior of the plasma membrane.


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Cell_Biology_Alberts. lipid bilayer of ER phosphatidic they can in principle be released from cells in soluble form in response to signals Figure 12–53 The synthesis of phosphatidylcholine. as illustrated, this that activate a specific phospholipase in the plasma membrane. Trypanosome phospholipid is synthesized from glycerol parasites, for example, use this mechanism to shed their coat of GPI-anchored 3-phosphate, cytidine-diphosphocholine surface proteins when attacked by the immune system. GPI anchors may also be (CDp-choline), and fatty acids delivered used to direct plasma membrane proteins into lipid rafts and thus segregate the to the er by a cytosolic fatty acid binding proteins from other membrane proteins (see Figure 10–13). protein. The er assembles most lipid Bilayers


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Cell_Biology_Alberts. The ER membrane is the site of synthesis of nearly all of the cell’s major classes of lipids, including both phospholipids and cholesterol, required for the production of new cell membranes. The major phospholipid made is phosphatidylcholine, which can be formed in three steps from choline, two fatty acids, and glycerol phosphate (Figure 12–53). Each step is catalyzed by enzymes in the ER membrane, which have their active sites facing the cytosol, where all of the required metabolites are found. Thus, phospholipid synthesis occurs exclusively in the cytosolic leaflet of the ER membrane. Because fatty acids are not soluble in water, they are shepherded from their sites of synthesis to the ER by a fatty acid binding protein in the cytosol. After arrival in the ER membrane and activation with CoA, acyl transferases successively add two fatty acids to glycerol phosphate to produce phosphatidic acid. Phosphatidic acid is sufficiently water-insoluble to remain in the lipid bilayer; it


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Cell_Biology_Alberts. with CoA, acyl transferases successively add two fatty acids to glycerol phosphate to produce phosphatidic acid. Phosphatidic acid is sufficiently water-insoluble to remain in the lipid bilayer; it cannot be extracted from the bilayer by the fatty acid binding proteins. It is therefore this first step that enlarges the ER lipid bilayer. The later steps determine the head group of a newly formed lipid molecule and therefore the chemical nature of the bilayer, but they do not result in net membrane growth. The two other major membrane phospholipids—phosphatidylethanolamine and phosphatidylserine (see Figure 10–3)—as well as the minor phospholipid phosphatidylinositol (PI), are all synthesized in this way.


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Cell_Biology_Alberts. Because phospholipid synthesis takes place in the cytosolic leaflet of the ER lipid bilayer, there needs to be a mechanism that transfers some of the newly formed phospholipid molecules to the lumenal leaflet of the bilayer. In synthetic lipid bilayers, lipids do not “flip-flop” in this way (see Figure 10–10). In the ER, however, phospholipids equilibrate across the membrane within minutes, which is almost 100,000 times faster than can be accounted for by spontaneous “flipflop.” This rapid trans-bilayer movement is mediated by a poorly characterized asymmetric lipid bilayer of plasma membrane phospholipid translocator called a scramblase, which nonselectively equilibrates phospholipids between the two leaflets of the lipid bilayer (Figure 12–54). Thus, the different types of phospholipids are thought to be equally distributed between the two leaflets of the ER membrane.


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Cell_Biology_Alberts. The plasma membrane contains a different type of phospholipid translocator that belongs to the family of P-type pumps (discussed in Chapter 11). These flip-pases specifically recognize those phospholipids that contain free amino groups in their head groups (phosphatidylserine and phosphatidylethanolamine—see Figure 10–3) and transfers them from the extracellular to the cytosolic leaflet, using the energy of ATP hydrolysis. The plasma membrane therefore has a highly asymmetric phospholipid composition, which is actively maintained by the flip-pases (see Figure 10–15). The plasma membrane also contains a scramblase but, in contrast to the ER scramblase, which is always active, the plasma membrane enzyme is regulated and only activated in some situations, such as in apoptosis and in activated platelets, where it acts to abolish the lipid bilayer asymmetry; the resulting exposure of phosphatidylserine on the surface of apoptotic cells serves as a signal for phagocytic cells to ingest and


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Cell_Biology_Alberts. platelets, where it acts to abolish the lipid bilayer asymmetry; the resulting exposure of phosphatidylserine on the surface of apoptotic cells serves as a signal for phagocytic cells to ingest and degrade the dead cell.


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Cell_Biology_Alberts. The ER also produces cholesterol and ceramide (Figure 12–55). Ceramide is made by condensing the amino acid serine with a fatty acid to form the amino alcohol sphingosine (see Figure 10–3); a second fatty acid is then covalently added to form ceramide. The ceramide is exported to the Golgi apparatus, where it serves as a precursor for the synthesis of two types of lipids: oligosaccharide chains are added to form glycosphingolipids (glycolipids; see Figure 10–16), and phosphocholine head groups are transferred from phosphatidylcholine to other ceramide molecules to form sphingomyelin (discussed in Chapter 10). Thus, both glycolipids and sphingomyelin are produced relatively late in the process of membrane synthesis. Because they are produced by enzymes that have their active sites exposed to the Golgi lumen, they are found exclusively in the noncytosolic leaflet of the lipid bilayers that contain them. Figure 12–54 The role of phospholipid translocators in lipid bilayer synthesis.


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Cell_Biology_Alberts. Figure 12–54 The role of phospholipid translocators in lipid bilayer synthesis. (a) Because new lipid molecules are added only to the cytosolic half of the er membrane bilayer and lipid molecules do not flip spontaneously from one monolayer to the other, a transmembrane phospholipid translocator (called a scramblase) is required to transfer lipid molecules from the cytosolic half to the lumenal half so that the membrane grows as a bilayer. The scramblase is not specific for particular phospholipid head groups and therefore equilibrates the different phospholipids between the two monolayers. (B) fueled by aTp hydrolysis, a head-group-specific flippase in the plasma membrane actively flips phosphatidylserine and phosphatidylethanolamine directionally from the extracellular to the cytosolic leaflet, creating the characteristically asymmetric lipid bilayer of the plasma membrane of animal cells (see figure 10–15). Figure 12–55 The structure of ceramide.


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Cell_Biology_Alberts. Figure 12–55 The structure of ceramide. As discussed in Chapter 13, the plasma membrane and the membranes of the Golgi apparatus, lysosomes, and endosomes all form part of a membrane system that communicates with the ER by means of transport vesicles, which transfer both proteins and lipids. Mitochondria and plastids, however, do not belong to this system, and they therefore require different mechanisms to import proteins and lipids for growth. We have already seen that they import most of their proteins from the cytosol. Although mitochondria modify some of the lipids they import, they do not synthesize lipids de novo; instead, their lipids have to be imported from the ER, either directly or indirectly by way of other cell membranes. In either case, special mechanisms are required for the transfer.


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Cell_Biology_Alberts. The details of how lipid distribution between different membranes is catalyzed and regulated are not known. Water-soluble carrier proteins called phospholipid exchange proteins (or phospholipid transfer proteins) are thought to transfer individual phospholipid molecules between membranes, functioning much like fatty acid binding proteins that shepherd fatty acids through the cytosol (see Figure 12–54). In addition, mitochondria are often seen in close juxtaposition to ER membranes in electron micrographs, and specific junction complexes have been identified that hold the ER and outer mitochondrial membrane in close proximity. It is thought that these junction complexes provide specific contact-dependent lipid transfer mechanisms that operate between these adjacent membranes.


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Cell_Biology_Alberts. The extensive ER network serves as a factory for the production of almost all of the cell’s lipids. In addition, a major portion of the cell’s protein synthesis occurs on the cytosolic surface of the rough ER: virtually all proteins destined for secretion or for the ER itself, the Golgi apparatus, the lysosomes, the endosomes, and the plasma membrane are first imported into the ER from the cytosol. In the ER lumen, the proteins fold and oligomerize, disulfide bonds are formed, and N-linked oligosaccharides are added. The pattern of N-linked glycosylation is used to indicate the extent of protein folding, so that proteins leave the ER only when they are properly folded. Proteins that do not fold or oligomerize correctly are translocated back into the cytosol, where they are deglycosylated, polyubiquitylated, and degraded in proteasomes. If misfolded proteins accumulate in excess in the ER, they trigger an unfolded protein response, which activates appropriate genes in the nucleus to


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Cell_Biology_Alberts. polyubiquitylated, and degraded in proteasomes. If misfolded proteins accumulate in excess in the ER, they trigger an unfolded protein response, which activates appropriate genes in the nucleus to help the ER cope.


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Cell_Biology_Alberts. Only proteins that carry a special ER signal sequence are imported into the ER. The signal sequence is recognized by a signal-recognition particle (SRP), which binds both the growing polypeptide chain and the ribosome and directs them to a receptor protein on the cytosolic surface of the rough ER membrane. This binding to the ER membrane initiates the translocation process that threads a loop of polypeptide chain across the ER membrane through the hydrophilic pore of a protein translocator.


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Cell_Biology_Alberts. Soluble proteins—destined for the ER lumen, for secretion, or for transfer to the lumen of other organelles—pass completely into the ER lumen. Transmembrane proteins destined for the ER or for other cell membranes are translocated part way across the ER membrane and remain anchored there by one or more membrane-spanning α-helical segments in their polypeptide chains. These hydrophobic portions of the protein can act either as start-transfer or stop-transfer signals during the translocation process. When a polypeptide contains multiple, alternating start-transfer and stop-transfer signals, it will pass back and forth across the bilayer multiple times as a multipass transmembrane protein. The asymmetry of protein insertion and glycosylation in the ER establishes the sidedness of the membranes of all the other organelles that the ER supplies with membrane proteins. how do nuclear import receptors negotiate the tangled gel-like interior of a nuclear pore complex so efficiently?


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Cell_Biology_Alberts. how do nuclear import receptors negotiate the tangled gel-like interior of a nuclear pore complex so efficiently? is the nuclear pore complex a rigid structure or can it expand and contract, depending on the cargo transported? sequence comparisons show that signal sequences for an individual protein such as insulin are quite conserved across species, much more so than would be expected from our current understanding that all that matters for their function are general structural features such as hydrophobicity. What other functions might signal sequences have that could account for their evolutionary sequence conservation? how are polyribosomes on the endoplasmic reticulum membrane arranged so that the next initiating ribosome will find an unoccupied translocator? Why does the signal-recognition particle have an indispensable rna subunit? Which statements are true? Explain why or why not.


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Cell_Biology_Alberts. Why does the signal-recognition particle have an indispensable rna subunit? Which statements are true? Explain why or why not. 12–1 Like the lumen of the ER, the interior of the nucleus is topologically equivalent to the outside of the cell. 12–2 ER-bound and free ribosomes, which are structurally and functionally identical, differ only in the proteins they happen to be making at a particular time. 12–3 To avoid the inevitable collisions that would occur if two-way traffic through a single pore were allowed, nuclear pore complexes are specialized so that some mediate import while others mediate export. 12–4 Peroxisomes are found in only a few specialized types of eukaryotic cell. Discuss the following problems. 12–5 What is the fate of a protein with no sorting signal?


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Cell_Biology_Alberts. 12–4 Peroxisomes are found in only a few specialized types of eukaryotic cell. Discuss the following problems. 12–5 What is the fate of a protein with no sorting signal? 12–6 The rough ER is the site of synthesis of many classes of membrane proteins. Some of these proteins remain in the ER, whereas others are sorted to compartments such as the Golgi apparatus, lysosomes, and the plasma membrane. One measure of the difficulty of the sorting problem is the degree of “purification” that must be achieved during transport from the ER. Are proteins bound for the plasma membrane common or rare among all ER membrane proteins?


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Cell_Biology_Alberts. A few simple considerations allow one to answer this question. In a typical growing cell that is dividing once every 24 hours, the equivalent of one new plasma membrane must transit the ER every day. If the ER membrane is 20 times the area of a plasma membrane, what is the ratio of plasma membrane proteins to other membrane proteins in the ER? (Assume that all proteins on their way to the plasma membrane remain in the ER for 30 minutes on average before exiting, and that the ratio of proteins to lipids in the ER and plasma membranes is the same.) 12–7 Before nuclear pore complexes were well understood, it was unclear whether nuclear proteins diffused passively into the nucleus and accumulated there by binding to residents of the nucleus such as chromosomes, or whether they were actively imported and accumulated regardless of their affinity for nuclear components.


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Cell_Biology_Alberts. A classic experiment that addressed this problem used several forms of radioactive nucleoplasmin, which is a large pentameric protein involved in chromatin assembly. In this experiment, either the intact protein or the nucleoplasmin heads, tails, or heads with a single tail were injected into the cytoplasm of a frog oocyte or into the nucleus (Figure Q12–1). All forms of nucleoplasmin, except heads, accumulated in the nucleus when injected into the cytoplasm, and all forms were retained in the nucleus when injected there. A. What portion of the nucleoplasmin molecule is responsible for localization in the nucleus? nucleoplasmin nuclear cytoplasmic Figure Q12–1 Cellular preparation injection injection location of injected nucleoplasmin components (problem 12–7). schematic diagrams of autoradiographs show the cytoplasm and nucleus with the location of nucleoplasmin indicated by the red areas.


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Cell_Biology_Alberts. B. How do these experiments distinguish between active transport, in which a nuclear localization signal triggers transport by the nuclear pore complex, and passive diffusion, in which a binding site for a nuclear component allows accumulation in the nucleus? 12–8 Assuming that 32 million histone octamers are required to package the human genome, how many his-tone molecules must be transported per second per nuclear pore complex in cells whose nuclei contain 3000 nuclear pores and are dividing once per day?


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Cell_Biology_Alberts. 12–9 The nuclear pore complex (NPC) creates a barrier to the free exchange of molecules between the nucleus and cytosol, but in a way that remains mysterious. In yeast, for example, the central pore of the NPC has a diameter of 35 nm and is 30 nm long, which is somewhat smaller than its vertebrate counterpart. Even so, it is large enough to accommodate virtually all components of the cytosol. Yet the pore allows passive diffusion of molecules only up to about 40 kd; entry of anything larger requires help from a nuclear import receptor. Selective permeability is controlled by protein components of the NPC that have unstructured, polar tails extending into the central pore. These tails are characterized by periodic repeats of the hydrophobic amino acids phenylalanine (F) and glycine (G).


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Cell_Biology_Alberts. At high enough concentration (~50 mM), the FG-repeat domains of these proteins can form a gel, with a meshwork of interactions between the hydrophobic FG repeats (Figure Q12–2A). These gels allow passive diffusion of small molecules, but they prevent entry of larger proteins such as the fluorescent protein mCherry fused to maltose binding protein (MBP) (Figure Q12–2B). (The fusion to MBP makes mCherry too large to enter the nucleus by passive diffusion.) However, if the nuclear import receptor, importin, is fused to a similar protein, MBP-GFP, the importin-MBP-GFP fusion readily enters the gel (Figure Q12–2B).


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Cell_Biology_Alberts. Figure Q12–2 fG-repeat gel and influx of proteins into the nucleus (problem 12–9). (a) Cartoon of the meshwork (gel) formed by pairwise interactions between hydrophobic fG repeats. for fG-repeats separated by 17 amino acids, as is typical, the mesh formed by extended amino acid side chains would correspond to about 4 nm on a side, which would be large enough to account for the characteristic passive diffusion of proteins through nuclear pores. (B) Diffusion of mBp-mCherry and importin-mBp-Gfp into a gel of fG-repeats. in each group, the solution is shown at left and the gel at right. The bright areas indicate regions that contain the fluorescent proteins.


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Cell_Biology_Alberts. A. FG-repeats only form gels in vitro at relatively high concentration (50 mM). Is this concentration reasonable for FG repeats in the NPC core? In yeast, there are about 5000 FG-repeats in each NPC. Given the dimensions of the yeast nuclear pore (35 nm diameter and 30 nm length), calculate the concentration of FG-repeats in the cylindrical volume of the pore. Is this concentration comparable to the one used in vitro?


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Cell_Biology_Alberts. B. A second question is whether the diffusion of importin-MBP-GFP through the FG-repeat gel is fast enough to account for the efficient flow of materials between the nucleus and cytosol. From experiments of the type shown in Figure Q12–2B, the diffusion coefficient (D) of importin-MBP-GFP through the FG-repeat gel was determined to be about 0.1 μm2/s. The equation for diffusion is t = x2/2D, where t is time and x is distance. Calculate the time it would take importin-MBP-GFP to diffuse through a yeast nuclear pore (30 nm) if the pore consisted of a gel of FG-repeats. Does this time seem fast enough for the needs of a eukaryotic cell?


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Cell_Biology_Alberts. 12–10 Components of the TIM complexes, the multi-subunit protein translocators in the mitochondrial inner membrane, are much less abundant than those of the TOM complex. They were initially identified using a genetic trick. The yeast Ura3 gene, whose product is an enzyme that is normally located in the cytosol where it is essential for synthesis of uracil, was modified so that the protein carried an import signal for the mitochondrial matrix. A population of cells carrying the modified Ura3 gene in place of the normal gene was then grown in the absence of uracil. Most cells died, but the rare cells that grew were shown to be defective for mitochondrial import. Explain how this selection identifies cells with defects in components required for import into the mitochondrial matrix. Why don’t normal cells with the modified Ura3 gene grow in the absence of uracil? Why do cells that are defective for mitochondrial import grow in the absence of uracil?


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Cell_Biology_Alberts. 12–11 If the enzyme dihydrofolate reductase (DHFR), which is normally located in the cytosol, is engineered to carry a mitochondrial targeting sequence at its N-terminus, it is efficiently imported into mitochondria. If the modified DHFR is first incubated with methotrexate, which binds tightly to the active site, the enzyme remains in the cytosol. How do you suppose that the binding of methotrexate interferes with mitochondrial import? 12–12 Why do mitochondria need a special translocator to import proteins across the outer membrane, when the membrane already has large pores formed by porins? 12–13 Examine the multipass transmembrane protein shown in Figure Q12–3. What would you predict would be the effect of converting the first hydrophobic trans-membrane segment to a hydrophilic segment? Sketch the arrangement of the modified protein in the ER membrane.


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Cell_Biology_Alberts. Figure Q12–3 arrangement of a multipass transmembrane protein in the er membrane (problem 12–13). Blue hexagons represent covalently attached oligosaccharides. The positions of positively and negatively charged amino acids flanking the second transmembrane segment are shown. 12–14 All new phospholipids are added to the cytosolic leaflet of the ER membrane, yet the ER membrane has a symmetrical distribution of different phospholipids in its two leaflets. By contrast, the plasma membrane, which receives all its membrane components ultimately from the ER, has a very asymmetrical distribution of phospholipids in the two leaflets of its lipid bilayer. How is the symmetry generated in the ER membrane, and how is the asymmetry generated and maintained in the plasma membrane? palade G (1975) intracellular aspects of the process of protein synthesis. Science 189, 347–358. The Compartmentalization of Cells


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Cell_Biology_Alberts. palade G (1975) intracellular aspects of the process of protein synthesis. Science 189, 347–358. The Compartmentalization of Cells Blobel G (1980) intracellular protein topogenesis. Proc. Natl Acad. Sci. USA 77, 1496–1500. Devos Dp, Gräf r & field mC (2014) evolution of the nucleus. Curr. Opin. Cell Biol. 28, 8–15. Warren G & Wickner W (1996) organelle inheritance. Cell 84, 395–400. The Transport of Molecules Between the Nucleus and the Cytosol adam sa & Gerace l (1991) Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell 66, 837–847. Burke B & stewart Cl (2013) The nuclear lamins: flexibility in function. Nat. Rev. Mol. Cell Biol. 14, 13–24. Cole Cn & scarcelli JJ (2006) Transport of messenger rna from the nucleus to the cytoplasm. Curr. Opin. Cell Biol. 18, 299–306. Güttinger s, laurell e & Kutay U (2009) orchestrating nuclear envelope disassembly and reassembly during mitosis. Nat. Rev. Mol. Cell Biol. 10, 178–191.


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Cell_Biology_Alberts. Güttinger s, laurell e & Kutay U (2009) orchestrating nuclear envelope disassembly and reassembly during mitosis. Nat. Rev. Mol. Cell Biol. 10, 178–191. hetzer mW & Wente sr (2009) Border control at the nucleus: biogenesis and organization of the nuclear membrane and pore complexes. Dev. Cell 17, 606–616. hoelz a, Debler eW & Blobel G (2011) The structure of the nuclear pore complex. Annu. Rev. Biochem. 80, 613–643. hülsmann BB, labokha aa & Görlich D (2012) The permeability of reconstituted nuclear pores provides direct evidence for the selective phase model. Cell 150, 738–751. Köhler a & hurt e (2007) exporting rna from the nucleus to the cytoplasm. Nat. Rev. Mol. Cell Biol. 8, 761–773. rothballer a & Kutay U (2013) poring over pores: nuclear pore complex insertion into the nuclear envelope. Trends Biochem. Sci. 38, 292–301.


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Cell_Biology_Alberts. rothballer a & Kutay U (2013) poring over pores: nuclear pore complex insertion into the nuclear envelope. Trends Biochem. Sci. 38, 292–301. strambio-De-Castilla C, niepel m & rout mp (2010) The nuclear pore complex: bridging nuclear transport and gene regulation. Nat. Rev. Mol. Cell Biol. 11, 490–501. Tran eJ & Wente sr (2006) Dynamic nuclear pore complexes: life on the edge. Cell 125, 1041–1053. The Transport of Proteins Into Mitochondria and Chloroplasts Chacinska a, Koehler Cm, milenkovic D et al. (2009) importing mitochondrial proteins: machineries and mechanisms. Cell 138, 628–644. Jarvis p & robinson C (2004) mechanisms of protein import and routing in chloroplasts. Curr. Biol. 14, r1064–r1077. Kessler f & schnell DJ (2009) Chloroplast biogenesis: diversity and regulation of the protein import apparatus. Curr. Opin. Cell Biol. 21, 494–500. prakash s & matouschek a (2004) protein unfolding in the cell. Trends Biochem. Sci. 29, 593–600.


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Cell_Biology_Alberts. prakash s & matouschek a (2004) protein unfolding in the cell. Trends Biochem. Sci. 29, 593–600. schleiff e & Becker T (2011) Common ground for protein translocation: access control for mitochondria and chloroplasts. Nat. Rev. Mol. Cell Biol. 12, 48–59. Dimitrov l, lam sK & schekman r (2013) The role of the endoplasmic reticulum in peroxisome biogenesis. Cold Spring Harb. Perspect. Biol. 5, a013243. fujiki Y, Yagita Y & matsuzaki T (2012) peroxisome biogenesis disorders. Biochim. Biophys. Acta 1822, 1337–1342. schliebs W, Girzalsky W& erdmann r (2010) peroxisomal protein import and eraD: variations on a common theme. Nat. Rev. Mol. Cell Biol. 11, 885–890. Tabak hf, Braakman i & van der zand a (2013) peroxisome formation and maintenance are dependent on the endoplasmic reticulum. Annu. Rev. Biochem. 82, 723–744.


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Cell_Biology_Alberts. Tabak hf, Braakman i & van der zand a (2013) peroxisome formation and maintenance are dependent on the endoplasmic reticulum. Annu. Rev. Biochem. 82, 723–744. The Endoplasmic Reticulum akopian D, shen K, zhang X & shan so (2013) signal recognition particle: an essential protein-targeting machine. Annu. Rev. Biochem. 82, 693–721. Blobel G & Dobberstein B (1975) Transfer of proteins across membranes. i. presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. Cell Biol. 67, 835–851. Borgese n, mok W, Kreibich G & sabatini DD (1974) ribosomal-membrane interaction: in vitro binding of ribosomes to microsomal membranes. J. Mol. Biol. 88, 559–580. Braakman i & Bulleid nJ (2011) protein folding and modification in the mammalian endoplasmic reticulum. Annu. Rev. Biochem. 80, 71–99.


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Cell_Biology_Alberts. Braakman i & Bulleid nJ (2011) protein folding and modification in the mammalian endoplasmic reticulum. Annu. Rev. Biochem. 80, 71–99. Brodsky Jl & skach Wr (2011) protein folding and quality control in the endoplasmic reticulum: recent lessons from yeast and mammalian cell systems. Curr. Opin. Cell Biol. 23, 464–475. Chen s, novick p & ferro-novick s (2013) er structure and function. Curr. Opin. Cell Biol. 25, 428–433. Clark mr (2011) flippin’ lipids. Nat. Immunol. 12, 373–375. Daleke Dl (2003) regulation of transbilayer plasma membrane phospholipid asymmetry. J. Lipid Res. 44, 233–242. Deshaies rJ, sanders sl, feldheim Da & schekman r (1991) assembly of yeast sec proteins involved in translocation into the endoplasmic reticulum into a membrane-bound multisubunit complex. Nature 349, 806–808. Gething mJ (1999) role and regulation of the er chaperone Bip. Semin. Cell Dev. Biol. 10, 465–472.


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Cell_Biology_Alberts. Gething mJ (1999) role and regulation of the er chaperone Bip. Semin. Cell Dev. Biol. 10, 465–472. Görlich D, prehn s, hartmann e et al. (1992) a mammalian homolog of seC61p and seCYp is associated with ribosomes and nascent polypeptides during translocation. Cell 71, 489–503. hegde rs & ploegh hl (2010) Quality and quantity control at the endoplasmic reticulum. Curr. Opin. Cell Biol. 22, 437–446. hegde rs & Keenan rJ (2011) Tail-anchored membrane protein insertion into the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol. 12, 787–798. levine T & loewen C (2006) inter-organelle membrane contact sites: through a glass, darkly. Curr. Opin. Cell Biol. 18, 371–378. lópez-marqués rl, holthuis JCm & pomorski TG (2011) pumping lipids with p4-aTpases. Biol. Chem. 392, 67–76. mamathambika Bs & Bardwell JC (2008) Disulfide-linked protein folding pathways. Annu. Rev. Cell Dev. Biol. 24, 211–235.


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Cell_Biology_Alberts. mamathambika Bs & Bardwell JC (2008) Disulfide-linked protein folding pathways. Annu. Rev. Cell Dev. Biol. 24, 211–235. marciniak sJ & ron D (2006) endoplasmic reticulum stress signaling in disease. Physiol. Rev. 86, 1133–1149. milstein C, Brownlee GG, harrison Tm & mathews mB (1972) a possible precursor of immunoglobulin light chains. Nat. New Biol. 239, 117–120. park e & rapoport Ta (2012) mechanisms of sec61/secY-mediated protein translocation across membranes. Annu. Rev. Biophys. 41, 21–40. römisch K (2005) endoplasmic reticulum-associated degradation. Annu. Rev. Cell Dev. Biol. 21, 435–456. rowland aa & Voeltz GK (2012) endoplasmic reticulum-mitochondria contacts: function of the junction. Nat. Rev. Mol. Cell Biol. 13, 607–625. Trombetta es & parodi aJ (2003) Quality control and protein folding in the secretory pathway. Annu. Rev. Cell Dev. Biol. 19, 649–676.


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Cell_Biology_Alberts. Trombetta es & parodi aJ (2003) Quality control and protein folding in the secretory pathway. Annu. Rev. Cell Dev. Biol. 19, 649–676. Tsai B, Ye Y & rapoport Ta (2002) retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat. Rev. Mol. Cell Biol. 3, 246–255. Walter p & ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334, 1081–1086. von heijne G (2011) introduction to theme “membrane protein folding and insertion”. Annu. Rev. Biochem. 80, 157–160.


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Cell_Biology_Alberts. Every cell must eat, communicate with the world around it, and quickly respond to changes in its environment. To help accomplish these tasks, cells continually adjust the composition of their plasma membrane and internal compartments in rapid response to need. They use an elaborate internal membrane system to add and remove cell-surface proteins, such as receptors, ion channels, and transporters (Figure 13–1). Through the process of exocytosis, the secretory pathway delivers newly synthesized proteins, carbohydrates, and lipids either to the plasma membrane or the extracellular space. By the converse process of endocytosis, cells remove plasma membrane components and deliver them to internal compartments called endosomes, from where they can be recycled to the same or different regions of the plasma membrane or be delivered to lysosomes for degradation. Cells also use endocytosis to capture important nutrients, such as vitamins, cholesterol, and iron; these are taken up together with


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Cell_Biology_Alberts. the plasma membrane or be delivered to lysosomes for degradation. Cells also use endocytosis to capture important nutrients, such as vitamins, cholesterol, and iron; these are taken up together with the macromolecules to which they bind and are then moved on to endosomes and lysosomes, from where they can be transported into the cytosol for use in various biosynthetic processes.


Cell_Biology_Alberts

Cell_Biology_Alberts. The interior space, or lumen, of each membrane-enclosed compartment along the secretory and endocytic pathways is equivalent to the lumen of most other membrane-enclosed compartments and to the cell exterior, in the sense that proteins can travel in this space without having to cross a membrane as they are passed from one compartment to another by means of numerous membrane-enclosed transport containers. These containers are formed from the donor compartment and are either small, spherical vesicles, larger irregular vesicles, or tubules. We shall use the term transport vesicle to apply to all forms of these containers.


Cell_Biology_Alberts

Cell_Biology_Alberts. Within a eukaryotic cell, transport vesicles continually bud off from one membrane and fuse with another, carrying membrane components and soluble lumenal molecules, which are referred to as cargo (Figure 13–2). This vesicular traffic flows along highly organized, directional routes, which allow the cell to secrete, eat, and remodel its plasma membrane and organelles. The secretory pathway leads outward from the endoplasmic reticulum (ER) toward the Golgi apparatus and cell surface, with a side route leading to lysosomes, while the endocytic pathway leads inward from the plasma membrane. In each case, retrieval pathways The Molecular MechanIsMs of MeMbrane TransporT and The MaInTenance of coMparTMenTal dIversITy TransporT froM The er Through The golgI apparaTus TransporT froM The Trans golgI neTwork To lysosoMes TransporT InTo The cell froM The plasMa MeMbrane: endocyTosIs TransporT froM The Trans golgI neTwork To The cell exTerIor: exocyTosIs


Cell_Biology_Alberts

Cell_Biology_Alberts. TransporT froM The Trans golgI neTwork To lysosoMes TransporT InTo The cell froM The plasMa MeMbrane: endocyTosIs TransporT froM The Trans golgI neTwork To The cell exTerIor: exocyTosIs Figure 13–1 Exocytosis and endocytosis. (a) In exocytosis, a transport vesicle fuses with the plasma membrane. Its content is released into the extracellular space, while the vesicle membrane (red) becomes continuous with the plasma membrane. (b) In endocytosis, a plasma membrane patch (red) is internalized, forming a transport vesicle. Its content derives from the extracellular space. 696 Chapter 13: Intracellular Membrane Traffic balance the flow of membrane between compartments in the opposite direction, Figure 13–2 Vesicle transport. Transport vesicles bud off from one compartment bringing membrane and selected proteins back to the compartment of origin and fuse with another. as they do so, they (Figure 13–3).


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Cell_Biology_Alberts. carry material as cargo from the lumen To perform its function, each transport vesicle that buds from a compartment (the space within a membrane-enclosed must be selective. It must take up only the appropriate molecules and must fuse compartment) and membrane of the donor only with the appropriate target membrane. A vesicle carrying cargo from the ER compartment to the lumen and membrane of the target compartment, as shown. to the Golgi apparatus, for example, must exclude most proteins that are to stay in the ER, and it must fuse only with the Golgi apparatus and not with any other organelle. We begin this chapter by considering the molecular mechanisms of budding and fusion that underlie all vesicle transport. We then discuss the fundamental problem of how, in the face of this transport, the cell maintains the molecular and


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Cell_Biology_Alberts. Figure 13–3 A “road-map” of the secretory and endocytic pathways. (a) In this schematic roadmap, which was introduced in chapter 12, the endocytic and secretory pathways are illustrated with green and red arrows, respectively. In addition, bluearrows denote retrieval pathways for the backflow of selected components. (b) The compartments of the eukaryotic cell involved in vesicle transport. The lumen of each membrane-enclosed compartment is topologically equivalent to the outside of the cell. all compartments shown communicate with one another and the outside of the cell by means of transport vesicles. In the secretory pathway (red arrows), protein molecules are transported from the er to the plasma membrane or (via endosomes) to lysosomes. In the endocytic pathway (green arrows), molecules are ingested in endocytic vesicles derived from the plasma membrane and delivered to early endosomes and then (via late endosomes) to lysosomes. Many endocytosed molecules are retrieved from early


Cell_Biology_Alberts

Cell_Biology_Alberts. are ingested in endocytic vesicles derived from the plasma membrane and delivered to early endosomes and then (via late endosomes) to lysosomes. Many endocytosed molecules are retrieved from early endosomes and returned (some via recycling endosomes) to the cell surface for reuse; similarly, some molecules are retrieved from the early and late endosomes and returned to the golgi apparatus, and some are retrieved from the golgi apparatus and returned to the er. all of these retrieval pathways are shown with blue arrows,as in part (a).


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Cell_Biology_Alberts. functional differences between its compartments. Finally, we consider the function of the Golgi apparatus, lysosomes, secretory vesicles, and endosomes, as we trace the pathways that connect these organelles. The Molecular MechanIsMs of MeMbrane TransporT and The MaInTenance of coMparTMenTal dIversITy


Cell_Biology_Alberts

Cell_Biology_Alberts. The Molecular MechanIsMs of MeMbrane TransporT and The MaInTenance of coMparTMenTal dIversITy Vesicle transport mediates a continuous exchange of components between the ten or more chemically distinct, membrane-enclosed compartments that collectively comprise the secretory and endocytic pathways. With this massive exchange, how can each compartment maintain its special identity? To answer this question, we must first consider what defines the character of a compartment. Above all, it is the composition of the enclosing membrane: molecular markers displayed on the cytosolic surface of the membrane serve as guidance cues for incoming traffic to ensure that transport vesicles fuse only with the correct compartment. Many of these membrane markers, however, are found on more than one compartment, and it is the specific combination of marker molecules that gives each compartment its molecular address.


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Cell_Biology_Alberts. How are these membrane markers kept at high concentration on one compartment and at low concentration on another? To answer this question, we need to consider how patches of membrane, enriched or depleted in specific membrane components, bud off from one compartment and transfer to another. We begin by discussing how cells segregate proteins into separate membrane domains by assembling a special protein coat on the membrane’s cytosolic face. We consider how coats form, what they are made of, and how they are used to extract specific cargo components from a membrane and compartment lumen for delivery to another compartment. Finally, we discuss how transport vesicles dock at the appropriate target membrane and then fuse with it to deliver their cargo. There are various Types of coated vesicles


Cell_Biology_Alberts

Cell_Biology_Alberts. There are various Types of coated vesicles Most transport vesicles form from specialized, coated regions of membranes. They bud off as coated vesicles, which have a distinctive cage of proteins covering their cytosolic surface. Before the vesicles fuse with a target membrane, they discard their coat, as is required for the two cytosolic membrane surfaces to interact directly and fuse. The coat performs two main functions that are reflected in a common two-layered structure. First, an inner coat layer concentrates specific membrane proteins in a specialized patch, which then gives rise to the vesicle membrane. In this way, the inner layer selects the appropriate membrane molecules for transport. Second, an outer coat layer assembles into a curved, basketlike lattice that deforms the membrane patch and thereby shapes the vesicle.


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Cell_Biology_Alberts. There are three well-characterized types of coated vesicles, distinguished by their major coat proteins: clathrin-coated, COPI-coated, and COPII-coated (Figure 13–4). Each type is used for different transport steps. Clathrin-coated vesicles, for example, mediate transport from the Golgi apparatus and from the plasma membrane, whereas COPIand COPII-coated vesicles most commonly mediate transport from the ER and from the Golgi cisternae (Figure 13–5). There is, however, much more variety in coated vesicles and their functions than this short list suggests. As we discuss below, there are several types of clathrin-coated vesicles, each specialized for a different transport step, and the COPIand COPII-coated vesicles may be similarly diverse. The assembly of a clathrin coat drives vesicle formation


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Cell_Biology_Alberts. The assembly of a clathrin coat drives vesicle formation Clathrin-coated vesicles, the first coated vesicles to be identified, transport material from the plasma membrane and between endosomal and Golgi compartments. COPI-coated vesicles and COPII-coated vesicles transport material early in the secretory pathway: COPI-coated vesicles bud from Golgi compartments, and COPII-coated vesicles bud from the ER (see Figure 13–5). We discuss clathrin-coated vesicles first, as they provide a good example of how vesicles form.


Cell_Biology_Alberts

Cell_Biology_Alberts. The major protein component of clathrin-coated vesicles is clathrin itself, which forms the outer layer of the coat. Each clathrin subunit consists of three large and three small polypeptide chains that together form a three-legged structure called a triskelion (Figure 13–6A,B). Clathrin triskelions assemble into a basketlike framework of hexagons and pentagons to form coated pits (buds) on the cytosolic surface of membranes (Figure 13–7). Under appropriate conditions, isolated triskelions spontaneously self-assemble into typical polyhedral cages in a test tube, even in the absence of the membrane vesicles that these baskets normally enclose (Figure 13–6C,D). Thus, the clathrin triskelions determine the geometry of the clathrin cage (Figure 13–6E).


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Cell_Biology_Alberts. Adaptor proteins, another major coat component in clathrin-coated vesicles, form a discrete inner layer of the coat, positioned between the clathrin cage and the membrane. They bind the clathrin coat to the membrane and trap various transmembrane proteins, including transmembrane receptors that capture soluble cargo molecules inside the vesicle—so-called cargo receptors. In this way, the adaptor proteins select a specific set of transmembrane proteins, together with the soluble proteins that interact with them, and package them into each newly formed clathrin-coated transport vesicle (Figure 13–8). KEY: Figure 13–4 Electron micrographs of clathrin-coated, COPI-coated, and COPII-coated vesicles. all are shown in electron micrographs at the same scale. (a clathrin-coated vesicles. (b) copIcoated vesicles and golgi cisternae (red arrows) from a cell-free system in which copI-coated vesicles bud in the test tube. copII-coated vesicles. (a and b, from l.


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Cell_Biology_Alberts. copII-coated vesicles. (a and b, from l. orci, b. glick and J. rothman, Cell 46:171–184, 1986. with permission from elsevier; c, courtesy of charles barlowe and lelio orci.) Figure 13–5 Use of different coats for different steps in vesicle traffic. different coat proteins select different cargo and shape the transport vesicles that mediate the various steps in the secretory and endocytic pathways. when the same coats function in different places in the cell, they usually incorporate different coat protein subunits that modify their properties (not shown). Many differentiated cells have additional pathways beside those shown here, including a sorting pathway from the trans golgi network to the apical surface of epithelial cells and a specialized recycling pathway for proteins of synaptic vesicles in the nerve terminals of neurons (see figure 11–36). The arrows are colored as in figure 13–3.


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Cell_Biology_Alberts. Figure 13–6 The structure of a clathrin coat. (a) electron micrograph of a clathrin triskelion shadowed with platinum.


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Cell_Biology_Alberts. each triskelion is composed of three clathrin heavy chains and three clathrin light chains, as shown in the diagram. (c and a cryoelectron micrograph taken of a clathrin coat composed of 36 triskelions organized in a network of 12 pentagons and 6 hexagons, with some heavy chains (c) and light chains (d) highlighted (Movie 13.1). The light chains link to the actin cytoskeleton, which helps generate force for membrane budding and vesicle movement, and their phosphorylation regulates clathrin coat assembly. The interwoven legs of the clathrin triskelions form an outer shell from which the n-terminal domains of the triskelions protrude inward. These domains bind to the adaptor proteins shown in figure 13–8. The coat shown was assembled biochemically from pure clathrin triskelions and is too small to enclose a membrane vesicle. (e) Images of clathrincoated vesicles isolated from bovine brain. The clathrin coats are constructed in a similar but less regular way, from pentagons, a larger


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Cell_Biology_Alberts. small to enclose a membrane vesicle. (e) Images of clathrincoated vesicles isolated from bovine brain. The clathrin coats are constructed in a similar but less regular way, from pentagons, a larger number of hexagons, and sometime heptagons, resembling the architecture of deformed soccer balls. The structures were determined by cryoelectron microscopy and tomographic reconstruction. (a, from e. ungewickell and d. branton, nature 289:420–422, 1981; c and d, from a. fotin et al., nature 432:573–579, 2004. all with permission from Macmillan publishers ltd; e, from y. cheng et al., J. Mol. Biol. 365:892–899, 2007. with permission from elsevier.)


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Cell_Biology_Alberts. There are several types of adaptor proteins. The best characterized have four different protein subunits; others are single-chain proteins. Each type of adaptor protein is specific for a different set of cargo receptors. Clathrin-coated vesicles budding from different membranes use different adaptor proteins and thus package different receptors and cargo molecules. The assembly of adaptor proteins on the membrane is tightly controlled, in part by the cooperative interaction of the adaptor proteins with other components of the coat. The adaptor protein AP2 serves as a well-understood example. When it binds to a specific phosphorylated phosphatidylinositol lipid (a phosphoinositide), it alters its conformation, exposing binding sites for cargo receptors in the membrane. The simultaneous binding to the cargo receptors and lipid head groups greatly enhances the binding of AP2 to the membrane (Figure 13–9).


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Cell_Biology_Alberts. Because several requirements must be met simultaneously to stably bind AP2 proteins to a membrane, the proteins act as coincidence detectors that only assemble at the right time and place. Upon binding, they induce membrane curvature, which makes the binding of additional AP2 proteins in its proximity more likely. The cooperative assembly of the AP2 coat layer then is further amplified by clathrin binding, which leads to the formation and budding of a transport vesicle. Adaptor proteins found in other coats also bind to phosphoinositides, which not only have a major role in directing when and where coats assemble in the cell, but also are used much more widely as molecular markers of compartment identity. This helps to control vesicular traffic, as we now discuss.


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Cell_Biology_Alberts. Figure 13–7 Clathrin-coated pits and vesicles. This rapid-freeze, deep- on the inner surface of the plasma membrane of cultured fibroblasts. The cells were rapidly frozen in liquid helium, fractured, and deep-etched to expose the cytoplasmic surface of the plasma membrane. (courtesy of John heuser.) 0.2 µm