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. Protein Sorting and Transport - The Endoplasmic Reticulum, Golgi Apparatus, and Lysosomes

In addition to the presence of a nucleus, eukaryotic cells are distinguished from prokaryotic cells by the presence of membrane-enclosed organelles within their cytoplasm. These organelles provide discrete compartments in which specific cellular activities take place, and the resulting subdivision of the cytoplasm allows eukaryotic cells to function efficiently in spite of their large size (about a thousand times the volume of bacteria).

Because of the complex internal organization of eukaryotic cells, the sorting and targeting of proteins to their appropriate destinations are considerable tasks. The first step of protein sorting takes place while translation is still in progress. Many proteins destined for the endoplasmic reticulum, the Golgi apparatus, lysosomes, the plasma membrane, and secretion from the cell are synthesized on ribosomes that are bound to the membrane of the endoplasmic reticulum. As translation proceeds, the polypeptide chains are transported into the endoplasmic reticulum, where protein folding and processing take place. From the endoplasmic reticulum, proteins are transported in vesicles to the Golgi apparatus, where they are further processed and sorted for transport to lysosomes, the plasma membrane, or secretion from the cell. The endoplasmic reticulum, Golgi apparatus, and lysosomes are thus distinguished from other cytoplasmic organelles by their common involvement in protein processing and connection by vesicular transport.

The Endoplasmic Reticulum

The endoplasmic reticulum (ER) is a network of membrane-enclosed tubules and sacs (cisternae) that extends from the nuclear membrane throughout the cytoplasm (Figure 9.1).

The entire endoplasmic reticulum is enclosed by a continuous membrane and is the largest organelle of most eukaryotic cells. Its membrane may account for about half of all cell membranes, and the space enclosed by the ER (the lumen, or cisternal space) may represent about 10% of the total cell volume. As discussed below, there are two distinct types of ER that perform different functions within the cell. The rough ER, which is covered by ribosomes on its outer surface, functions in protein processing. The smooth ER is not associated with ribosomes and is involved in lipid, rather than protein, metabolism.

The Endoplasmic Reticulum and Protein Secretion

The role of the endoplasmic reticulum in protein processing and sorting was first demonstrated by George Palade and his colleagues in the 1960s (Figure 9.2).

Figure 9.2. The secretory pathway Pancreatic acinar cells, which secrete most of their newly synthesized proteins into the digestive tract, were labeled with radioactive amino acids to study the intracellular pathway taken by secreted proteins. After a short incubation with radioactive amino acids (3-minute label), autoradiography revealed that newly synthesized proteins were localized to the rough ER. Following further incubation with nonradioactive amino acids (a chase), proteins were found to move from the ER to the Golgi apparatus and then, within secretory vesicles, to the plasma membrane and cell exterior.

These investigators studied the fate of newly synthesized proteins in specialized cells of the pancreas (pancreatic acinar cells) that secrete digestive enzymes into the small intestine. Because most proteins synthesized by these cells are secreted, Palade and coworkers were able to study the pathway taken by secreted proteins simply by labeling newly synthesized proteins with radioactive amino acids. The location of the radiolabeled proteins within the cell was then determined by autoradiography, revealing the cellular sites involved in the events leading to protein secretion. After a brief exposure of pancreatic acinar cells to radioactive amino acids, newly synthesized proteins were detected in the rough ER, which was therefore identified as the site of synthesis of proteins destined for secretion. If the cells were then incubated for a short time in media containing nonradioactive amino acids (a process known as a chase), the radiolabeled proteins were detected in the Golgi apparatus. Following longer chase periods, the radiolabeled proteins traveled from the Golgi apparatus to the cell surface in secretory vesicles, which then fused with the plasma membrane to release their contents outside of the cell.

These experiments defined a pathway taken by secreted proteins, the secretory pathway: rough ER [pic]Golgi [pic]secretory vesicles [pic]cell exterior. Further studies extended these results and demonstrated that this pathway is not restricted to proteins destined for secretion from the cell. Plasma membrane and lysosomal proteins also travel from the rough ER to the Golgi and then to their final destinations. Still other proteins travel through the initial steps of the secretory pathway but are then retained and function within either the ER or the Golgi apparatus.

The entrance of proteins into the ER thus represents a major branch point for the traffic of proteins within eukaryotic cells. Proteins destined for secretion or incorporation into the ER, Golgi apparatus, lysosomes, or plasma membrane are initially targeted to the ER. In mammalian cells, most proteins are transferred into the ER while they are being translated on membrane-bound ribosomes (Figure 9.3).

Figure 9.3. Overview of protein sorting In mammalian cells, the initial sorting of proteins to the ER takes place while translation is in progress. Proteins synthesized on free ribosomes either remain in the cytosol or are transported to the nucleus, mitochondria, chloroplasts, or peroxisomes. In contrast, proteins synthesized on membrane-bound ribosomes are translocated into the ER while their translation is in progress. They may be either retained within the ER or transported to the Golgi apparatus and, from there, to lysosomes, the plasma membrane, or the cell exterior via secretory vesicles.

In contrast, proteins destined to remain in the cytosol or to be incorporated into the nucleus, mitochondria, chloroplasts, or peroxisomes are synthesized on free ribosomes and released into the cytosol when their translation is complete.

Targeting Proteins to the Endoplasmic Reticulum

Proteins can be translocated into the ER either during their synthesis on membrane-bound ribosomes (cotranslational translocation) or after their translation has been completed on free ribosomes in the cytosol (posttranslational translocation). In mammalian cells, most proteins enter the ER co-translationally, whereas both cotranslational and posttranslational pathways are used in yeast. The first step in the cotranslational pathway is the association of ribosomes with the ER. Ribosomes are targeted for binding to the ER membrane by the amino acid sequence of the polypeptide chain being synthesized, rather than by intrinsic properties of the ribosome itself. Free and membrane-bound ribosomes are functionally indistinguishable, and all protein synthesis initiates on ribosomes that are free in the cytosol. Ribosomes engaged in the synthesis of proteins that are destined for secretion are then targeted to the endoplasmic reticulum by a signal sequence at the amino terminus of the growing polypeptide chain. These signal sequences are short stretches of hydrophobic amino acids that are cleaved from the polypeptide chain during its transfer into the ER lumen.

The general role of signal sequences in targeting proteins to their appropriate locations within the cell was first elucidated by studies of the import of secretory proteins into the ER. These experiments used in vitro preparations of rough ER, which were isolated from cell extracts by density-gradient centrifugation (Figure 9.4).

Figure 9.4. Isolation of rough ER When cells are disrupted, the ER fragments into small vesicles called microsomes. The microsomes derived from the rough ER (rough microsomes) are lined with ribosomes on their outer surface. Because ribosomes contain large amounts of RNA, the rough microsomes are denser than smooth microsomes and can be isolated by equilibrium density-gradient centrifugation.

When cells are disrupted, the ER breaks up into small vesicles called microsomes. Because the vesicles derived from the rough ER are covered with ribosomes, they can be separated from similar vesicles derived from the smooth ER or from other membranes (e.g., the plasma membrane). In particular, the large amount of RNA within ribosomes increases the density of the membrane vesicles to which they are attached, allowing purification of vesicles derived from the rough ER (rough microsomes) by equilibrium centrifugation in density gradients.

David Sabatini and Günter Blobel first proposed in 1971 that the signal for ribosome attachment to the ER was an amino acid sequence near the amino terminus of the growing polypeptide chain. This hypothesis was supported by the results of in vitro translation of mRNAs encoding secreted proteins, such as immunoglobulins. If an mRNA encoding a secreted protein was translated on free ribosomes in vitro, it was found that the protein produced was slightly larger than the normal secreted protein. If microsomes were added to the system, however, the in vitro-translated protein was incorporated into the microsomes and cleaved to the correct size. These experiments led to a more detailed formulation of the signal hypothesis, which proposed that an amino-terminal leader sequence targets the polypeptide chain to the microsomes and is then cleaved by a microsomal protease. Many subsequent findings have substantiated this model, including recombinant DNA experiments demonstrating that addition of a signal sequence to a normally nonsecreted protein is sufficient to direct the incorporation of the recombinant protein into the rough ER.

The mechanism by which secretory proteins are targeted to the ER during their translation (the cotranslational pathway) is now well understood. The signal sequences span about 20 amino acids, including a stretch of hydrophobic residues, usually at the amino terminus of the polypeptide chain (Figure 9.6).

Figure 9.6. The signal sequence of growth hormone Most signal sequences contain a stretch of hydrophobic amino acids, preceded by basic residues (e.g., arginine).

As they emerge from the ribosome, signal sequences are recognized and bound by a signal recognition particle (SRP) consisting of six polypeptides and a small cytoplasmic RNA (7SL RNA). SRP binds the ribosome as well as the signal sequence, inhibiting further translation and targeting the entire complex (the SRP, ribosome, and growing polypeptide chain) to the rough ER by binding to the SRP receptor on the ER membrane (Figure 9.7).

Figure 9.7. Cotranslational targeting of secretory proteins to the ER Step 1: As the signal sequence emerges from the ribosome, it is recognized and bound by the signal recognition particle (SRP). Step 2: The SRP escorts the complex to the ER membrane, where it binds to the SRP receptor. Step 3: The SRP is released, the ribosome binds to a membrane translocation complex of Sec61 proteins, and the signal sequence is inserted into a membrane channel. Step 4: Translation resumes, and the growing polypeptide chain is translocated across the membrane. Step 5: Cleavage of the signal sequence by signal peptidase releases the polypeptide into the lumen of the ER.

Binding to the receptor releases the SRP from both the ribosome and the signal sequence of the growing polypeptide chain. The ribosome then binds to a protein translocation complex in the ER membrane, and the signal sequence is inserted into a membrane channel. In both yeast and mammalian cells, the translocation channels through the ER membrane are complexes of three transmembrane proteins, called the Sec61 proteins. The yeast and mammalian Sec61 proteins are closely related to the plasma membrane proteins that translocate secreted polypeptides in bacteria, demonstrating a striking conservation of the protein secretion machinery in prokaryotic and eukaryotic cells. Transfer of the ribosome from the SRP to the Sec61 complex allows translation to resume, and the growing polypeptide chain is transferred directly into the Sec61 channel and across the ER membrane as translation proceeds. Thus, the process of protein synthesis directly drives the transfer of growing polypeptide chains through the Sec61 channel and into the ER. As translocation proceeds, the signal sequence is cleaved by signal peptidase and the polypeptide is released into the lumen of the ER.

Many proteins in yeast, as well as a few proteins in mammalian cells, are targeted to the ER after their translation is complete (posttranslational translocation), rather than being transferred into the ER during synthesis on membrane-bound ribosomes. These proteins are synthesized on free cytosolic ribosomes, and their posttranslational incorporation into the ER does not require SRP. Instead, their signal sequences are recognized by distinct receptor proteins (the Sec62/63 complex) associated with the Sec61 complex in the ER membrane (Figure 9.8).

Figure 9.8. Posttranslational translocation of proteins into the ER Proteins destined for posttranslational import to the ER are synthesized on free ribosomes and maintained in an unfolded conformation by cytosolic chaperones. Their signal sequences are recognized by the Sec62/63 complex, which is associated with the Sec61 translocation channel in the ER membrane. The Sec63 protein is also associated with a chaperone protein (BiP), which acts as a molecular ratchet to drive protein translocation into the ER.

Cytosolic chaperones are required to maintain the polypeptide chains in an unfolded conformation so they can enter the Sec61 channel, and another chaperone within the ER (called BiP) is required to pull the polypeptide chain through the channel and into the ER. It appears that the binding of polypeptide chains to BiP is needed to drive the posttranslational translocation of proteins into the ER, whereas the cotranslational translocation of growing polypeptide chains is driven directly by the process of protein synthesis.

Insertion of Proteins into the ER Membrane

Proteins destined for secretion or residence within the lumen of the ER, Golgi apparatus, or lysosomes are translocated across the ER membrane and released into the lumen of the ER as already described. However, proteins destined for incorporation into the plasma membrane or the membranes of the ER, Golgi, or lysosomes are initially inserted into the ER membrane instead of being released into the lumen. From the ER membrane, they proceed to their final destination along the same pathway as that of secretory proteins: ER[pic] Golgi[pic] plasma membrane or lysosomes. These proteins are transported along this pathway as membrane components, however, rather than as soluble proteins.

Integral membrane proteins are embedded in the membrane by hydrophobic regions that span the phospholipid bilayer (see Figure 2.48). The membrane-spanning portions of these proteins are usually α-helical regions consisting of 20 to 25 hydrophobic amino acids. The formation of an α helix maximizes hydrogen bonding between the peptide bonds, and the hydrophobic amino acid side chains interact with the fatty acid tails of the phospholipids. However, different integral membrane proteins differ in how they are inserted (Figure 9.9).

Figure 9.9. Orientations of membrane proteins Integral membrane proteins span the membrane via α-helical regions of 20 to 25 hydrophobic amino acids, which can be inserted in a variety of orientations. The two proteins at left and center each span the membrane once, but they differ in whether the amino (N) or carboxy (C) terminus is on the cytosolic side. On the right is an example of a protein that has multiple membrane-spanning regions.

For example, whereas some integral membrane proteins span the membrane only once, others have multiple membrane-spanning regions. In addition, some proteins are oriented in the membrane with their amino terminus on the cytosolic side; others have their carboxy terminus exposed to the cytosol. These orientations of proteins inserted into the ER, Golgi, lysosomal, and plasma membranes are established as the growing polypeptide chains are translocated into the ER. The lumen of the ER is topologically equivalent to the exterior of the cell, so the domains of plasma membrane proteins that are exposed on the cell surface correspond to the regions of polypeptide chains that are translocated into the ER (Figure 9.10).

Figure 9.10. Topology of the secretory pathway The lumens of the endoplasmic reticulum and Golgi apparatus are topologically equivalent to the exterior of the cell. Consequently, those portions of polypeptide chains that are translocated into the ER are exposed on the cell surface following transport to the plasma membrane.

The most straightforward mode of insertion into the ER membrane results in the synthesis of transmembrane proteins oriented with their carboxy termini exposed to the cytosol (Figure 9.11).

Figure 9.11. Insertion of a membrane protein with a cleavable signal sequence and a single stop-transfer sequence The signal sequence is cleaved as the polypeptide chain crosses the membrane, so the amino terminus of the polypeptide chain is exposed in the ER lumen. However, translocation of the polypeptide chain across the membrane is halted by a transmembrane stop-transfer sequence that closes the Sec61 translocation channel and exits the channel laterally to anchor the protein in the ER membrane. Continued translation results in a membrane-spanning protein with its carboxy terminus on the cytosolic side.

These proteins have a normal amino-terminal signal sequence, which is cleaved by signal peptidase during translocation of the polypeptide chain across the ER membrane through the Sec61 channel. They are then anchored in the membrane by a second membrane-spanning α helix in the middle of the protein. This transmembrane sequence, called a stop-transfer sequence, signals closure of the Sec61 channel. Further translocation of the polypeptide chain across the ER membrane is thus blocked, so the carboxy-terminal portion of the growing polypeptide chain is synthesized in the cytosol. The transmembrane domain then exits the translocation channel laterally to enter the lipid bilayer. The insertion of these proteins in the membrane thus involves the sequential action of two distinct elements: a cleavable amino-terminal signal sequence that initiates translocation across the membrane and a transmembrane stop-transfer sequence that anchors the protein in the membrane.

Proteins can also be anchored in the ER membrane by internal signal sequences that are not cleaved by signal peptidase (Figure 9.12).

Figure 9.12. Insertion of membrane proteins with internal noncleavable signal sequences Internal noncleavable signal sequences can lead to the insertion of polypeptide chains in either orientation in the ER membrane. (A) The signal sequence directs insertion of the polypeptide such that its amino terminus is exposed on the cytosolic side. The remainder of the polypeptide chain is translocated into the ER as translation proceeds. The signal sequence is not cleaved, so it acts as a membranespanning sequence that anchors the protein in the membrane with its carboxy terminus in the lumen of the ER. (B) Other internal signal sequences are oriented to direct the transfer of the amino-terminal portion of the polypeptide across the membrane. Continued translation results in a protein that spans the ER membrane with its amino terminus in the lumen and its carboxy terminus in the cytosol. Note that this orientation is the same as that resulting from insertion of a protein that contains a cleavable signal sequence followed by a stop-transfer sequence

These internal signal sequences are recognized by the SRP and brought to the ER membrane as already discussed. Because they are not cleaved by signal peptidase, however, these signal sequences act as transmembrane α helices that exit the translocation channel and anchor proteins in the ER membrane. Importantly, internal signal sequences can be oriented so as to direct the translocation of either the amino or carboxy terminus of the polypeptide chain across the membrane. Therefore, depending on the orientation of the signal sequence, proteins inserted into the membrane by this mechanism can have either their amino or carboxy terminus exposed to the cytosol.

Proteins that span the membrane multiple times are thought to be inserted as a result of an alternating series of internal signal sequences and transmembrane stop-transfer sequences. For example, an internal signal sequence can result in membrane insertion of a polypeptide chain with its amino terminus on the cytosolic side (Figure 9.13).

Figure 9.13. Insertion of a protein that spans the membrane multiple times In this example, an internal signal sequence results in insertion of the polypeptide chain with its amino terminus on the cytosolic side of the membrane. A stop-transfer sequence then signals closure of the translocation channel, causing the polypeptide chain to form a loop within the lumen of the ER, and translation continues in the cytosol. A second internal signal sequence reopens the channel, triggering reinsertion of the polypeptide chain into the ER membrane and forming a loop in the cytosol. The process can be repeated many times, resulting in the insertion of proteins with multiple membrane-spanning regions.

If a stop-transfer sequence is then encountered, the polypeptide will form a loop in the ER lumen, and protein synthesis will continue on the cytosolic side of the membrane. If a second signal sequence is encountered, the growing polypeptide chain will again be inserted into the ER, forming another looped domain on the cytosolic side of the membrane. This can be followed by yet another stop-transfer sequence and so forth, so that an alternating series of signal and stop-transfer sequences can result in the insertion of proteins that span the membrane multiple times, with looped domains exposed on both the lumenal and cytosolic sides.

Protein Folding and Processing in the ER

The folding of polypeptide chains into their correct three-dimensional conformations, the assembly of polypeptides into multisubunit proteins, and the covalent modifications involved in protein processing were discussed in Chapter 7. For proteins that enter the secretory pathway, many of these events occur either during translocation across the ER membrane or within the ER lumen. One such processing event is the proteolytic cleavage of the signal peptide as the polypeptide chain is translocated across the ER membrane. The ER is also the site of protein folding, assembly of multisubunit proteins, disulfide bond formation, the initial stages of glycosylation, and the addition of glycolipid anchors to some plasma membrane proteins. Indeed, the primary role of lumenal ER proteins is to catalyze the folding and assembly of newly translocated polypeptides.

As already discussed, proteins are translocated across the ER membrane as unfolded polypeptide chains while their translation is still in progress. These polypeptides, therefore, fold into their three-dimensional conformations within the ER, assisted by the molecular chaperones that facilitate the folding of polypeptide chains. For example, one of the major proteins within the ER lumen is a member of the Hsp70 family of chaperones called BiP. BiP is thought to bind to the unfolded polypeptide chain as it crosses the membrane and then mediates protein folding and the assembly of multisubunit proteins within the ER (Figure 9.14).

Figure 9.14. Protein folding in the ER The molecular chaperone BiP binds to polypeptide chains as they cross the ER membrane and facilitates protein folding and assembly within the ER.

Correctly assembled proteins are released from BiP and are available for transport to the Golgi apparatus. Abnormally folded or improperly assembled proteins, however, remain bound to BiP and are consequently retained within the ER or degraded, rather than being transported farther along the secretory pathway.

The formation of disulfide bonds between the side chains of cysteine residues is an important aspect of protein folding and assembly within the ER. These bonds do not form in the cytosol, which is characterized by a reducing environment that maintains cysteine residues in their reduced ([pic]SH) state. In the ER, however, an oxidizing environment promotes disulfide (S[pic]S) bond formation, and disulfide bonds formed in the ER play important roles in the structure of secreted and cell surface proteins. Disulfide bond formation is facilitated by the enzyme protein disulfide isomerase (see Figure 7.21), which is located in the ER lumen.

Figure 7.21. The action of protein disulfide isomerase Protein disulfide isomerase (PDI) catalyzes the breakage and rejoining of disulfide bonds, resulting in exchanges between paired disulfides in a polypeptide chain. The enzyme forms a disulfide bond with a cysteine residue of the polypeptide and then exchanges its paired disulfide with another cysteine residue. In this example, PDI catalyzes the conversion of two incorrect disulfide bonds (1-2 and 3-4) to the correct pairing (1-3 and 2-4).

Proteins are also glycosylated on specific asparagine residues (N-linked glycosylation) within the ER while their translation is still in process (Figure 9.15).

Figure 9.15. Protein glycosylation in the ER

As discussed in Chapter 7, oligosaccharide units consisting of 14 sugar residues are added to acceptor asparagine residues of growing polypeptide chains as they are translocated into the ER. The oligosaccharide is synthesized on a lipid (dolichol) carrier anchored in the ER membrane. It is then transferred as a unit to acceptor asparagine residues in the consensus sequence Asn-X-Ser/Thr by a membrane-bound enzyme called oligosaccharyl transferase. Four sugar residues (three glucose and one mannose) are removed while the protein is still within the ER, and the protein is modified further after being transported to the Golgi apparatus. Some proteins are anchored in the plasma membrane by glycolipids rather than by membrane-spanning regions of the polypeptide chain. Because these membrane-anchoring glycolipids contain phosphatidylinositol, they are called glycosylphosphatidylinositol (GPI) anchors, the structure of which was illustrated in Figure 7.32.

Figure 7.32. Structure of a GPI anchor The GPI anchor, attached to the C terminus, anchors the protein in the plasma membrane. The anchor is joined to the C-terminal amino acid by an ethanolamine, which is linked to an oligosaccharide that consists of mannose, N-acetylgalactosamine, and glucosamine residues. The oligosaccharide is in turn joined to the inositol head group of phosphatidylinositol. The two fatty acid chains of the lipid are embedded in the plasma membrane. The GPI anchor shown here is that of a rat protein, Thy-1.

The GPI anchors are assembled in the ER membrane. They are then added immediately after completion of protein synthesis to the carboxy terminus of some proteins anchored in the membrane by a C-terminal membrane-spanning region (Figure 9.16). The transmembrane region of the protein is exchanged for the GPI anchor, so these proteins remain attached to the membrane only by their associated glycolipid. Like transmembrane proteins, they are transported to the cell surface as membrane components via the secretory pathway. Their orientation within the ER dictates that GPI-anchored proteins are exposed on the outside of the cell, with the GPI anchor mediating their attachment to the plasma membrane.

Figure 9.16. Addition of GPI anchors Glycosylphosphatidylinositol (GPI) anchors contain two fatty acid chains, an oligosaccharide portion consisting of inositol and other sugars, and ethanolamine (see Figure 7.32 for a more detailed structure). The GPI anchors are assembled in the ER and added to polypeptides anchored in the membrane by a carboxy-terminal membrane- spanning region. The membranespanning region is cleaved, and the new carboxy terminus is joined to the NH2 group of ethanolamine immediately after translation is completed, leaving the protein attached to the membrane by the GPI anchor.

Export of Proteins and Lipids from the ER

Both proteins and lipids travel along the secretory pathway in transport vesicles, which bud from the membrane of one organelle and then fuse with the membrane of another. Thus, molecules are exported from the ER in vesicles that bud from the ER and carry their cargo first to the ER-Golgi intermediate compartment and then to the Golgi apparatus (Figure 9.20).

Figure 9.20. Vesicular transport from the ER to the Golgi Proteins and lipids are carried from the ER to the Golgi in transport vesicles that bud from the membrane of the ER and then fuse to form the vesicles and tubules of the ER-Golgi intermediate compartment (ERGIC). Lumenal ER proteins are taken up by the vesicles and released into the lumen of the Golgi. Membrane proteins maintain the same orientation in the Golgi as in the ER.

Subsequent steps in the secretory pathway involve vesicular transport between different compartments of the Golgi and from the Golgi to lysosomes or the plasma membrane. In each case, proteins within the lumen of one organelle are packaged into the budding transport vesicle and then released into the lumen of the recipient organelle following vesicle fusion. Membrane proteins and lipids are transported similarly, and it is noteworthy that their topological orientation is maintained as they travel from one membrane-enclosed organelle to another. For example, the domains of a protein exposed on the cytosolic side of the ER membrane will also be exposed on the cytosolic side of the Golgi and plasma membranes, whereas protein domains exposed on the lumenal side of the ER membrane will be exposed on the lumenal side of the Golgi and on the exterior of the cell (see Figure 9.10).

While most proteins travel from the ER to the Golgi, some proteins must be retained within the ER rather than proceeding along the secretory pathway. In particular, proteins that function within the ER (including BiP, signal peptidase, protein disulfide isomerase, and other enzymes discussed earlier) must be retained within that organelle. Export to the Golgi versus retention in the ER is thus the first branch point encountered by proteins being sorted to their correct destinations in the secretory pathway. Similar branch points arise at each subsequent stage of transport, such as retention in the Golgi versus export to lysosomes or the plasma membrane. In each case, specific localization signals target proteins to their correct intracellular destinations.

The distinction between proteins exported from and those retained in the ER appears to be governed by two distinct types of targeting sequences that specifically mark proteins as either (1) destined for transport to the Golgi or (2) destined for retention in the ER. Many proteins are retained in the ER lumen as a result of the presence of the targeting sequence Lys-Asp-Glu-Leu (KDEL, in the single-letter code) at their carboxy terminus. If this sequence is deleted from a protein that is normally retained in the ER (e.g., BiP), the mutated protein is instead transported to the Golgi and secreted from the cell. Conversely, addition of the KDEL sequence to the carboxy terminus of proteins that are normally secreted causes them to be retained in the ER. The retention of some transmembrane proteins in the ER is similarly dictated by short C-terminal sequences that contain two lysine residues (KKXX sequences).

Interestingly, the KDEL and KKXX signals do not prevent soluble ER proteins from being packaged into vesicles and carried to the Golgi. Instead, these signals cause resident ER proteins to be selectively retrieved from the ER-Golgi intermediate compartment or the Golgi complex and returned to the ER via a recycling pathway (Figure 9.21).

Figure 9.21. Retrieval of resident ER proteins Proteins destined to remain in the lumen of the ER are marked by the sequence Lys-Asp-Glu-Leu (KDEL) at their carboxy terminus. These proteins are exported from the ER to the Golgi in the nonselective bulk flow of proteins through the secretory pathway, but they are recognized by a receptor in the ER-Golgi intermediate compartment (ERGIC) or the Golgi apparatus and selectively returned to the ER.

Proteins bearing the KDEL and KKXX sequences appear to bind to specific recycling receptors in the membranes of these compartments and are then selectively transported back to the ER.

The action of the KDEL and KKXX sequences as retention/retrieval signals indicates that there is a nonselective bulk flow of proteins through the secretory pathway leading from the ER to the cell surface. This bulk flow from the ER to the Golgi may be responsible for the export of many proteins from the ER. However, it also appears that some proteins destined for secretion are marked by signals that actively direct their export from the ER. Protein export from the ER can thus take place not only by bulk flow, but also by a regulated pathway that specifically recognizes targeting signals that mediate selective transport of proteins to the Golgi apparatus

The Golgi Apparatus

The Golgi apparatus, or Golgi complex, functions as a factory in which proteins received from the ER are further processed and sorted for transport to their eventual destinations: lysosomes, the plasma membrane, or secretion. In addition, as noted earlier, glycolipids and sphingomyelin are synthesized within the Golgi. In plant cells, the Golgi apparatus further serves as the site at which the complex polysaccharides of the cell wall are synthesized. The Golgi apparatus is thus involved in processing the broad range of cellular constituents that travel along the secretory pathway.

Organization of the Golgi

Morphologically the Golgi is composed of flattened membrane-enclosed sacs (cisternae) and associated vesicles. A striking feature of the Golgi apparatus is its distinct polarity in both structure and function. Proteins from the ER enter at its cis face (entry face), which is convex and usually oriented toward the nucleus. They are then transported through the Golgi and exit from its concave trans face (exit face). As they pass through the Golgi, proteins are modified and sorted for transport to their eventual destinations within the cell.

Figure 9.23. Regions of the Golgi apparatus Vesicles from the ER fuse to form the ER-Golgi intermediate compartment, and proteins from the ER are then transported to the cis Golgi network. Resident ER proteins are returned from the ER-Golgi intermediate compartment and the cis Golgi network via the recycling pathway. The medial and trans compartments of the Golgi stack correspond to the cisternae in the middle of the Golgi complex and are the sites of most protein modifications. Proteins are then carried to the trans Golgi network, where they are sorted for transport to the plasma membrane, secretion, or lysosomes.

Distinct processing and sorting events appear to take place in an ordered sequence within different regions of the Golgi complex, so the Golgi is usually considered to consist of multiple discrete compartments. Although the number of such compartments has not been established, the Golgi is most commonly viewed as consisting of four functionally distinct regions: the cisGolgi network, the Golgi stack (which is divided into the medial and trans subcompartments), and the transGolgi network (Figure 9.23). Proteins from the ER are transported to the ER-Golgi intermediate compartment and then enter the Golgi apparatus at the cis Golgi network. They then progress to the medial and trans compartments of the Golgi stack, within which most metabolic activities of the Golgi apparatus take place. The modified proteins, lipids, and polysaccharides then move to the trans Golgi network, which acts as a sorting and distribution center, directing molecular traffic to lysosomes, the plasma membrane, or the cell exterior.

Although the Golgi apparatus was first described over 100 years ago, the mechanism by which proteins move through the Golgi apparatus has still not been established and is an area of controversy among cell biologists. One possibility is that transport vesicles carry proteins between the cisternae of the Golgi compartments. However, there is considerable experimental support for an alternative model proposing that proteins are simply carried through compartments of the Golgi within the Golgi cisternae, which gradually mature and progressively move through the Golgi in the cis to trans direction.

Protein Glycosylation within the Golgi

Protein processing within the Golgi involves the modification and synthesis of the carbohydrate portions of glycoproteins. One of the major aspects of this processing is the modification of the N-linked oligosaccharides that were added to proteins in the ER. As discussed earlier in this chapter, proteins are modified within the ER by the addition of an oligosaccharide consisting of 14 sugar residues (see Figure 9.15). Three glucose residues and one mannose are then removed while the polypeptides are still in the ER. Following transport to the Golgi apparatus, the N-linked oligosaccharides of these glycoproteins are subject to extensive further modifications.

Figure 9.24. Processing of N-linked oligosaccharides in the Golgi The N-linked oligosaccharides of glycoproteins transported from the ER are further modified by an ordered sequence of reactions in the Golgi.

N-linked oligosaccharides are processed within the Golgi apparatus in an ordered sequence of reactions (Figure 9.24). The first modification of proteins destined for secretion or for the plasma membrane is the removal of three additional mannose residues. This is followed by the sequential addition of an N-acetylglucosamine, the removal of two more mannoses, and the addition of a fucose and two more N-acetylglucosamines. Finally, three galactose and three sialic acid residues are added. As noted in Chapter 7, different glycoproteins are modified to different extents during their passage through the Golgi, depending on both the structure of the protein and on the amount of processing enzymes that are present within the Golgi complexes of different types of cells. Consequently, proteins can emerge from the Golgi with a variety of different N-linked oligosaccharides.

The processing of the N-linked oligosaccharide of lysosomal proteins differs from that of secreted and plasma membrane proteins. Rather than the initial removal of three mannose residues, proteins destined for incorporation into lysosomes are modified by mannose phosphorylation. In the first step of this reaction, N-acetylglucosamine phosphates are added to specific mannose residues, probably while the protein is still in the cis Golgi network (Figure 9.25).

Figure 9.25. Targeting of lysosomal proteins by phosphorylation of mannose residues Proteins destined for incorporation into lysosomes are specifically recognized and modified by the addition of phosphate groups to the 6 position of mannose residues. In the first step of the reaction, N-acetylglucosamine phosphates are transferred to mannose residues from UDP-N-acetylglucosamine. The N-acetylglucosamine groups are then removed, leaving mannose-6-phosphates.

This is followed by removal of the N-acetylglucosamine group, leaving mannose-6-phosphate residues on the N-linked oligosaccharide. Because of this modification, these residues are not removed during further processing. Instead, these phosphorylated mannose residues are specifically recognized by a mannose-6-phosphate receptor in the trans Golgi network, which directs the transport of these proteins to lysosomes.

The phosphorylation of mannose residues is thus a critical step in sorting lysosomal proteins to their correct intracellular destination. The specificity of this process resides in the enzyme that catalyzes the first step in the reaction sequence[pic]the selective addition of N-acetylglucosamine phosphates to lysosomal proteins. This enzyme recognizes a structural determinant that is present on lysosomal proteins but not on proteins destined for the plasma membrane or secretion. This recognition determinant is not a simple sequence of amino acids; rather, it is formed in the folded protein by the juxtaposition of amino acid sequences from different regions of the polypeptide chain. In contrast to the signal sequences that direct protein translocation to the ER, the recognition determinant that leads to mannose phosphorylation, and thus ultimately targets proteins to lysosomes, depends on the three-dimensional conformation of the folded protein. Such determinants are called signal patches, in contrast to the linear targeting signals discussed earlier in this chapter.

Figure 7.28. Examples of O-linked oligosaccharides O-linked oligosaccharides usually consist of only a few carbohydrate residues, which are added one sugar at a time.

Proteins can also be modified by the addition of carbohydrates to the side chains of acceptor serine and threonine residues within specific sequences of amino acids (O-linked glycosylation) (see Figure 7.28). These modifications take place in the Golgi apparatus by the sequential addition of single sugar residues. The serine or threonine is usually linked directly to N-acetylgalactosamine, to which other sugars can then be added. In some cases, these sugars are further modified by the addition of sulfate groups.

Protein Sorting and Export from the Golgi Apparatus

Proteins, as well as lipids and polysaccharides, are transported from the Golgi apparatus to their final destinations through the secretory pathway. This involves the sorting of proteins into different kinds of transport vesicles, which bud from the trans Golgi network and deliver their contents to the appropriate cellular locations (Figure 9.27).

Figure 9.27. Transport from the Golgi apparatus Proteins are sorted in the trans Golgi network and transported in vesicles to their final destinations. In the absence of specific targeting signals, proteins are carried to the plasma membrane by constitutive secretion. Alternatively, proteins can be diverted from the constitutive secretion pathway and targeted to other destinations, such as lysosomes or regulated secretion from the cells.

Some proteins are carried from the Golgi to the plasma membrane by a constitutive secretory pathway, which accounts for the incorporation of new proteins and lipids into the plasma membrane, as well as for the continuous secretion of proteins from the cell. Other proteins are transported to the cell surface by a distinct pathway of regulated secretion or are specifically targeted to other intracellular destinations, such as lysosomes in animal cells or vacuoles in yeast.

Proteins that function within the Golgi apparatus must be retained within that organelle, rather than being transported along the secretory pathway. In contrast to the ER, all of the proteins retained within the Golgi complex are associated with the Golgi membrane rather than being soluble proteins within the lumen. The signals responsible for retention of some proteins within the Golgi have been localized to their transmembrane domains, which retain proteins within the Golgi apparatus by preventing them from being packaged in the transport vesicles that leave the trans Golgi network. In addition, like the KKXX sequences of resident ER membrane proteins, signals in the cytoplasmic tails of some Golgi proteins mediate the retrieval of these proteins from subsequent compartments along the secretory pathway.

The constitutive secretory pathway, which operates in all cells, leads to continual unregulated protein secretion. However, some cells also possess a distinct regulated secretory pathway in which specific proteins are secreted in response to environmental signals. Examples of regulated secretion include the release of hormones from endocrine cells, the release of neurotransmitters from neurons, and the release of digestive enzymes from the pancreatic acinar cells discussed at the beginning of this chapter (see Figure 9.2). Proteins are sorted into the regulated secretory pathway in the trans Golgi network, where they are packaged into specialized secretory vesicles. These secretory vesicles, which are larger than other transport vesicles, store their contents until specific signals direct their fusion with the plasma membrane. For example, the digestive enzymes produced by pancreatic acinar cells are stored in secretory vesicles until the presence of food in the stomach and small intestine triggers their secretion. The sorting of proteins into the regulated secretory pathway appears to involve the recognition of signal patches shared by multiple proteins that enter this pathway. These proteins selectively aggregate in the trans Golgi network and are then released by budding as secretory vesicles.

A further complication in the transport of proteins to the plasma membrane arises in many epithelial cells, which are polarized when they are organized into tissues. The plasma membrane of such cells is divided into two separate regions, the apical domain and the basolateral domain, that contain specific proteins related to their particular functions. For example, the apical membrane of intestinal epithelial cells faces the lumen of the intestine and is specialized for the efficient absorption of nutrients; the remainder of the cell is covered by the basolateral membrane (Figure 9.28).

Figure 9.28. Transport to the plasma membrane of polarized cells The plasma membranes of polarized epithelial cells are divided into apical and basolateral domains. In this example (intestinal epithelium), the apical surface of the cell faces the lumen of the intestine, the lateral surfaces are in contact with neighboring cells, and the basal surface rests on a sheet of extracellular matrix (the basal lamina). The apical membrane is characterized by the presence of microvilli, which facilitate the absorption of nutrients by increasing surface area. Specific proteins are targeted to either the apical or basolateral membranes in the trans Golgi network. Tight junctions between neighboring cells maintain the identity of the apical and basolateral membranes by preventing the diffusion of proteins between these domains.

Distinct domains of the plasma membrane are present not only in epithelial cells, but also in other cell types. Thus, the constitutive secretory pathway must selectively transport proteins from the trans Golgi network to these distinct domains of the plasma membrane. This is accomplished by the selective packaging of proteins into at least two types of constitutive secretory vesicles that leave the trans Golgi network targeted specifically for either the apical or basolateral plasma membrane domains of the cell.

The best-characterized pathway of protein sorting in the Golgi is the selective transport of proteins to lysosomes. As already discussed, lumenal lysosomal proteins are marked by mannose-6-phosphates that are formed by modification of their N-linked oligosaccharides shortly after entry into the Golgi apparatus. A specific receptor in the membrane of the trans Golgi network then recognizes these mannose-6-phosphate residues. The resulting complexes of receptor plus lysosomal enzyme are packaged into transport vesicles destined for lysosomes. Lysosomal membrane proteins are targeted by sequences in their cytoplasmic tails, rather than by mannose-6-phosphates.

In yeasts and plant cells, which lack lysosomes, proteins are transported from the Golgi apparatus to an additional destination: the vacuole. Vacuoles assume the functions of lysosomes in these cells as well as performing a variety of other tasks, such as the storage of nutrients and the maintenance of turgor pressure and osmotic balance. In contrast to lysosomal targeting, proteins are directed to vacuoles by short peptide sequences instead of carbohydrate markers.

The Mechanism of Vesicular Transport

As is evident from the preceding sections of this chapter, transport vesicles play a central role in the traffic of molecules between different membrane-enclosed compartments of the secretory pathway. As discussed in Chapter 12, vesicles are similarly involved in the transport of materials taken up at the cell surface. Vesicular transport is thus a major cellular activity, responsible for molecular traffic between a variety of specific membrane-enclosed compartments. The selectivity of such transport is therefore key to maintaining the functional organization of the cell. For example, lysosomal enzymes must be transported specifically from the Golgi apparatus to lysosomes[pic]not to the plasma membrane or to the ER. Some of the signals that target proteins to specific organelles, such as lysosomes, were discussed earlier in this chapter. These proteins are transported within vesicles, so the specificity of transport is based on the selective packaging of the intended cargo into vesicles that recognize and fuse only with the appropriate target membrane. Because of the central importance of vesicular transport to the organization of eukaryotic cells, understanding the molecular mechanisms that control vesicle packaging, budding, and fusion is a major area of research in cell biology.

Experimental Approaches to Understanding Vesicular Transport

Progress toward elucidating the molecular mechanisms of vesicular transport has been made by three distinct experimental approaches: (1) isolation of yeast mutants that are defective in protein transport and sorting; (2) reconstitution of vesicular transport in cell-free systems; and (3) biochemical analysis of synaptic vesicles, which are responsible for the regulated secretion of neurotransmitters by neurons. Each of these experimental systems has distinct advantages for understanding particular aspects of the transport process. Most important, however, is the fact that results from all three of these avenues of investigation have converged, indicating that similar molecular mechanisms regulate secretion in cells as different as yeasts and mammalian neurons.

As in other areas of cell biology, yeasts have proved to be advantageous in studying the secretory pathway because they are readily amenable to genetic analysis. In particular, Randy Schekman and his colleagues have pioneered the isolation of yeast mutants defective in vesicular transport. These include mutants that are defective at various stages of protein secretion (sec mutants), mutants that are unable to transport proteins to the vacuole, and mutants that are unable to retain resident ER proteins. The isolation of such mutants in yeasts led directly to the molecular cloning and analysis of the corresponding genes, thereby identifying a number of proteins involved in various steps of the secretory pathway. For example, the role of Sec61 as a major component of the protein translocation channel in the endoplasmic reticulum was discussed earlier in this chapter.

Biochemical studies of vesicular transport using reconstituted systems have complemented these genetic studies and have enabled the direct isolation of transport proteins from mammalian cells. The first cell-free transport system was developed by James Rothman and colleagues, who analyzed protein transport between compartments of the Golgi apparatus (Figure 9.30).

Figure 9.30. Reconstituted vesicular transport Golgi stacks prepared from a virus-infected mutant cell line unable to catalyze the addition of N-acetylglucosamine to N-linked oligosaccharides are mixed with Golgi stacks from a normal cell line. Because the mutant cell line is infected with a virus, the proteins it synthesizes can be specifically detected. Transport of these proteins to normal Golgi stacks is signaled by the addition of N-acetylglucosamine.

The experimental design exploited a mutant mammalian cell line that lacked the enzyme required to transfer N-acetylglucosamine residues to the N-linked oligosaccharide at an early stage of its modification in the Golgi apparatus (see Figure 9.24). Consequently, the glycoproteins produced by this mutant cell line lacked added N-acetylglucosamine units. However, if Golgi stacks isolated from the mutant cell line were incubated with stacks isolated from normal cells, N-acetylglucosamine residues were added to glycoproteins synthesized by the mutant cells. A variety of experiments established that this resulted from vesicular transport of proteins from the Golgi stacks of the mutant cell line to the Golgi stacks of normal cells, so the addition of N-acetylglucosamine provided a readily detectable marker for vesicular transport in this reconstituted system. Similar reconstituted systems have been developed to analyze transport between other compartments, including transport from the ER to the Golgi and transport from the Golgi to secretory vesicles, vacuoles, and the plasma membrane. The development of these in vitro systems has enabled biochemical studies of the transport process and functional analysis of proteins identified by mutations in yeasts, as well as direct isolation of some of the proteins involved in vesicle budding and fusion.

Critical insights into the molecular mechanisms of vesicular transport have also come from studies of synaptic transmission in neurons, which represents a highly specialized form of regulated secretion. A synapse is the junction of a neuron with another cell, which may be either another neuron or an effector, such as a muscle cell. Information is transmitted across the synapse by chemical neurotransmitters, such as acetylcholine, which are stored in synaptic vesicles. Stimulation of the transmitting neuron triggers the fusion of synaptic vesicles with the plasma membrane, causing neurotransmitters to be released and stimulating the postsynaptic neuron or effector cell. Synaptic vesicles are extremely abundant in the brain, allowing them to be purified in large amounts for biochemical analysis. Some of the proteins isolated from synaptic vesicles are closely related to proteins that have been shown to play critical roles in vesicular transport by yeast genetics and reconstitution experiments, so biochemical analysis of these proteins have provided important insights into the molecular mechanisms of vesicle fusion.

Coat Proteins and Vesicle Budding

The first step in vesicular transport is the formation of a vesicle by budding from the membrane. The cytoplasmic surfaces of transport vesicles are coated with proteins, and it appears to be the assembly of these protein coats that drives vesicle budding by distorting membrane conformation. Three kinds of coated vesicles, which appear to function in different types of vesicular transport, have been characterized. The first to be described were the clathrin-coated vesicles, which are responsible for the uptake of extracellular molecules from the plasma membrane by endocytosis as well as the transport of molecules from the trans Golgi network to lysosomes. Two other types of coated vesicles have been identified as budding from the ER and Golgi complex. These vesicles are called nonclathrin-coated or COP-coated vesicles (COP stands for coat protein). One class of these vesicles (COPII-coated vesicles) bud from the ER and carry their cargo forward along the secretory pathway, to the Golgi apparatus. In contrast, COPI-coated vesicles bud from the ER-Golgi intermediate compartment or the Golgi apparatus and function in the retrieval pathways that serve to retain resident proteins in the Golgi and ER. For example, COPI-coated vesicles transport resident ER proteins marked by the KDEL or KKXX retrieval signals back to the ER from the ER-Golgi intermediate compartment or the cis Golgi network.

The coats of clathrin-coated vesicles are composed of two types of protein complexes, clathrin and adaptor proteins, which assemble on the cytosolic side of membranes (Figure 9.31).

Figure 9.31. Incorporation of lysosomal proteins into clathrin-coated vesicles Proteins targeted for lysosomes are marked by mannose-6-phosphates, which bind to mannose-6-phosphate receptors in the trans Golgi network. The mannose-6-phosphate receptors span the Golgi membrane and serve as binding sites for cytosolic adaptor proteins, which in turn bind clathrin. Clathrins consist of three protein chains that associate with each other to form a basketlike lattice that distorts the membrane and drives vesicle budding.

Clathrin plays a structural role by assembling into a basketlike lattice structure that distorts the membrane and drives vesicle budding. The binding of clathrin to membranes is mediated by a second class of proteins, called adaptor proteins. Different adaptor proteins are responsible for the assembly of clathrin-coated vesicles at the plasma membrane and at the trans Golgi network, and it is the adaptor proteins that are involved in selecting the specific molecules to be incorporated into the vesicles. For example, the AP-1 adaptor protein involved in budding from the trans Golgi network binds to the cytosolic portion of the mannose-6-phosphate receptor, thereby directing proteins destined for lysosomes into clathrin-coated vesicles.

The coats of COPI- and COPII-coated vesicles are composed of distinct protein complexes, which function analogously to clathrin and adaptor proteins in vesicle budding. Interestingly, components of the COPI coat interact with the KKXX motif responsible for the retrieval of ER proteins from the Golgi apparatus, consistent with a role for COPI-coated vesicles in recycling from the Golgi to the ER.

The assembly of vesicle coats also requires GTP-binding proteins, which appear to regulate the binding of coat proteins to the membrane. The budding of both clathrin-coated and COPI-coated vesicles from the Golgi complex requires a GTP-binding protein called ARF (ADP-ribosylation factor), while the budding of COPII-coated vesicles from the ER requires a distinct GTP-binding protein called Sar1. The role of these proteins is illustrated by the function of ARF in assembly of COPI-coated vesicles (Figure 9.32).

Figure 9.32. Role of ARF in the formation of COP-coated vesicles ARF alternates between GTP-bound and GDP-bound states. When bound to GDP, ARF associates with the membrane of the trans Golgi network, where guanine nucleotide exchange factors (GEF) promote the exchange of the bound GDP for GTP. In its GTP-bound state, ARF promotes the binding of COPI coat protein (coatomer), leading to vesicle budding. Hydrolysis of the bound GTP then converts ARF to the GDP-bound state, leading to disassembly of the vesicle coat prior to fusion with the target membrane.

The first step in vesicle formation is the association of ARF bound to GDP with the Golgi membrane. Proteins in the Golgi membrane then stimulate the exchange of the GDP bound to ARF for GTP, and the COPI coat proteins bind to the ARF/GTP complex. Assembly of the coat is then followed by deformation of the membrane and vesicle budding. ARF then hydrolyzes its bound GTP, leading to the conversion of ARF to the GDP-bound state and the dissociation of coat proteins from the vesicle membrane.

Vesicle Fusion

The fusion of a transport vesicle with its target involves two types of events. First, the transport vesicle must specifically recognize the correct target membrane; for example, a vesicle carrying lysosomal enzymes has to deliver its cargo only to lysosomes. Second, the vesicle and target membranes must fuse, thereby delivering the contents of the vesicle to the target organelle. Research over the last several years has led to development of a model of vesicle fusion in which specific recognition between a vesicle and its target is mediated by interactions between unique pairs of transmembrane proteins, followed by fusion between the phospholipid bilayers of the vesicle and target membranes.

Proteins involved in vesicle fusion were initially identified in James Rothman's laboratory by biochemical analysis of reconstituted vesicular transport systems from mammalian cells (see Figure 9.30). Analysis of the proteins involved in vesicle fusion in these systems led Rothman and his colleagues to propose a general model, called the SNARE hypothesis, in which vesicle fusion is mediated by interactions between specific pairs of proteins, called SNAREs, on the vesicle and target membranes (v-SNAREs and t-SNAREs, respectively) (Figure 9.33).

Figure 9.33. Vesicle fusion Vesicle fusion is mediated by binding between specific pairs of v-SNAREs and t-SNAREs on the vesicle and target membranes, respectively. Rab GTP-binding proteins are required to facilitate formation of v-SNARE/t-SNARE complexes. Following membrane fusion, the NSF/SNAP proteins disassemble the SNARE complex.

This hypothesis was supported by the identification of SNAREs that were present on synaptic vesicles and by the finding of yeast secretion mutants that appeared to encode SNAREs required for a variety of vesicle transport events. For example, transport from the ER to the Golgi in yeast requires specific SNAREs that are located on both the vesicle and target membranes. The formation of complexes between v-SNAREs on the vesicle and t-SNAREs on the target membranes then leads to membrane fusion, by mechanisms which remain to be fully understood.

In addition to SNAREs, vesicle fusion requires at least two other types of proteins. The Rab proteins are a family of small GTP-binding proteins that are related to the Ras proteins, which were discussed in Chapter 7. More than 30 different Rab proteins have been identified and shown to function in specific vesicle transport processes. They may function in several steps of vesicle trafficking, including interacting with SNAREs to regulate and facilitate the formation of v-SNARE/t-SNARE complexes.

Following the formation of complexes between complementary SNAREs and membrane fusion, a complex of two additional proteins (the NSF/ SNAP complex) is needed to complete the process of vesicle transport. The NSF/SNAP proteins are recruited to membranes following the formation of v-SNARE/t-SNARE complexes, and they are not required directly for either vesicle/target pairing or for the fusion of paired membranes. Instead, the NSF/SNAP proteins act after membrane fusion to disassemble the SNARE complex, thereby allowing the SNAREs to be reutilized for subsequent rounds of vesicle transport.

Lysosomes

Lysosomes are membrane-enclosed organelles that contain an array of enzymes capable of breaking down all types of biological polymers[pic]proteins, nucleic acids, carbohydrates, and lipids. Lysosomes function as the digestive system of the cell, serving both to degrade material taken up from outside the cell and to digest obsolete components of the cell itself. In their simplest form, lysosomes are visualized as dense spherical vacuoles, but they can display considerable variation in size and shape as a result of differences in the materials that have been taken up for digestion. Lysosomes thus represent morphologically diverse organelles defined by the common function of degrading intracellular material.

Lysosomal Acid Hydrolases

Lysosomes contain about 50 different degradative enzymes that can hydrolyze proteins, DNA, RNA, polysaccharides, and lipids. Mutations in the genes that encode these enzymes are responsible for more than 30 different human genetic diseases, which are called lysosomal storage diseases because undegraded material accumulates within the lysosomes of affected individuals. Most of these diseases result from deficiencies in single lysosomal enzymes. For example, Gaucher's disease (the most common of these disorders) results from a mutation in the gene that encodes a lysosomal enzyme required for the breakdown of glycolipids. An intriguing exception is I-cell disease, which is caused by a deficiency in the enzyme that catalyzes the first step in the tagging of lysosomal enzymes with mannose-6-phosphate in the Golgi apparatus (see Figure 9.25). The result is a general failure of lysosomal enzymes to be incorporated into lysosomes.

All of the lysosomal enzymes are acid hydrolases, which are active at the acidic pH (about 5) that is maintained within lysosomes but not at the neutral pH (about 7.2) characteristic of the rest of the cytoplasm (Figure 9.35).

Figure 9.35. Organization of the lysosome Lysosomes contain a variety of acid hydrolases that are active at the acidic pH maintained within the lysosome, but not at the neutral pH of the cytosol. The acidic internal pH of lysosomes results from the action of a proton pump in the lysosomal membrane, which imports protons from the cytosol coupled to ATP hydrolysis.

The requirement of these lysosomal hydrolases for acidic pH provides double protection against uncontrolled digestion of the contents of the cytosol; even if the lysosomal membrane were to break down, the released acid hydrolases would be inactive at the neutral pH of the cytosol. To maintain their acidic internal pH, lysosomes must actively concentrate H+ ions (protons). This is accomplished by a proton pump in the lysosomal membrane, which actively transports protons into the lysosome from the cytosol. This pumping requires expenditure of energy in the form of ATP hydrolysis, since it maintains approximately a hundredfold higher H+ concentration inside the lysosome.

Endocytosis and Lysosome Formation

One of the major functions of lysosomes is the digestion of material taken up from outside the cell by endocytosis, which is discussed in detail in Chapter 12. However, the role of lysosomes in the digestion of material taken up by endocytosis relates not only to the function of lysosomes but also to their formation. In particular, lysosomes are formed by the fusion of transport vesicles budded from the trans Golgi network with endosomes, which contain molecules taken up by endocytosis at the plasma membrane.

The formation of lysosomes thus represents an intersection between the secretory pathway, through which lysosomal proteins are processed, and the endocytic pathway, through which extracellular molecules are taken up at the cell surface (Figure 9.36).

Material from outside the cell is taken up in clathrin-coated endocytic vesicles, which bud from the plasma membrane and then fuse with early endosomes. Membrane components are then recycled to the plasma membrane (discussed in detail in Chapter 12) and the early endosomes gradually mature into late endosomes, which are the precursors to lysosomes. One of the important changes during endosome maturation is the lowering of the internal pH to about 5.5, which plays a key role in the delivery of lysosomal acid hydrolases from the trans Golgi network.

Figure 9.36. Endocytosis and lysosome formation Molecules are taken up from outside the cell in endocytic vesicles, which fuse with early endosomes. Membrane components are recycled as the early endosomes mature into late endosomes. Transport vesicles carrying acid hydrolases from the Golgi apparatus then fuse with late endosomes, which mature into lysosomes as they acquire a full complement of lysosomal enzymes. The acid hydrolases dissociate from the mannose-6-phosphate receptor when the transport vesicles fuse with late endosomes, and the receptors are recycled to the Golgi apparatus.

As discussed earlier, acid hydrolases are targeted to lysosomes by mannose-6-phosphate residues, which are recognized by mannose-6-phosphate receptors in the trans Golgi network and packaged into clathrin-coated vesicles. Following removal of the clathrin coat, these transport vesicles fuse with late endosomes, and the acidic internal pH causes the hydrolases to dissociate from the mannose-6-phosphate receptor (see Figure 9.36). The hydrolases are thus released into the lumen of the endosome, while the receptors remain in the membrane and are eventually recycled to the Golgi. Late endosomes then mature into lysosomes as they acquire a full complement of acid hydrolases, which digest the molecules originally taken up by endocytosis.

Phagocytosis and Autophagy

In addition to degrading molecules taken up by endocytosis, lysosomes digest material derived from two other routes: phagocytosis and autophagy (Figure 9.37). In phagocytosis, specialized cells, such as macrophages, take up and degrade large particles, including bacteria, cell debris, and aged cells that need to be eliminated from the body. Such large particles are taken up in phagocytic vacuoles (phagosomes), which then fuse with lysosomes, resulting in digestion of their contents. The lysosomes formed in this way (phagolysosomes) can be quite large and heterogeneous, since their size and shape is determined by the content of material that is being digested.

Lysosomes are also responsible for autophagy, the gradual turnover of the cell's own components. The first step of autophagy appears to be the enclosure of an organelle (e.g., a mitochondrion) in membrane derived from the ER. The resulting vesicle (an autophagosome) then fuses with a lysosome, and its contents are digested (see Figure 9.37). As discussed in Chapter 7, autophagy is responsible for the gradual turnover of cytoplasmic organelles

Figure 9.37. Lysosomes in phagocytosis and autophagy In phagocytosis, large particles (such as bacteria) are taken up into phagocytic vacuoles or phagosomes. In autophagy, internal organelles (such as mitochondria) are enclosed by membrane fragments from the ER, forming autophagosomes. Both phagosomes and autophagosomes fuse with lysosomes to form large phagolysosomes, in which their contents are digested.

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