cell and then recycle to the plasma membrane, once again to mediate internalization of ligand molecules. For instance, the LDL receptor makes one round trip into and out of the cell every 10-20 minutes, for a total of several hundred trips in its 20-hour life span.
Internalized receptor-ligand complexes commonly follow the pathway depicted for the M6P receptor in Figure 17-23 and the LDL receptor in Figure 17-28. Endocytosed cell-surface receptors typically dissociate from their ligands within late endosomes, which appear as spherical vesicles with tubular branching membranes located a few micrometers from the cell surface. The original experiments that defined the late endosome sorting vesicle utilized the asialoglycoprotein receptor. This liver-specific protein mediates the binding and internal-ization of abnormal glycoproteins whose oligosaccharides terminate in galactose rather than the normal sialic acid, hence the name asialoglycoprotein. Electron microscopy of liver cells perfused with asialoglycoprotein reveal that 5-10 minutes after internalization, ligand molecules are found in the lumen of late endosomes, while the tubular membrane extensions are rich in receptor and rarely contain ligand (Figure 17-29). These findings indicate that the late endosome is the organelle in which receptors and ligands are uncoupled.
The dissociation of receptor-ligand complexes in late en-dosomes occurs not only in the endocytic pathway but also in the delivery of soluble lysosomal enzymes via the secretory pathway (see Figure 17-23). As discussed in Chapter 7, the membranes of late endosomes and lysosomes contain V-class proton pumps that act in concert with CP channels to acidify the vesicle lumen (see Figure 7-10). Most receptors, including the M6P receptor and cell-surface receptors for LDL particles and asialoglycoprotein, bind their ligands tightly at neutral pH but release their ligands if the pH is lowered to 6.0 or below. The late endosome is the first vesicle encountered by receptor-ligand complexes whose luminal pH is sufficiently acidic to promote dissociation of most endocytosed receptors from their tightly bound ligands.
The mechanism by which the LDL receptor releases bound LDL particles is now understood in detail (Figure 17-30). At the endosomal pH of 5.0-5.5, histidine residues in the P-propeller domain of the receptor become protonated, forming a site that can bind with high affinity to the negatively charged repeats in the ligand-binding domain. This intramolecular interaction sequesters the repeats in a conformation that cannot simultaneously bind to apoB-100, thus causing release of the bound LDL particle.
The Endocytic Pathway Delivers Iron to Cells Without Dissociation of Receptor-Transferrin Complex in Endosomes
An exception to the general theme of pH-dependent recep-tor-ligand dissociation in the late endosome occurs in the en-docytic pathway that delivers transferrin-bound iron to cells. A major glycoprotein in the blood, transferrin transports iron to all tissue cells from the liver (the main site of iron storage in the body) and from the intestine (the site of iron absorption). The iron-free form, apotransferrin, binds two Fe3+ ions very tightly to form ferrotransferrin. All mammalian cells contain cell-surface transferrin receptors that avidly bind ferrotransferrin at neutral pH, after which the receptor-bound ferrotransferrin is subjected to endocytosis. Like the components of a LDL particle, the two bound Fe3+ atoms remain in the cell, but the apotransferrin part of the ligand does not dissociate from the receptor and is secreted from the cell within minutes after being endocytosed.
Although apotransferrin remains bound to the transfer-rin receptor at the low pH of late endosomes, changes in pH are critical to functioning of the transferrin endocytic pathway. At a pH below 6.0, the two bound Fe3+ atoms dissociate from ferrotransferrin, are reduced to Fe2+ by an unknown mechanism, and then are exported into the cytosol by an endosomal transporter specific for divalent metal ions. The receptor-apotransferrin complex remaining after dissociation of the iron atoms is recycled back to the cell surface. Although apotransferrin binds tightly to its receptor at a pH of 5.0 or 6.0, it does not bind at neutral pH. Hence the bound apotransferrin dissociates from the transferrin receptor when the recycling vesicles fuse with the plasma membrane and the receptor-ligand complex encounters the neutral pH of the extracellular interstitial fluid or growth medium. The recycled receptor is then free to bind another molecule of ferrotransferrin, and the released apotransferrin is carried in the bloodstream to the liver or intestine to be reloaded with iron.
Specialized Vesicles Deliver Cell Components to the Lysosome for Degradation
The major function of lysosomes is to degrade extracellular materials taken up by the cell and intracellular components under certain conditions. Materials to be degraded must be delivered to the lumen of the lysosome where the various degradative enzymes reside. As just discussed, endocytosed ligands (e.g., LDL particles) that dissociate from their receptors in the late endosome subsequently enter the lysosomal lumen when the membrane of the late endosome fuses with the membrane of the lysosome (see Figure 17-28). Likewise, phagosomes carrying bacteria or other particulate matter can fuse with lysosomes, releasing their contents into the lumen for degradation. However, the delivery of endocytosed membrane proteins and of cytoplasmic materials to lysosomes for degradation poses special problems and involves two unusual types of vesicles.
Transport vesicle I1I ^
Multivesicular endosomal pathway
Multivesicular endosomal pathway
▲ FIGURE 17-31 Delivery of plasma-membrane proteins and cytoplasmic components to the lysosomal interior for degradation. Left: Early endosomes carrying endocytosed plasma-membrane proteins (blue) and vesicles carrying lysosomal membrane proteins (red) from the trans-Golgi network fuse with the late endosome, transferring their membrane proteins to the endosomal membrane (step 1 ). Proteins to be degraded are incorporated into vesicles that bud into the interior of the late endosome, eventually forming a multivesicular endosome containing many such internal vesicles (step 2| ). Fusion of a multivesicular endosome directly with a lysosome releases the internal vesicles into the lumen of the lysosome where they can be degraded (step 3 ). Because proton pumps and other lysomal membrane proteins normally are not incorporated into internal endosomal vesicles, they are delivered to the lysosomal membrane and are protected from degradation. Right: In the autophagic pathway, a cup-shaped structure forms around portions of the cytosol or an organelle such as a peroxisome, as shown here. Continued addition of membrane eventually leads to the formation of an autophagic vesicle that envelopes its contents by two complete membranes (step 1). Fusion of the outer membrane with the membrane of a lysosome releases a single-layer vesicle and its contents into the lysosome interior (step &). [See F. Reggiori and D. J. Klionsky, 2002, Eukaryot. Cell 1:11, and D. J. Katzmann et al., 2002, Nature Rev. Mol. Cell Biol. 3:893.]
Multivesicular Endosomes Resident lysosomal proteins, such as V-class proton pumps and other lysosomal membrane proteins, can carry out their functions and remain in the lysosomal membrane where they are protected from degradation by the soluble hydrolytic enzymes in the lumen. Such proteins are delivered to the lysosomal membrane by transport vesicles that bud from the trans-Golgi network by the same basic mechanisms described in earlier sections. In contrast, endocytosed membrane proteins to be degraded are transferred in their entirety to the interior of the lysosome by a specialized delivery mechanism. Lysosomal degradation of cell-surface receptors for extracellular signaling molecules is a common mechanism for controlling the sensitivity of cells to such signals (Chapter 13). Receptors that become damaged also are targeted for lysosomal degradation.
Early evidence that membranes can be delivered to the lumen of compartments came from electron micrographs showing membrane vesicles and fragments of membranes within endosomes and lysosomes (see Figure 5-20c). Parallel experiments in yeast revealed that endocytosed receptor proteins targeted to the vacuole (the yeast organelle equivalent to the lysosome) were primarily associated with membrane fragments and small vesicles within the interior of the vacuole rather than with the vacuole surface membrane.
These observations suggest that endocytosed membrane proteins can be incorporated into specialized vesicles that form at the endosomal membrane (Figure 17-31, left). Although these vesicles are similar in size and appearance to transport vesicles, they differ topologically. Transport vesicles bud outward from the surface of a donor organelle into the cytosol, whereas vesicles within the endosome bud inward from the surface into the lumen (away from the cytosol). Mature endosomes containing numerous vesicles in their interior are usually called multivesicular endosomes (or bodies). Eventually the surface membrane of a multivesicular endosome fuses with the membrane of a lysosome, thereby delivering its internal vesicles and the membrane proteins they contain into the lysosome interior for degradation. Thus the sorting of proteins in the endosomal membrane determines which ones will remain on the lysosome surface (e.g., pumps and transporters) and which ones will be incorporated into internal vesicles and ultimately degraded in lysosomes.
Autophagic Vesicles The delivery of bulk amounts of cy-tosol or entire organelles to lysosomes and their subsequent degradation is known as autophagy ("eating oneself"). Au-tophagy is often a regulated process and is typically induced in cells placed under conditions of starvation or other types of stress, allowing the cell to recycle macromolecules for use as nutrients.
The autophagic pathway begins with the formation of a flattened double-membraned cup-shaped structure (Figure 17-31, right). This structure can grow by vesicle fusion and eventually seals to form an autophagic vesicle that envelops a region of the cytosol or an entire organelle (e.g., peroxi-some, mitochondrion). Unknown at this time is the origin of the membranes that form the initial cup-shaped organelle and the vesicles that are added to it, but the endosome itself is a likely candidate. The outer membrane of an autophagic vesicle can fuse with the lysosome delivering a large vesicle, bounded by a single membrane bilayer, to the interior of the lysosome. The lipases and proteases within the lysosome eventually will degrade this vesicle and its contents into their molecular components.
Retroviruses Bud from the Plasma Membrane by a Process Similar to Formation of Multivesicular Endosomes
The vesicles that bud into the interior of endosomes have a topology similar to that of enveloped virus particles that bud from the plasma membrane of virus-infected cells. Moreover, recent experiments demonstrate that a common set of proteins are required for both types of membrane-budding events. In fact, the two processes so closely parallel one another in mechanistic detail as to suggest that enveloped viruses have evolved mechanisms to recruit the cellular proteins used in inward endosomal budding for their own purposes.
Many of the proteins required for inward budding of the endosomal membrane were first identified by mutations in yeast that blocked delivery of membrane proteins to the interior of the vacuole. More than 10 such "budding" proteins have been identified in yeast, most with significant similarities to mammalian proteins that evidently perform the same function in mammalian cells. The current model of endosomal budding to form multivesicular endosomes in mammalian cells is based primarily on studies in yeast (Figure 17-32). A ubiquitin-tagged peripheral membrane protein of the endosome, known as Hrs, facilitates loading of specific ubiquitinated membrane cargo proteins into vesicle buds directed into the interior of the endosome. The ubiquitinated Hrs protein then recruits a set of three different protein complexes to the membrane. These ESCRT (endosomal sorting complexes required for transport) complexes include the ubiquitin-binding protein Tsg101. The membrane-associated ESCRT complexes act to complete vesicle budding, leading to release of a vesicle carrying specific membrane cargo into the interior of the endosome. Finally, an ATPase, known as Vps4, uses the energy from ATP hydrolysis to disassemble the ESCRT complexes, releasing them into the cytosol for another round of budding. In the fusion event that pinches off a completed endosomal vesicle, the ESCRT proteins and Vps4 may function like SNAREs and NSF, respectively, in the typical membrane-fusion process discussed previously (see Figure 17-11).
The human immunodeficiency virus (HIV) is an enveloped retrovirus that buds from the plasma membrane of infected cells in a process driven by viral Gag protein, the major structural component of completed virus particles. Gag protein binds to the plasma membrane of an infected cell and «4000 Gag molecules polymerize into a spherical
► FIGURE 17-32 Model of the common mechanism for formation of multivesicular endosomes and budding of HIV from the plasma membrane.
Bottom: In endosomal budding, ubiquitinated Hrs on the endosomal membrane directs loading of specific membrane cargo proteins (blue) into vesicle buds and then recruits cytosolic ESCRT complexes to the membrane (step 1). Note that both Hrs and the recruited cargo proteins are tagged with ubiquitin. After the set of bound ESCRT complexes mediate membrane fusion and pinching off of the completed vesicle (step 2|), they are disasssembled by the ATPase Vps4 and returned to the cytosol (step 3). Top: Budding of HIV particles from HIV-infected cells occurs by a similar mechanism using the virally encoded Gag protein and cellular ESCRT complexes and Vps4 (steps 4|- 6). Ubiquitinated Gag near a budding particle functions like Hrs. See text for discussion. [Adapted from O. Pornillos et al., 2002, Trends Cell Biol. 12:569.]
shell, producing a structure that looks like a vesicle bud protruding outward from the plasma membrane. Mutational studies with HIV have revealed that the N-terminal segment of Gag protein is required for association with the plasma membrane, whereas the C-terminal segment is required for pinching off of complete HIV particles. For instance, if the portion of the viral genome encoding the C-terminus of Gag is removed, HIV buds will form in infected cells, but pinching off does not occur and thus no free virus particles are released.
The first indication that HIV budding employs the same molecular machinery as vesicle budding into endosomes
► FIGURE 17-33 Electron micrographs of virus budding from wild-type and ESCRT-deficient HIV-infected cells. (a) In wild-type cells infected with HIV, virus particles bud from the plasma membrane and are rapidly released into the extracellular space. (b) In cells that lack the functional ESCRT protein Tsg101, the viral Gag protein forms dense virus-like structures, but budding of these structures from the plasma membrane cannot be completed and chains of incomplete viral buds still attached to the plasma membrane accumulate. [Wes Sundquist, University of Utah.]
came from the observation that Tsg101, a component of the ESCRT complex, binds to the C-terminus of Gag protein. Subsequent findings have clearly established the mechanistic parallels between the two processes (Figure 17-32). For example, Gag is ubiquitinated as part of the process of virus budding, and in cells with mutations in Tsg101 or Vps4, HIV virus buds accumulate but cannot pinch off from the membrane (Figure 17-33). Moreover, when a segment from the cellular Hrs protein is added to a truncated Gag protein, proper budding and release of virus particles is restored. Taken together, these results indicate that Gag protein mimics the function of Hrs, redirecting ESCRT complexes to the
plasma membrane where they can function in the budding of virus particles.
Other enveloped retroviruses such as murine leukemia virus and Rous sarcoma virus also have been shown to require ESCRT complexes for their budding, although each virus appears to have evolved a somewhat different mechanism to recruit ESCRT complexes to the site of virus budding.
Transcytosis Moves Some Endocytosed Ligands Across an Epithelial Cell Layer
As noted previously, transcytosis is used by some cells in the apical-basolateral sorting of certain membrane proteins (see Figure 17-26). This process of transcellular transport, which combines endocytosis and exocytosis, also can be employed to import an extracellular ligand from one side of a cell, transport it across the cytoplasm, and secrete it from the plasma membrane at the opposite side. Transcytosis occurs mainly in sheets of polarized epithelial cells.
Maternal immunoglobulins (antibodies) contained in ingested breast milk are transported across the intestinal epithelial cells of the newborn mouse and human by transcytosis (Figure 17-34). The Fc receptor that mediates this movement binds antibodies at the acidic pH of 6 found in the intestinal lumen but not at the neutral pH of the extracellular fluid on the basal side of the intestinal epithelium. This difference in the pH of the extracellular media on the two sides of intes-
Fc region Fc receptor Membrane
Blood and interstitial fluid (pH ~7)
Fc region Fc receptor Membrane
Blood and interstitial fluid (pH ~7)
Intestinal lumen (pH ~6)
Intestinal lumen (pH ~6)
▲ FIGURE 17-34 Transcytosis of maternal IgG immunoglobulins across the intestinal epithelial cells of newborn mice. This transcellular movement of a ligand involves both endocytosis and exocytosis. The one-way movement of ligand from the intestinal lumen to the blood depends on the differential affinity of the Fc receptor for antibody at pH 6 (strong binding) and at pH 7 (weak binding). Transcytosis in the opposite direction returns the empty Fc receptor to the luminal membrane. See text for discussion.
tinal epithelial cells allows maternal immunoglobulins to move in one direction—from the lumen to the blood. The same process also moves circulating maternal immunoglobulins across mammalian yolk-sac cells into the fetus.
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